**Membranous Nephropathy**

 Abdelaziz Elsanjak and Sharma S. Prabhakar  *Department of Medicine, Texas Tech University Health Sciences Center USA* 

### **1. Introduction**

Membranous nephropathy (MN), a very common cause of nephrotic syndrome, is a glomerulopathy defined histopathologically by the presence of immune complexes on the extracapillary side of the glomerular basement membrane (GBM). Idiopathic membranous nephropathy (IMN) is an antibody-mediated glomerular disease with no defined etiology, histologically characterized by uniform thickening of glomerular basement membrane (GBM), caused by subepithelial immune complex deposits. Most cases of MN are idiopathic, for instance approximately 75% of the cases of MN in developed countries are idiopathic, or primary membranous nephropathy (IMN). MN can be secondary to a wide spectrum of infections, tumors, autoimmune diseases or exposure to drugs or toxic agents. Examples include systemic lupus erythematosus, hepatitis B antigenemia or other chronic infections, and historically graft vs. host disease, sickle cell anemia, a number of drugs and toxins such as therapeutic gold salts, penicillamine, tumors, and agents containing mercury

Idiopathic MN is a glomerulus-specific autoimmune disease and second only to focal glomerulosclerosis, is a leading primary cause of the nephrotic syndrome in adults. The name, '*membranous* nephropathy' reflects the pathological observation in light microscopy of thickening in the GBM between and around immune deposits that occur beneath the podocyte foot processes. The histological hallmarks of the disease were first described by Jones and Mellors and Ortega' over 60 years ago. These include "spikes," stained by methenamine silver, of normal GBM that extend between the immune deposits, a fine granular distribution of immunoglobulin (lg) G and the complement component C3 in a capillary-loop pattern revealed by immunofluorescence, and the presence of electron-dense subepithelial immune deposits indicated by electron microscopy (EM). Idiopathic MN most commonly occurs in patients between the ages of 30 and 60 years, with men twice as likely to be affected as women. However, MN does occur in children as well as in the very elderly. Up to 70% of patients present with the nephrotic syndrome and the others garner clinical attention due to abnormalities in urine sediment such as proteinuria. Microscopic hematuria is observed in up to 50% of cases although red cell casts are rare. Hypertension and impaired renal function are uncommon at the outset of the disease and are more likely to occur with disease progression

### **2. Pathogenesis**

Idiopathic membranous nephropathy (IMN) is an antibody-mediated glomerular disease that is histologically characterized by uniform thickening of glomerular basement membrane (GBM), caused by subepithelial immune complex deposits. The immune

Membranous Nephropathy 5

T cells play a significant role in the pathogenesis. The presence of IgG4, which is a product of the type 2 response T helper cells (Th2) and an upregulation of cytokines, such as interleukins (IL) -4 and -10, suggest Th2 involvement. This CD4, T-cell dependent humoral

More recently Stanescu et al (2011) performed independent genome-wide association studies of single-nucleotide polymorphisms (SNPs) in patients with idiopathic membranous nephropathy from three populations of white ancestry (75 French, 146 Dutch, and 335 British patients). The patients were compared with racially matched control subjects; population stratification and quality controls were carried out according to standard criteria. In a joint analysis of data from the 556 patients studied (398 men), they identified significant alleles at two genomic loci associated with idiopathic membranous nephropathy. Chromosome 2q24 contains the gene encoding M-type phospholipase A2 receptor (PLA2R1) (SNP rs4664308, P=8.6×10−29), previously shown to be the target of an autoimmune response. Chromosome 6p21 contains the gene encoding HLA complex class II HLA-DQ alpha chain 1 (HLA-DQA1) (SNP rs2187668, P=8.0×10−93). The association with HLA-DQA1 was significant in all three populations (P=1.8×10−9, P=5.6×10−27, and P=5.2×10−36 in the French, Dutch, and British groups, respectively). The odds ratio for idiopathic membranous nephropathy with homozygosity for both risk alleles was 78.5 (95% confidence interval, 34.6 to 178.2). They concluded that an HLA-DQA1 allele on chromosome 6p21 is most closely associated with idiopathic membranous nephropathy in persons of white ancestry. This allele may facilitate an autoimmune response against targets such as variants of *PLA2R1*, findings which suggest a basis for understanding this disease and illuminate how adaptive

MN is a chronic disease, with spontaneous remission and relapses. In the United States and Europe, MN remains the second or third leading cause of ESRD among the primary glomerulonephritis types. Spontaneous remissions occur in up to 30% of cases and usually occur within the first 2 yrs after presentation. The percentage of patients going into spontaneous remission is much lower in patients with higher grades of proteinuria at presentation (*e.g.*, proteinuria >8 g/24 h). The remaining two thirds are divided into those with persistent proteinuria who maintain renal function long term, or who progress to renal failure. In white patients with NS, 10-yr kidney survival of 70% has been reported. Although the percentage of the IMGN population that progresses to end-stage renal failure remains relatively small, the absolute numbers are large. It affects people predominantly in their 30s and 40s, and has an enormous long-term impact on their quality of life and productivity. Because they have single-organ disease rather than multisystem organ failure (as is seen in diabetes), they survive longer on dialysis and after renal transplantation. However, even though these patients survive longer, they continue to function at a lower level in comparison with the age and gender matched normal population, and rarely returns to the same level of productivity or quality of life as their peers. Even in patients who do not progress to ESRD, complications often occur, including life-threatening thromboembolic phenomena and accelerated vascular disease. These may be due to an underlying specific defect in coagulation and/or tissue repair and/or the long-term sequelae of their prolonged nephrotic condition. Today, once the diagnosis is made, the management of edema, BP, and hyperlipidemia is effective in almost all IMGN patients. The impact of the control of these factors alone on the

response leads to subsequent Ig deposition and complement activation.

immunity is regulated by HLA.

**2.1 Natural history and prognosis of idiopathic MN** 

deposits consist of IgG, mainly IgG4 and IgG1 of antigens that have long escaped identification, and the membrane attack complex of complement C5b-9 (MAC). The formation of subepithelial immune deposits and complement activation are responsible for functional impairment of the glomerular capillary wall, causing proteinuria .Most data on the pathogenesis of MN comes from an animal model, the Heymann model of experimental MN in rats, which suggests that the podocyte is the target of injury. Studies show that there is in-situ binding of a circulating antibody to antigen in the subepithelial space. In the Heymann nephritis model, megalin was identified as the antigenic target. However, megalin, which is a member of the low-density lipoprotein receptor family and is expressed with clathrin at the base of podocyte foot processes (the site of immune complex formation) in rats, is an unlikely antigen for human MN.

In Heymann nephritis model of MN, rats are immunized against an antigenic fraction derived from rat proximal tubular brush border and develop subepithelial deposits virtually identical to those observed in human disease. The target antigen is a large transmembrane endocytic receptor known as megalin. In the rat (but not in human beings) megalin is additionally present on the foot processes of podocytes, allowing circulating antimegalin antibodies to cross the GBM, bind megalin at the podocyte cell surface, and ultimately form subepithelial immune deposits in situ. Complement, activated by the immune deposits, leads to insertion of the terminal complement components C5b-9 (the membrane attack complex) into the podocyte cell membrane, causing cell injury, effacement of the foot processes, and proteinuria.

In 2002, Debiec and Ronco identified neutral endopeptidase (NEP) as the responsible antigen in a rare subset of patients with alloimmune antenatal membranous nephropathy. This discovery supplied proof of concept that a human podocyte antigen could serve as a target for nephritogenic antibodies, as shown some 20 years earlier for the rat podocyte megalin in Heymann nephritis, the well established experimental model of membranous nephropathy.

Debiec and Ronco studied the development of neonatal MN in infants born of mothers genetically lacking neutral endopeptidase (NEP), a membrane-associated podocyte antigen that digests peptides. Because the fetus did not lack NEP, fetomaternal alloimmunization occurred and anti-NEP antibodies (often in very high titers) developed in the mothers. These antibodies (often of the IgG4 or IgG1 subclasses, similar to human idiopathic MN) crossed the placental barrier and interacted with the NEP, heavily expressed on the normal fetal podocyte. In situ immune complexes (containing both IgG1 and IgG4) developed in the newborn infant (or soon after birth) and typical MN ensued, along with proteinuria and nephrotic syndrome. The finding of the C5b-C9 membrane attack complex in the deposits, suggesting that this spontaneous human alloimmune disease also might be complementdependent, was similar to what had been proposed for Heymann nephritis.

In 2009 Beck and et al identified circulating autoantibodies reactive with the transmembrane glycoprotein M-type phospholipase A2 receptor (PLA2R) in the majority of cases of adult IMN. This protein is expressed by the human podocyte, again suggesting a mechanism of disease that fits the paradigm established in Heymann nephritis. These anti-PLA2R autoantibodies were highly specific for IMN, and were not present in normal individuals, in patients with other causes of the nephrotic syndrome, or in cases of secondary MN. Levels of circulating anti- PLA2R antibodies parallel the course of clinical disease, declining or disappearing before a partial or complete remission of proteinuria, and reappearing with recurrence of nephrotic syndrome

deposits consist of IgG, mainly IgG4 and IgG1 of antigens that have long escaped identification, and the membrane attack complex of complement C5b-9 (MAC). The formation of subepithelial immune deposits and complement activation are responsible for functional impairment of the glomerular capillary wall, causing proteinuria .Most data on the pathogenesis of MN comes from an animal model, the Heymann model of experimental MN in rats, which suggests that the podocyte is the target of injury. Studies show that there is in-situ binding of a circulating antibody to antigen in the subepithelial space. In the Heymann nephritis model, megalin was identified as the antigenic target. However, megalin, which is a member of the low-density lipoprotein receptor family and is expressed with clathrin at the base of podocyte foot processes (the site of immune complex formation)

In Heymann nephritis model of MN, rats are immunized against an antigenic fraction derived from rat proximal tubular brush border and develop subepithelial deposits virtually identical to those observed in human disease. The target antigen is a large transmembrane endocytic receptor known as megalin. In the rat (but not in human beings) megalin is additionally present on the foot processes of podocytes, allowing circulating antimegalin antibodies to cross the GBM, bind megalin at the podocyte cell surface, and ultimately form subepithelial immune deposits in situ. Complement, activated by the immune deposits, leads to insertion of the terminal complement components C5b-9 (the membrane attack complex) into the podocyte cell membrane, causing cell injury, effacement of the foot

In 2002, Debiec and Ronco identified neutral endopeptidase (NEP) as the responsible antigen in a rare subset of patients with alloimmune antenatal membranous nephropathy. This discovery supplied proof of concept that a human podocyte antigen could serve as a target for nephritogenic antibodies, as shown some 20 years earlier for the rat podocyte megalin in Heymann nephritis, the well established experimental model of membranous

Debiec and Ronco studied the development of neonatal MN in infants born of mothers genetically lacking neutral endopeptidase (NEP), a membrane-associated podocyte antigen that digests peptides. Because the fetus did not lack NEP, fetomaternal alloimmunization occurred and anti-NEP antibodies (often in very high titers) developed in the mothers. These antibodies (often of the IgG4 or IgG1 subclasses, similar to human idiopathic MN) crossed the placental barrier and interacted with the NEP, heavily expressed on the normal fetal podocyte. In situ immune complexes (containing both IgG1 and IgG4) developed in the newborn infant (or soon after birth) and typical MN ensued, along with proteinuria and nephrotic syndrome. The finding of the C5b-C9 membrane attack complex in the deposits, suggesting that this spontaneous human alloimmune disease also might be complement-

In 2009 Beck and et al identified circulating autoantibodies reactive with the transmembrane glycoprotein M-type phospholipase A2 receptor (PLA2R) in the majority of cases of adult IMN. This protein is expressed by the human podocyte, again suggesting a mechanism of disease that fits the paradigm established in Heymann nephritis. These anti-PLA2R autoantibodies were highly specific for IMN, and were not present in normal individuals, in patients with other causes of the nephrotic syndrome, or in cases of secondary MN. Levels of circulating anti- PLA2R antibodies parallel the course of clinical disease, declining or disappearing before a partial or complete remission of proteinuria, and reappearing with

dependent, was similar to what had been proposed for Heymann nephritis.

in rats, is an unlikely antigen for human MN.

processes, and proteinuria.

recurrence of nephrotic syndrome

nephropathy.

T cells play a significant role in the pathogenesis. The presence of IgG4, which is a product of the type 2 response T helper cells (Th2) and an upregulation of cytokines, such as interleukins (IL) -4 and -10, suggest Th2 involvement. This CD4, T-cell dependent humoral response leads to subsequent Ig deposition and complement activation.

More recently Stanescu et al (2011) performed independent genome-wide association studies of single-nucleotide polymorphisms (SNPs) in patients with idiopathic membranous nephropathy from three populations of white ancestry (75 French, 146 Dutch, and 335 British patients). The patients were compared with racially matched control subjects; population stratification and quality controls were carried out according to standard criteria. In a joint analysis of data from the 556 patients studied (398 men), they identified significant alleles at two genomic loci associated with idiopathic membranous nephropathy. Chromosome 2q24 contains the gene encoding M-type phospholipase A2 receptor (PLA2R1) (SNP rs4664308, P=8.6×10−29), previously shown to be the target of an autoimmune response. Chromosome 6p21 contains the gene encoding HLA complex class II HLA-DQ alpha chain 1 (HLA-DQA1) (SNP rs2187668, P=8.0×10−93). The association with HLA-DQA1 was significant in all three populations (P=1.8×10−9, P=5.6×10−27, and P=5.2×10−36 in the French, Dutch, and British groups, respectively). The odds ratio for idiopathic membranous nephropathy with homozygosity for both risk alleles was 78.5 (95% confidence interval, 34.6 to 178.2). They concluded that an HLA-DQA1 allele on chromosome 6p21 is most closely associated with idiopathic membranous nephropathy in persons of white ancestry. This allele may facilitate an autoimmune response against targets such as variants of *PLA2R1*, findings which suggest a basis for understanding this disease and illuminate how adaptive immunity is regulated by HLA.

#### **2.1 Natural history and prognosis of idiopathic MN**

MN is a chronic disease, with spontaneous remission and relapses. In the United States and Europe, MN remains the second or third leading cause of ESRD among the primary glomerulonephritis types. Spontaneous remissions occur in up to 30% of cases and usually occur within the first 2 yrs after presentation. The percentage of patients going into spontaneous remission is much lower in patients with higher grades of proteinuria at presentation (*e.g.*, proteinuria >8 g/24 h). The remaining two thirds are divided into those with persistent proteinuria who maintain renal function long term, or who progress to renal failure. In white patients with NS, 10-yr kidney survival of 70% has been reported. Although the percentage of the IMGN population that progresses to end-stage renal failure remains relatively small, the absolute numbers are large. It affects people predominantly in their 30s and 40s, and has an enormous long-term impact on their quality of life and productivity. Because they have single-organ disease rather than multisystem organ failure (as is seen in diabetes), they survive longer on dialysis and after renal transplantation. However, even though these patients survive longer, they continue to function at a lower level in comparison with the age and gender matched normal population, and rarely returns to the same level of productivity or quality of life as their peers. Even in patients who do not progress to ESRD, complications often occur, including life-threatening thromboembolic phenomena and accelerated vascular disease. These may be due to an underlying specific defect in coagulation and/or tissue repair and/or the long-term sequelae of their prolonged nephrotic condition.

Today, once the diagnosis is made, the management of edema, BP, and hyperlipidemia is effective in almost all IMGN patients. The impact of the control of these factors alone on the

Membranous Nephropathy 7

Finally, MN may briefly occur early in infancy as a result of feto-maternal alloimmunization. Idiopathic MN must be distinguished from the various secondary causes, since treating or eliminating those underlying conditions are often sufficient to cause nephrotic syndrome

The most common secondary form of MN in the United States (US) is membranous lupus nephritis (LN), designated class V LN by the International Society of Nephrology-Renal Pathology Society, and is seen in -10%-20% of LN cases (picture of LMN). The disease may occur in isolation and pre-date other symptoms or serological abnormalities suggestive of lupus. Thus, even in the absence of positive serological markers such as antinuclear antibodies (ANAs), membranous LN should remain a possibility in any young woman with a biopsy diagnosis of MN. Features that distinguish idiopathic MN from membranous LN and other secondary forms of MN include the glomerular location of the immune deposits, the predominance of a particular IgG subclass, and other pathological features. Clues to the diagnosis of membranous LN include the presence of subendothelial and mesangial deposits, in addition to the predominant subepithelial deposits, and a "full house" pattern of staining for IgG, IgA, IgM, C3, and Clq on immunofluorescence. In idiopathic MN the predominant IgG subclass found in the glomerular deposits is IgG4, whereas in many secondary forms, IgGl, IgG2, and IgG3 predorninate. Finally, an ultrastructural finding of tubuloreticular structures in the glomerular endothelium suggests lupus, although these

Currently, renal biopsy is the sole means for diagnosis of MN and distinguishing it from other causes of nephrotic syndrome. The results of routine serological studies, including complement levels, are all normal in idiopathic MN. Possibly, antibodies to the human phospholipase A2 receptor (PLA2R) found in many patients with idiopathic MN may allow a serological diagnosis of MN, but this test is only available in research settings. Secondary causes of MN may be suggested by the presence of ANA, hepatitis B virus (HBV) antigenemia, or concurrent infection with schistosomiasis or secondary syphilis. Hypocomplementemia may occur in lupus or HBV-associated MN, but normal complement levels do not rule out these diagnoses. Associations of MN with malignancy have been found in older individuals seemingly more frequent than chance. Therefore, in older

individuals with newly diagnosed MN, tests to exclude malignancy is reasonable.

A number of secondary processes can also cause MN that are clinically and histologically similar to IMN. Worldwide, chronic infections such as hepatitis B, malaria, syphilis, and schistosomiasis are the most important causes of secondary MN. Systemic lupus erythematosus can give rise to a membranous form of glomerular disease, classified as class V lupus nephritis. Other autoimmune diseases such as rheumatoid arthritis, autoimmune thyroid diseases, and Sjogren's syndrome can all be associated with MN. Historically, certain medications used for the treatment of rheumatoid arthritis such as gold salts, penicillamine, and some NSAIDs were causally linked to MN. Solid tumors are associated with secondary MN more often than chance alone would predict, and on rare occasions remissions and relapses of the glomerular disease have been noted to occur with removal or

Finally, MN can occur de novo after renal transplantation or allogeneic hematopoietic stem cell transplantation, perhaps reflecting alloimmunization to a minor histocompatibility antigen expressed in the glomerulus. Secondary forms of MN often exhibit histopathological clues that distinguish them from IMN, although this is not always the case. As opposed to the exclusively subepithelial and intra-membranous deposits seen in IMN, secondary forms,

structures can also be found in other non-idiopathic forms of MN.

remission.

relapse of the malignancy.

natural history is expected to be positive but is currently unknown. This is partly due to the unusual phenomena of up to 30% of IMGN patients experiencing spontaneous remission. This wide variation in outcome is one of the factors that has led meta-analysis and systematic reviews of this disease to reach varying conclusions about the impact of immunosuppressive treatment on patient and renal survival and on proteinuria remission rates.

Female gender and low grade proteinuria is associated with good prognosis and associated with spontaneous remission. End-stage renal disease occurs at a 2-3:1 male:female ratio. Also, Asians with IMN appear to have a more favorable long-term prognosis than their non-Asian counterparts.

The Toronto Glomerulonephritis Registry created a model for identifying patients at risk for progression of renal insufficiency, taking into account the initial creatinine clearance (CrCl), the slope of the CrCl, and the lowest amount of proteinuria during a 6-month period. According to this model, patients who present with a normal CrCl (proteinuria <4 g/24 h), and stable renal function over 6 months are considered to be at low risk for progression. On the other hand, patients with persistent proteinuria (>8 g/24 h) have a 66−80% probability of progression to ESRD within 10 years, independent of the degree of renal dysfunction. Other factors associated with poor prognosis include older age, tubular interstitial changes on kidney biopsy and a high degree of glomerulosclerosis.

### **2.2 Clinical manifestations**

MN affects patients of all ages and races, but is generally more common in men than women. It most commonly occurs in middle age, with peak incidence between the ages of 40-60. In contrast to primary MN, secondary forms of MN are most commonly encountered in young children and in individuals who are older than 60. At presentation, 60-70% of patients have nephrotic syndrome with the remaining 30-40% of patients presenting with subnephrotic range proteinuria (<3.5 g/24 h). 60 % of patients who present with subnephrotic range proteinuria will progress to full nephrotic syndrome in 1-2 years (Daniel Cattran 2006). Microscopic hematuria is common in MN (30 to 40%), but macroscopic hematuria and red cell casts are rare and should suggest other diagnoses .The majority of patients with MN are normotensive at presentation, however hypertension is present in 10- 20 % of patients. Less than 20% present with renal insufficiency.

Serologic evaluation of all patients with MN should include anti-nuclear antibody HbsAg and hepatitis C virus antibody studies. Workup for malignancy is also warranted, to the extent that testing should be guided by the patient's age and whether there is a history of tobacco use.

#### **2.3 Primary (idiopathic) vs. secondary forms of membraneous nephropathy**

In developed countries, MN is primarily idiopathic, implying that known secondary causes have been effectively ruled out. Secondary forms of MN have been linked to multiple different agents and conditions. MN occurring post-hematopoietic stem cell transplantation (HSCT) may be a humoral manifestation of chronic graft-versus-host disease; it is the most common cause of post-HSCT nephrotic syndrome, and like idiopathic, post-HSCT MN disproportionately affects males. MN may recur in up to 42% of renal allografts with slowly progressive proteinuria; it is also possible for *de novo* MN to occur, perhaps as *an*  alloimmune reactivation to minor histocompatibility antigens on the allograft podocytes.

natural history is expected to be positive but is currently unknown. This is partly due to the unusual phenomena of up to 30% of IMGN patients experiencing spontaneous remission. This wide variation in outcome is one of the factors that has led meta-analysis and systematic reviews of this disease to reach varying conclusions about the impact of immunosuppressive treatment on patient and renal survival and on proteinuria remission

Female gender and low grade proteinuria is associated with good prognosis and associated with spontaneous remission. End-stage renal disease occurs at a 2-3:1 male:female ratio. Also, Asians with IMN appear to have a more favorable long-term prognosis than their non-

The Toronto Glomerulonephritis Registry created a model for identifying patients at risk for progression of renal insufficiency, taking into account the initial creatinine clearance (CrCl), the slope of the CrCl, and the lowest amount of proteinuria during a 6-month period. According to this model, patients who present with a normal CrCl (proteinuria <4 g/24 h), and stable renal function over 6 months are considered to be at low risk for progression. On the other hand, patients with persistent proteinuria (>8 g/24 h) have a 66−80% probability of progression to ESRD within 10 years, independent of the degree of renal dysfunction. Other factors associated with poor prognosis include older age, tubular interstitial changes

MN affects patients of all ages and races, but is generally more common in men than women. It most commonly occurs in middle age, with peak incidence between the ages of 40-60. In contrast to primary MN, secondary forms of MN are most commonly encountered in young children and in individuals who are older than 60. At presentation, 60-70% of patients have nephrotic syndrome with the remaining 30-40% of patients presenting with subnephrotic range proteinuria (<3.5 g/24 h). 60 % of patients who present with subnephrotic range proteinuria will progress to full nephrotic syndrome in 1-2 years (Daniel Cattran 2006). Microscopic hematuria is common in MN (30 to 40%), but macroscopic hematuria and red cell casts are rare and should suggest other diagnoses .The majority of patients with MN are normotensive at presentation, however hypertension is present in 10-

Serologic evaluation of all patients with MN should include anti-nuclear antibody HbsAg and hepatitis C virus antibody studies. Workup for malignancy is also warranted, to the extent that testing should be guided by the patient's age and whether there is a history of

In developed countries, MN is primarily idiopathic, implying that known secondary causes have been effectively ruled out. Secondary forms of MN have been linked to multiple different agents and conditions. MN occurring post-hematopoietic stem cell transplantation (HSCT) may be a humoral manifestation of chronic graft-versus-host disease; it is the most common cause of post-HSCT nephrotic syndrome, and like idiopathic, post-HSCT MN disproportionately affects males. MN may recur in up to 42% of renal allografts with slowly progressive proteinuria; it is also possible for *de novo* MN to occur, perhaps as *an*  alloimmune reactivation to minor histocompatibility antigens on the allograft podocytes.

**2.3 Primary (idiopathic) vs. secondary forms of membraneous nephropathy** 

on kidney biopsy and a high degree of glomerulosclerosis.

20 % of patients. Less than 20% present with renal insufficiency.

rates.

Asian counterparts.

**2.2 Clinical manifestations** 

tobacco use.

Finally, MN may briefly occur early in infancy as a result of feto-maternal alloimmunization. Idiopathic MN must be distinguished from the various secondary causes, since treating or eliminating those underlying conditions are often sufficient to cause nephrotic syndrome remission.

The most common secondary form of MN in the United States (US) is membranous lupus nephritis (LN), designated class V LN by the International Society of Nephrology-Renal Pathology Society, and is seen in -10%-20% of LN cases (picture of LMN). The disease may occur in isolation and pre-date other symptoms or serological abnormalities suggestive of lupus. Thus, even in the absence of positive serological markers such as antinuclear antibodies (ANAs), membranous LN should remain a possibility in any young woman with a biopsy diagnosis of MN. Features that distinguish idiopathic MN from membranous LN and other secondary forms of MN include the glomerular location of the immune deposits, the predominance of a particular IgG subclass, and other pathological features. Clues to the diagnosis of membranous LN include the presence of subendothelial and mesangial deposits, in addition to the predominant subepithelial deposits, and a "full house" pattern of staining for IgG, IgA, IgM, C3, and Clq on immunofluorescence. In idiopathic MN the predominant IgG subclass found in the glomerular deposits is IgG4, whereas in many secondary forms, IgGl, IgG2, and IgG3 predorninate. Finally, an ultrastructural finding of tubuloreticular structures in the glomerular endothelium suggests lupus, although these structures can also be found in other non-idiopathic forms of MN.

Currently, renal biopsy is the sole means for diagnosis of MN and distinguishing it from other causes of nephrotic syndrome. The results of routine serological studies, including complement levels, are all normal in idiopathic MN. Possibly, antibodies to the human phospholipase A2 receptor (PLA2R) found in many patients with idiopathic MN may allow a serological diagnosis of MN, but this test is only available in research settings. Secondary causes of MN may be suggested by the presence of ANA, hepatitis B virus (HBV) antigenemia, or concurrent infection with schistosomiasis or secondary syphilis. Hypocomplementemia may occur in lupus or HBV-associated MN, but normal complement levels do not rule out these diagnoses. Associations of MN with malignancy have been found in older individuals seemingly more frequent than chance. Therefore, in older individuals with newly diagnosed MN, tests to exclude malignancy is reasonable.

A number of secondary processes can also cause MN that are clinically and histologically similar to IMN. Worldwide, chronic infections such as hepatitis B, malaria, syphilis, and schistosomiasis are the most important causes of secondary MN. Systemic lupus erythematosus can give rise to a membranous form of glomerular disease, classified as class V lupus nephritis. Other autoimmune diseases such as rheumatoid arthritis, autoimmune thyroid diseases, and Sjogren's syndrome can all be associated with MN. Historically, certain medications used for the treatment of rheumatoid arthritis such as gold salts, penicillamine, and some NSAIDs were causally linked to MN. Solid tumors are associated with secondary MN more often than chance alone would predict, and on rare occasions remissions and relapses of the glomerular disease have been noted to occur with removal or relapse of the malignancy.

Finally, MN can occur de novo after renal transplantation or allogeneic hematopoietic stem cell transplantation, perhaps reflecting alloimmunization to a minor histocompatibility antigen expressed in the glomerulus. Secondary forms of MN often exhibit histopathological clues that distinguish them from IMN, although this is not always the case. As opposed to the exclusively subepithelial and intra-membranous deposits seen in IMN, secondary forms,

Membranous Nephropathy 9

Idiopathic MN recurs in 10–30% of patients after kidney transplantation. De novo MN, which is the most common de novo glomerulopathy in renal allografts, affects 2–9% of renal allografts. De novo MN occurs 2-3 years post-transplantation, while recurrent MN occurs after 1-2 years. The exact pathogenesis of de novo MN is unknown. Recurrence of membranous nephropathy is preceded by nephrotic range proteinuria. Recurrent membranous nephropathy usually presents sooner after transplantation (within 2 years) than *de novo* membranous nephropathy (after 2 years). Some data suggests that the actual risk of recurrence reaches 29% 3 years after transplantation. Half of the cases of recurrent membranous nephropathy progressed to end-stage renal disease within a decade. There is one case report in literature where the de novo MN was linked to antibody mediated rejection: the patient had donor specific antibody-DQ7. Remission of proteinuria was associated with a fall in the anti-DQ7 titer. This raised the possibility that *de novo*  membranous nephropathy could be a particular manifestation of chronic antibody -

The subtle nature of the light microscopic findings in some cases of Membranous Glomerulonephritis (MGN) and presence of basement membrane thickening in other glomerular diseases lead to the uncertainty in the diagnosis of MGN in its earlier days of evolution as a pathologic entity (Heptinstall R ). Only after the development of electron microscopy and immunologic techniques MGN was distinguished with certainty from other causes of the nephrotic syndrome including minimal change disease and its variants and

The characteristic changes of MGN are seen in glomerular capillary walls. The other compartments may have secondary changes but are usually minor until the advanced stages of disease. The light microscopic appearance may be subtle, especially in early cases; however, in these cases immunopathology and electron microscopy can easily establish the diagnosis. Capillary loops may appear round and rigid in more advanced stages on

The earliest sign by light microscopy is "moth-eaten" appearance of the GBM on silver

The immunofluorescence characteristic of MGN is granular capillary wall staining for immunoglobulins and complement. IgG is present in almost all cases, and C3 staining is seen in approximately three quarter of cases (Fig. 3). The most important and invariable deposit is IgG, and even when other immunoglobulin or complement reactants are seen,

Mesangial deposits are usually not seen. The presence of prominent mesangial deposits or full house positivity with other immunoglobulins or complements should suggest MGN

they have a weaker staining and only segmental presence (Jenette JC 1983).

**2.4 Post-transplantation membranous glomerulopathy** 

mediated rejection (Menon, Shina et al 2010)

**3. Light microscopy** 

hematoxylin and eosin stain (Fig.1).

secondary to systemic lupus erythematosus.

**3.1 Glomeruli** 

stains (fig.2).

**3.2 Immunopathology** 

**2.5 Histopathologic considerations of membranous nephropathy** 

certain forms of chronic glomerulonephritis (Heptinstall).

especially membranous lupus nephritis, often have mesangial and subendothelial deposits. Tubuloreticular inclusions may also been seen within the glomerular endothelium on electron microscopy in lupus-associated MN. The IgG subclasses found within the glomerular deposits also differ. In contrast to the predominant IgG4 found in IMN, IgG2 and IgG3 are typically most abundant in secondary (lupus- and malignancy-associated) forms of MN. Finally, the nature of the electron dense material itself may herald a secondary cause. A form of MN characterized by spherular structures within the subepithelial deposits has been described that appears to be distinct from its idiopathic cousin.

#### Infectious


Drugs


Autoimmune Diseases


Neoplasms


Others


Table 1. Secondary Causes of MN

especially membranous lupus nephritis, often have mesangial and subendothelial deposits. Tubuloreticular inclusions may also been seen within the glomerular endothelium on electron microscopy in lupus-associated MN. The IgG subclasses found within the glomerular deposits also differ. In contrast to the predominant IgG4 found in IMN, IgG2 and IgG3 are typically most abundant in secondary (lupus- and malignancy-associated) forms of MN. Finally, the nature of the electron dense material itself may herald a secondary cause. A form of MN characterized by spherular structures within the subepithelial deposits

has been described that appears to be distinct from its idiopathic cousin.

Infectious

Drugs

 Hepatitis B Hepatitis C

Malaria

 Syphilis Leprosy Tuberculosis Cytomegalovirus

 Captopril Clopidogrel Mercury Penicillamine NSAIDs Gold Autoimmune Diseases

Schistosomiasis

Streptococcal Infections

Systemic Lupus erythematosis

Mixed Connective Tissue Disease

Hematopoietic Stem-cell transplant

Carcinomas of the bladder, breast, pancreas, prostate, stomach cancer, lung

Hematological malignancies: Lymphoma, Chronic Lymphoctic Leukemia

Rheumatoid Arthritis

 Thyroiditis Sjogren's Disease

 Psoriasis Sarcoidosis

cancer.

 Sickle Cell Diabetes Mellitus Post-Transplant

Table 1. Secondary Causes of MN

Neoplasms

Others

#### **2.4 Post-transplantation membranous glomerulopathy**

Idiopathic MN recurs in 10–30% of patients after kidney transplantation. De novo MN, which is the most common de novo glomerulopathy in renal allografts, affects 2–9% of renal allografts. De novo MN occurs 2-3 years post-transplantation, while recurrent MN occurs after 1-2 years. The exact pathogenesis of de novo MN is unknown. Recurrence of membranous nephropathy is preceded by nephrotic range proteinuria. Recurrent membranous nephropathy usually presents sooner after transplantation (within 2 years) than *de novo* membranous nephropathy (after 2 years). Some data suggests that the actual risk of recurrence reaches 29% 3 years after transplantation. Half of the cases of recurrent membranous nephropathy progressed to end-stage renal disease within a decade. There is one case report in literature where the de novo MN was linked to antibody mediated rejection: the patient had donor specific antibody-DQ7. Remission of proteinuria was associated with a fall in the anti-DQ7 titer. This raised the possibility that *de novo*  membranous nephropathy could be a particular manifestation of chronic antibody mediated rejection (Menon, Shina et al 2010)

#### **2.5 Histopathologic considerations of membranous nephropathy**

The subtle nature of the light microscopic findings in some cases of Membranous Glomerulonephritis (MGN) and presence of basement membrane thickening in other glomerular diseases lead to the uncertainty in the diagnosis of MGN in its earlier days of evolution as a pathologic entity (Heptinstall R ). Only after the development of electron microscopy and immunologic techniques MGN was distinguished with certainty from other causes of the nephrotic syndrome including minimal change disease and its variants and certain forms of chronic glomerulonephritis (Heptinstall).

#### **3. Light microscopy**

#### **3.1 Glomeruli**

The characteristic changes of MGN are seen in glomerular capillary walls. The other compartments may have secondary changes but are usually minor until the advanced stages of disease. The light microscopic appearance may be subtle, especially in early cases; however, in these cases immunopathology and electron microscopy can easily establish the diagnosis. Capillary loops may appear round and rigid in more advanced stages on hematoxylin and eosin stain (Fig.1).

The earliest sign by light microscopy is "moth-eaten" appearance of the GBM on silver stains (fig.2).

#### **3.2 Immunopathology**

The immunofluorescence characteristic of MGN is granular capillary wall staining for immunoglobulins and complement. IgG is present in almost all cases, and C3 staining is seen in approximately three quarter of cases (Fig. 3). The most important and invariable deposit is IgG, and even when other immunoglobulin or complement reactants are seen, they have a weaker staining and only segmental presence (Jenette JC 1983).

Mesangial deposits are usually not seen. The presence of prominent mesangial deposits or full house positivity with other immunoglobulins or complements should suggest MGN secondary to systemic lupus erythematosus.

Membranous Nephropathy 11

Fig.3. Membranous glomerulonephropathy. IgG is positive (2+) by immunofluorescence.

Electron microscopy findings helped define MGN by demonstrating the subepithelial and intramembranous (depending on the stage) location of electron-dense deposits (Gartner HV 1974). Electron-dense deposits are seen on the epithelial side of glomerular capillary loops (subepithelial). The location of electron-dense deposits at different levels in the GBM in the course of the disease led to the hypothesis that there is a sequence of changes in the GBM

Tubular changes in MGN include progressive atrophy as the glomerular lesion progresses.

In uncomplicated cases, interstitial fibrosis may be seen without prominent inflammation or tubular atrophy. Development of interstitial fibrosis may reflect a progression of the

The light microscopic differential includes all glomerular diseases that have thickening of the glomerular basement membrane. In context of diseases associated with the nephrotic syndrome, the differential diagnosis includes minimal change disease, focal segmental glomerulosclerosis, membranoproliferative glomerulonephritis, diabetes mellitus and amyloidosis. In the past, before the development of electron microscopy and immunologic

**3.3 Electron microscopic findings** 

glomerular lesion (Magill AB 1995).

**3.6 Differential diagnosis** 

**3.4 Tubules** 

**3.5 Interstitium** 

following initial subepithelial deposition (Ehrenreich T 1968).

Fig. 1. Membranous glomerulonephropathy. Capillary loops may appear round and rigid in advanced cases. (H&E, 40X)

Fig. 2. Membranous glomerulonephropathy. Linear projections or "spikes" protrude from the outer surface of the GBM on silver stains (Periodic acid methenamine silver, 100X)

Fig. 1. Membranous glomerulonephropathy. Capillary loops may appear round and rigid in

Fig. 2. Membranous glomerulonephropathy. Linear projections or "spikes" protrude from the outer surface of the GBM on silver stains (Periodic acid methenamine silver, 100X)

advanced cases. (H&E, 40X)

Fig.3. Membranous glomerulonephropathy. IgG is positive (2+) by immunofluorescence.

#### **3.3 Electron microscopic findings**

Electron microscopy findings helped define MGN by demonstrating the subepithelial and intramembranous (depending on the stage) location of electron-dense deposits (Gartner HV 1974). Electron-dense deposits are seen on the epithelial side of glomerular capillary loops (subepithelial). The location of electron-dense deposits at different levels in the GBM in the course of the disease led to the hypothesis that there is a sequence of changes in the GBM following initial subepithelial deposition (Ehrenreich T 1968).

### **3.4 Tubules**

Tubular changes in MGN include progressive atrophy as the glomerular lesion progresses.

### **3.5 Interstitium**

In uncomplicated cases, interstitial fibrosis may be seen without prominent inflammation or tubular atrophy. Development of interstitial fibrosis may reflect a progression of the glomerular lesion (Magill AB 1995).

### **3.6 Differential diagnosis**

The light microscopic differential includes all glomerular diseases that have thickening of the glomerular basement membrane. In context of diseases associated with the nephrotic syndrome, the differential diagnosis includes minimal change disease, focal segmental glomerulosclerosis, membranoproliferative glomerulonephritis, diabetes mellitus and amyloidosis. In the past, before the development of electron microscopy and immunologic

Membranous Nephropathy 13

tubulointerstitial disease on renal biopsy, and a tubulointerstitial disease score has been included as a prognostic variable in several studies. Although the rate of renal decline may not differ in comparison with MN patients having preserved renal function, patients with a higher serum creatinine or increased interstitial disease at presentation will reach ESRD in a shorter time; therefore, it is advisable to consider early treatment in these patients. Asians with IMN appear to have a more favorable long-term prognosis than their non-Asian

Achieving complete remission predicts an excellent long-term renal prognosis and those patients have nearly universal renal survival at 10 years, whereas the number falls to 90% with partial remission, and 45% with no remission. Cattran and his colleagues (Cattran 2005) proposed a prognostic model dividing patients with into low-, moderate-, and highrisk groups based on their degree of proteinuria and clinical course over 6 months of observation. Those with normal renal function and lower amounts of proteinuria <4 g daily) over 6 months constitute a group at low risk for developing progressive renal insufficiency from the disease. Intermediate levels of proteinuria (4-8 g daily) with stable renal function over 6 months define a group at moderate risk. The highest-risk patients are those with >8 g of daily proteinuria for 6 months, and/or reduced renal function at outset or deterioration of renal function over 6 months. The risk of further renal deterioration in this group is at least

A number of adult studies have allowed practitioners to characterize prognostic factors in adult patients with MN. Laluck et al. showed that low-grade, subnephrotic proteinuria and female gender were associated with spontaneous remission. Ideally, only patients unlikely to spontaneously remit and those at risk for significant renal deterioration should be treated. Male gender, age >50 years, persistent high-grade proteinuria, impaired renal function at onset, presence of segmental glomerular sclerosis, and tubulointerstitial damage on the kidney biopsy have been considered to be poor prognostic factors in adult idiopathic MN . The Toronto Glomerulonephritis Registry created a model for identifying patients at risk for progression of renal insufficiency, taking into account the initial creatinine clearance (CrCl), the slope of the CrCl, and the lowest amount of proteinuria during a 6-month period. According to this model, patients who present with a normal CrCl, proteinuria <4 g/24 h, and stable renal function over 6 months are considered to be at low risk for progression. On the other hand, patients with persistent proteinuria (>8 g/24 h) have a 66−80% probability of progression to ESRD within10 years, independent of the degree of renal dysfunction

Treatment goals in IMN are to prevent loss of renal function and to prevent the complications of the nephrotic syndrome (eg, hyperlipidemia, volume overload hypertension, and thrombophilia). Opinions vary on how best to obtain the desired results, and the literature concerning the treatment of IMN is still unclear. The relatively low incidence of MN hampers recruitment into clinical trials, and the variable natural history of the disease adds further treatment complications. In addition, substantial risks for treatment are associated with established immunosuppressive agents and newer, potentially less toxic agents (eg, mycophenolate or rituximab) have been introduced for the treatment of MN without the benefit of long-term clinical trials. A meta-analysis on 1025 patients with MN from 18 randomized clinical trials concluded that immunosuppressive treatment had no

**5. Treatment of idiopathic membranous nephropathy** 

counterparts.

75%.

**5.1 General outlines** 

techniques, many of these distinctions were made on clinical grounds or not at all. However, currently, characteristic histologic, immunopathologic, and ultrastructural findings can reliably distinguish it from other causes of nephrotic syndrome.

Fig. 4. Electron-dense deposits are present in subepithelial location.

### **3.7 Prognostic indicators**

Ehrenreich and Churg (5) described the stages of membranous transformation as the morphologic representations of progression of the disease. Favorable outcomes are generally related to early stages (I and II) of membranous transformation. However, several later studies (Wahrmann 1989) did not find a relation between glomerular stage and outcome.

## **4. Natural history and prognosis of idiopathic MN**

Although spontaneous remission of nephrotic syndrome occurs in about a third of patients, end-stage renal failure is observed in about 40% of patients after 10 years. Predicting the clinical course of a patient with MN at disease presentation is impossible given the variable and fluctuating disease course. A widely appreciated yet oversimplified view is that onethird of all patients will spontaneously remit without treatment, another third will remain proteinuric with preserved renal function and the final third will progress to end-stage renal disease (ESRD). Young females and those with subnephrotic levels of proteinuria are most likely to experience spontaneous remission, justifying several months of observation prior to any initiation of treatment in the absence of problematic clinical features. Baseline demographic differences in natural-history studies lead to a blurred prognostic picture. Several risk factors for MN progression have been proposed: older age at onset, male sex, nephrotic-range proteinuria (especially >8 g), and increased serum creatinine at presentation. As with most renal diseases, progression correlates with the amount of

techniques, many of these distinctions were made on clinical grounds or not at all. However, currently, characteristic histologic, immunopathologic, and ultrastructural findings can

reliably distinguish it from other causes of nephrotic syndrome.

Fig. 4. Electron-dense deposits are present in subepithelial location.

**4. Natural history and prognosis of idiopathic MN** 

Ehrenreich and Churg (5) described the stages of membranous transformation as the morphologic representations of progression of the disease. Favorable outcomes are generally related to early stages (I and II) of membranous transformation. However, several later studies (Wahrmann 1989) did not find a relation between glomerular stage and

Although spontaneous remission of nephrotic syndrome occurs in about a third of patients, end-stage renal failure is observed in about 40% of patients after 10 years. Predicting the clinical course of a patient with MN at disease presentation is impossible given the variable and fluctuating disease course. A widely appreciated yet oversimplified view is that onethird of all patients will spontaneously remit without treatment, another third will remain proteinuric with preserved renal function and the final third will progress to end-stage renal disease (ESRD). Young females and those with subnephrotic levels of proteinuria are most likely to experience spontaneous remission, justifying several months of observation prior to any initiation of treatment in the absence of problematic clinical features. Baseline demographic differences in natural-history studies lead to a blurred prognostic picture. Several risk factors for MN progression have been proposed: older age at onset, male sex, nephrotic-range proteinuria (especially >8 g), and increased serum creatinine at presentation. As with most renal diseases, progression correlates with the amount of

**3.7 Prognostic indicators** 

outcome.

tubulointerstitial disease on renal biopsy, and a tubulointerstitial disease score has been included as a prognostic variable in several studies. Although the rate of renal decline may not differ in comparison with MN patients having preserved renal function, patients with a higher serum creatinine or increased interstitial disease at presentation will reach ESRD in a shorter time; therefore, it is advisable to consider early treatment in these patients. Asians with IMN appear to have a more favorable long-term prognosis than their non-Asian counterparts.

Achieving complete remission predicts an excellent long-term renal prognosis and those patients have nearly universal renal survival at 10 years, whereas the number falls to 90% with partial remission, and 45% with no remission. Cattran and his colleagues (Cattran 2005) proposed a prognostic model dividing patients with into low-, moderate-, and highrisk groups based on their degree of proteinuria and clinical course over 6 months of observation. Those with normal renal function and lower amounts of proteinuria <4 g daily) over 6 months constitute a group at low risk for developing progressive renal insufficiency from the disease. Intermediate levels of proteinuria (4-8 g daily) with stable renal function over 6 months define a group at moderate risk. The highest-risk patients are those with >8 g of daily proteinuria for 6 months, and/or reduced renal function at outset or deterioration of renal function over 6 months. The risk of further renal deterioration in this group is at least 75%.

A number of adult studies have allowed practitioners to characterize prognostic factors in adult patients with MN. Laluck et al. showed that low-grade, subnephrotic proteinuria and female gender were associated with spontaneous remission. Ideally, only patients unlikely to spontaneously remit and those at risk for significant renal deterioration should be treated. Male gender, age >50 years, persistent high-grade proteinuria, impaired renal function at onset, presence of segmental glomerular sclerosis, and tubulointerstitial damage on the kidney biopsy have been considered to be poor prognostic factors in adult idiopathic MN .

The Toronto Glomerulonephritis Registry created a model for identifying patients at risk for progression of renal insufficiency, taking into account the initial creatinine clearance (CrCl), the slope of the CrCl, and the lowest amount of proteinuria during a 6-month period. According to this model, patients who present with a normal CrCl, proteinuria <4 g/24 h, and stable renal function over 6 months are considered to be at low risk for progression. On the other hand, patients with persistent proteinuria (>8 g/24 h) have a 66−80% probability of progression to ESRD within10 years, independent of the degree of renal dysfunction

### **5. Treatment of idiopathic membranous nephropathy**

#### **5.1 General outlines**

Treatment goals in IMN are to prevent loss of renal function and to prevent the complications of the nephrotic syndrome (eg, hyperlipidemia, volume overload hypertension, and thrombophilia). Opinions vary on how best to obtain the desired results, and the literature concerning the treatment of IMN is still unclear. The relatively low incidence of MN hampers recruitment into clinical trials, and the variable natural history of the disease adds further treatment complications. In addition, substantial risks for treatment are associated with established immunosuppressive agents and newer, potentially less toxic agents (eg, mycophenolate or rituximab) have been introduced for the treatment of MN without the benefit of long-term clinical trials. A meta-analysis on 1025 patients with MN from 18 randomized clinical trials concluded that immunosuppressive treatment had no

Membranous Nephropathy 15

those with reduced GFR. Cancer risk is increased when alkylating agents are used for a long time. There are some reports of increased risk of wegener granulomatosis with cyclophosphamide when the dose is more than 36 gmr (equivalent to 100 mg daily for one year) were associated with a 9.5-fold increased risk of bladder cancer. Use of cyclophosphamide for long time have also been associated with an increased risk of lympho-proliferative disorders. Relapses occur in 25–30% of patients within 5 years of discontinuation of therapy with alkylating agents. While this rate of relapse is lower than that observed after discontinuation of cyclosporine, it is still disconcerting since relapses generally necessitate increased immunosuppression. Despite reduction in proteinuria, these

Cyclosporine is an alternative, clinically validated immunosuppressive agent used in the treatment of IMN. In one randomized clinical trial (RCT), 51 patients with steroidresistant IMN, treatment with cyclosporine plus steroids for 6 months with tapering over 4 weeks resulted in a 75% complete or partial remission rate, versus only 22% in the placebo (steroids alone) group. Typically, many patients in cyclosporine based treatment regimens achieved partial remissions, and many relapsed after discontinuing treatment. Another similarly-sized trial compared 12 months of cyclosporine and corticosteroids to cyclosporine alone." Although both groups achieved -80% remission rates at 12 months, the relapse rate was lower in the group receiving adjunctive corticosteroids from the beginning. Longer courses of cyclosporine (1-2 years) with a slow taper may be necessary to avoid a high rate of relapse. Other investigators demonstrated that treatment with tacrolimus in heavily nephrotic patients resulted in higher remission rates compared with conservative treatment alone; however, nearly half of these patients had a nephritic relapse within several months of tapering tacrolimus. Cyclosporine reduces proteinuria and the rate of decline in renal function in patients with IMN. These effects have been demonstrated in patients with preserved renal function, in those with declining or impaired renal function and also in patients resistant to other immunosuppressants. In some studies almost 50% of patients who had achieved remission relapsed within 1 year of cyclosporine withdrawal, especially in the first 6 months. In high-risk patients with declining renal function a 12 month treatment of cyclosporine led to a 50% reduction in proteinuria in half of the patients, and slowed the rate of renal deterioration compared with placebo. Notably, no prospective, randomized, head-to-head comparisons of

On the basis of the available data, extended therapy seems to enhance the likelihood of remission. In one analysis, the majority of complete remissions occurred after at least 6 months of therapy, and the number increased as treatment continued for more than 12 months. Thereafter, the combination of low-dose cyclosporine (1.4–1.5 mg/kg per day; trough levels >100 ng/ml) and prednisolone (0.1 mg/kg per day) might be more beneficial

Several investigators have evaluated whether tacrolimus could provide similar efficacy to cyclosporine in IMN. Tacrolimus is considered to be more potent than cyclosporine, has a more favorable cardiovascular risk profile and leads to better long-term renal function after renal transplantation. Studies showed the overall remission rate achieved with tacrolimus is similar to that reported with cyclosporine but the rate of complete remission is higher with tacrolimus. This difference might be, in part, related to the long duration of therapy used in

than cyclosporin monotherapy for maintaining remission and preventing relapse.

agents failed to show beneficial effects on overall mortality or risk of ESRD.

**5.3 The calcineurin inhibitors (CNls): Cyclosporine and tacrolimus** 

cyclosporine and alkylating agents have been conducted in IMN.

benefits in patient or renal survival; however some data suggest that the treatment is warranted. Because of the high rate of spontaneous remission in MN, newly diagnosed patients with nephritic syndrome and normal renal function should initially receive conservative therapy with an ACE inhibitor or ARB, diuretics, salt restriction, and statins. If a patient remains proteinuric with normal renal function, such conservative treatment can be continued, but those patients who remain frankly nephrotic after 6 months or who initially present with (or develop) renal dysfunction should be treated with an immunosuppressive agent.

The treatment of MN depends on patient presentation and disease progression after diagnosis is made by biopsy. The two leading immunomodulatory therapies used are alkylating agents (cyclophosphamide or chlorambucil) and calcineurin inhibitors (cyclosporine or tacrolimus), both typically given orally or intravenous with corticosteroids . Given the limited efficacy, high rate of relapse, and toxicities of alkylating agents, calcineurin inhibitors, and corticosteroids, other therapies for MN are needed. Recently, rituximab has surfaced as a potential treatment option for MN. This monoclonal antibody directed against the B cell antigen CD20 may be beneficial in MN on the basis of experimental evidence that B cell activation is a key step in the pathogenesis of MN. Furthermore, rituximab is generally well tolerated with a limited short-term toxicity profile. A significant amount of literature is emerging on the benefits of rituximab in MN as primary treatment and as treatment for IMN refractory to other immunosuppressant regimens.

The treatment of membranous nephropathy in patients with normal renal function remains controversial. However in patients with deteriorating renal function, a combination therapy with steroids and cytotoxic agents is considered beneficial . It remains unclear if therapy is effective in more advanced renal failure since there is no published data on such cases. There are some cases reported in literature with near end-stage renal failure in whom treatment resulted in clinical improvement. Thus therapy is effective in patients with primary membranous nephropathy and advanced azotemia especially in those who had never been treated.( (Prabhakar S et al 1996)

#### **5.2 Alkylating agents**

Corticosteroids as monotherapy for treatment of IMN is not effective, instead, typical immunosuppressive regimens for idiopathic MN combine corticosteroids with alkylating agents for 6-12 months. Treatment with cyclophosphamide or chlorambucil in conjunction with corticosteroids is supported by randomized controlled trials (RCTs) cumulative data suggest that 30%-40% of those treated will achieve complete remission, with 30%-50% attaining partial remission and only 10% developing progressive renal disease," Relapse occurs in approximately 25%- 30% within 5 years of discontinuing the alkylating agent, but often responds to a repeat course of immunosuppressive therapy arm. A 6-month regimen consisting of alternating months of corticosteroids and alkylating agents has both short-term and long-term beneficial effects on proteinuria and renal survival. Ponticelli and colleagues found that this regimen increased remission rates at the final follow-up visit from 36% in untreated patients to 76%, and improved 10-year renal survival from 60% to 92%. The longterm outcomes of a randomized, controlled trial from India (Jha et al) found the same result. Some studies indicated that a delay in therapy did not lead to differences in efficacy. Studies showed immunosuppressive therapy markedly lessened the decline in renal function.

Despite the favorable results of alkylating agents in IMN, many physicians are reluctant to use these drugs, because of increased risk of infection and myelosuppression, particularly

benefits in patient or renal survival; however some data suggest that the treatment is warranted. Because of the high rate of spontaneous remission in MN, newly diagnosed patients with nephritic syndrome and normal renal function should initially receive conservative therapy with an ACE inhibitor or ARB, diuretics, salt restriction, and statins. If a patient remains proteinuric with normal renal function, such conservative treatment can be continued, but those patients who remain frankly nephrotic after 6 months or who initially present with (or develop) renal dysfunction should be treated with an

The treatment of MN depends on patient presentation and disease progression after diagnosis is made by biopsy. The two leading immunomodulatory therapies used are alkylating agents (cyclophosphamide or chlorambucil) and calcineurin inhibitors (cyclosporine or tacrolimus), both typically given orally or intravenous with corticosteroids . Given the limited efficacy, high rate of relapse, and toxicities of alkylating agents, calcineurin inhibitors, and corticosteroids, other therapies for MN are needed. Recently, rituximab has surfaced as a potential treatment option for MN. This monoclonal antibody directed against the B cell antigen CD20 may be beneficial in MN on the basis of experimental evidence that B cell activation is a key step in the pathogenesis of MN. Furthermore, rituximab is generally well tolerated with a limited short-term toxicity profile. A significant amount of literature is emerging on the benefits of rituximab in MN as primary treatment and as treatment for IMN refractory to other immunosuppressant regimens. The treatment of membranous nephropathy in patients with normal renal function remains controversial. However in patients with deteriorating renal function, a combination therapy with steroids and cytotoxic agents is considered beneficial . It remains unclear if therapy is effective in more advanced renal failure since there is no published data on such cases. There are some cases reported in literature with near end-stage renal failure in whom treatment resulted in clinical improvement. Thus therapy is effective in patients with primary membranous nephropathy and advanced azotemia especially in those who had

Corticosteroids as monotherapy for treatment of IMN is not effective, instead, typical immunosuppressive regimens for idiopathic MN combine corticosteroids with alkylating agents for 6-12 months. Treatment with cyclophosphamide or chlorambucil in conjunction with corticosteroids is supported by randomized controlled trials (RCTs) cumulative data suggest that 30%-40% of those treated will achieve complete remission, with 30%-50% attaining partial remission and only 10% developing progressive renal disease," Relapse occurs in approximately 25%- 30% within 5 years of discontinuing the alkylating agent, but often responds to a repeat course of immunosuppressive therapy arm. A 6-month regimen consisting of alternating months of corticosteroids and alkylating agents has both short-term and long-term beneficial effects on proteinuria and renal survival. Ponticelli and colleagues found that this regimen increased remission rates at the final follow-up visit from 36% in untreated patients to 76%, and improved 10-year renal survival from 60% to 92%. The longterm outcomes of a randomized, controlled trial from India (Jha et al) found the same result. Some studies indicated that a delay in therapy did not lead to differences in efficacy. Studies showed immunosuppressive therapy markedly lessened the decline in renal function. Despite the favorable results of alkylating agents in IMN, many physicians are reluctant to use these drugs, because of increased risk of infection and myelosuppression, particularly

immunosuppressive agent.

never been treated.( (Prabhakar S et al 1996)

**5.2 Alkylating agents** 

those with reduced GFR. Cancer risk is increased when alkylating agents are used for a long time. There are some reports of increased risk of wegener granulomatosis with cyclophosphamide when the dose is more than 36 gmr (equivalent to 100 mg daily for one year) were associated with a 9.5-fold increased risk of bladder cancer. Use of cyclophosphamide for long time have also been associated with an increased risk of lympho-proliferative disorders. Relapses occur in 25–30% of patients within 5 years of discontinuation of therapy with alkylating agents. While this rate of relapse is lower than that observed after discontinuation of cyclosporine, it is still disconcerting since relapses generally necessitate increased immunosuppression. Despite reduction in proteinuria, these agents failed to show beneficial effects on overall mortality or risk of ESRD.

### **5.3 The calcineurin inhibitors (CNls): Cyclosporine and tacrolimus**

Cyclosporine is an alternative, clinically validated immunosuppressive agent used in the treatment of IMN. In one randomized clinical trial (RCT), 51 patients with steroidresistant IMN, treatment with cyclosporine plus steroids for 6 months with tapering over 4 weeks resulted in a 75% complete or partial remission rate, versus only 22% in the placebo (steroids alone) group. Typically, many patients in cyclosporine based treatment regimens achieved partial remissions, and many relapsed after discontinuing treatment. Another similarly-sized trial compared 12 months of cyclosporine and corticosteroids to cyclosporine alone." Although both groups achieved -80% remission rates at 12 months, the relapse rate was lower in the group receiving adjunctive corticosteroids from the beginning. Longer courses of cyclosporine (1-2 years) with a slow taper may be necessary to avoid a high rate of relapse. Other investigators demonstrated that treatment with tacrolimus in heavily nephrotic patients resulted in higher remission rates compared with conservative treatment alone; however, nearly half of these patients had a nephritic relapse within several months of tapering tacrolimus. Cyclosporine reduces proteinuria and the rate of decline in renal function in patients with IMN. These effects have been demonstrated in patients with preserved renal function, in those with declining or impaired renal function and also in patients resistant to other immunosuppressants. In some studies almost 50% of patients who had achieved remission relapsed within 1 year of cyclosporine withdrawal, especially in the first 6 months. In high-risk patients with declining renal function a 12 month treatment of cyclosporine led to a 50% reduction in proteinuria in half of the patients, and slowed the rate of renal deterioration compared with placebo. Notably, no prospective, randomized, head-to-head comparisons of cyclosporine and alkylating agents have been conducted in IMN.

On the basis of the available data, extended therapy seems to enhance the likelihood of remission. In one analysis, the majority of complete remissions occurred after at least 6 months of therapy, and the number increased as treatment continued for more than 12 months. Thereafter, the combination of low-dose cyclosporine (1.4–1.5 mg/kg per day; trough levels >100 ng/ml) and prednisolone (0.1 mg/kg per day) might be more beneficial than cyclosporin monotherapy for maintaining remission and preventing relapse.

Several investigators have evaluated whether tacrolimus could provide similar efficacy to cyclosporine in IMN. Tacrolimus is considered to be more potent than cyclosporine, has a more favorable cardiovascular risk profile and leads to better long-term renal function after renal transplantation. Studies showed the overall remission rate achieved with tacrolimus is similar to that reported with cyclosporine but the rate of complete remission is higher with tacrolimus. This difference might be, in part, related to the long duration of therapy used in

Membranous Nephropathy 17

In contrast to the above-mentioned studies, responses to MMF in other multicenter randomized controlled trials the have been poor. Firm recommendations regarding the use of this agent as initial therapy are difficult to make. MMF might be a reasonable option when the toxic effects of alkylating agents and high-dose steroids are of particular concern or when severe azotemia prohibits use of calcineurin inhibitors. Studies in large numbers of patients with prolonged follow-up are needed to determine the long-term effectiveness of MMF for maintenance of remission and preservation of renal function. Additional information is also needed to fully evaluate the adverse effect profile of MMF. MMF is also associated with pregnancy loss and congenital malformations and it can also increase the risk of lymphoma and infection. Cases of JC-virus-associated progressive multifocal leukoencephalopathy in patients with systemic lupus erythematosus receiving MMF have elicited concern. All these considerations must be weighed in the decision to use MMF in

Before the use of MMF became widespread, azathioprine was tested as a treatment for IMN in several small studies, with mixed results. A combination of azathioprine and corticosteroids was reported to be beneficial in high-risk patients with declining renal function. Some patients experienced reduction in proteinuria and stabilization or improvement of renal function. However, these studies were case series with no control groups and the combined number of patients analyzed was small. In contrast to these favorable findings, a recent retrospective review indicated that azathioprine had no longterm benefit in IMN. Due to the conflicting evidence regarding the efficacy of azathioprine in IMN and the popularity of MMF, azathioprine is unlikely to be tested in future

Due to the often severe adverse or nephrotoxic effects associated with cyclophosphamide and cyclosporine, several newer and potentially less toxic agents are under evaluation for the treatment of MN. Several small studies indicate the potential efficacy of rituximab, mycophenolate, or synthetic adrenocorticotrophic hormone (ACTH) in MN; unfortunately,

Rituximab is a monoclonal anti-CD20 antibody that depletes B cells, Its rationale for use is provided by the suggested pathophysiological basis for MN of autoantibodies targeting a suspected glomerular antigen. Rituximab has been used in treatment of non-Hodgkin Lymphoma and others diseases. Although Rituximab appears to induce remission with an initial efficacy comparable to alkylating agents and corticosteroids, long-term data on dialysis-free survival have not been reported. In an open label trial of rituximab with a group of 15 high-risk idiopathic MN patients, there were 2 complete and 6 partial remissions at final follow-up. Others reported the effects of treatment with 4 weekly doses of rituximab on 50 consecutive patients with persistent nephrotic levels of proteinuria despite 6 months of conservative therapy," Ten patients achieved a full remission after treatment; however, they were more likely female and with lower baseline serum creatinine values, which is a population of high spontaneous remission. Recently, a small RCT study

none are large RCTs nor do they provide long-term follow-up data

IMN,

**6.2 Azathioprine** 

randomized trials in this setting.

**7. Alternative agents** 

**7.1 Rituximab** 

these studies (18 months, compared with 26 weeks in the study of cyclosporine by Cattran et al). The nephrotoxic effects of calcineurin inhibitors are of concern, particularly if long-term treatment is required as a result of relapses. Managing the use of these agents in patients with reduced GFR can be difficult. Due to this issue, Ponticelli and Villa recommend alternative agents in patients with impaired renal function (creatinine clearance <60 ml/min), severe hypertension or severe interstitial fibrosis and tubular atrophy. Finally, the extent to which calcineurin inhibitors affect the underlying immune process rather than merely modifying disease expression is unclear. In view of the broad range of toxic effects and the high rates of relapse associated with the use of steroids, alkylating agents and calcineurin inhibitors, alternative treatments have been investigated

### **6. Antimetabolites**

#### **6.1 Mycophenolate mofetil**

Mycophenolate is another agent used for MN treatment with varying results. Initial studies suggested that mycophenolate could reduce proteinuria in patients with MN resistant to other conventional therapies. However, a recent RCT detected no effect of mycophenolate monotherapy in patients with normal renal function and nephrotic levels of proteinuria, when compared to conservative antiproteinuric therapy. Corticosteroid treatment with mycophenolate therapy achieved a 1-year cumulative remission rate of 66% in a group of MN patients with moderate renal dysfunction, but was inferior to alkalating agents and steroids in a historically treated control group and demonstrated a relapse rate of nearly 40%. However, a small RCT revealed similar effects from 6 months of mycophenolate and steroids compared with chlorambucil and steroids at 15 months of follow-up. Given these small studies and lack of consistently demonstrated superior efficacy, mycophenolate is not a first-line agent for the treatment of MN, but may be considered with adjunctive corticosteroids, if standard therapies are not effective or cannot be tolerated.

Clinical efficacy studies of mycophenolate mofetil (MMF) in IMN have produced mixed results in a multicenter study (Chan *et al.)* randomized 20 newly diagnosed patients with persistent proteinuria ≥3 g per day to undergo 6 months of treatment with either MMF plus prednisolone or with a regimen of chlorambucil alternating monthly with corticosteroids. The groups achieved similar remission rates (65%) and experienced few relapses, which suggests that MMF in conjunction with steroids has similar efficacy to a modified Ponticelli regimen. An open-label trial in the Netherlands evaluated the efficacy of MMF in patients considered to be at high risk of disease progression. The outcomes of 32 patients treated for 1 year with MMF 2 g per day and steroids were compared with those of historic matched controls treated with oral cyclophosphamide plus corticosteroids for 1 year. Patients in both groups had reduced GFR at baseline (median approximately 40 ml/min) and median proteinuria was >8 g/g creatinine. The two groups achieved similar remission rates (approximately 70%), but the relapse was higher in the MMF group such that by the end of follow-up, patients in the MMF arm were less likely to be in remission than those in the cyclophosphamide . Both treatments resulted in stabilization or improvement of renal function in the majority of patients, and infections and hospitalization occurred at a similar frequency in the two groups. Although the investigators concluded that MMF did not seem to be as effective as, or any better tolerated than cyclophosphamide, this study does suggest that a prolonged course of MMF might be of benefit even in patients with unfavorable baseline characteristics.

these studies (18 months, compared with 26 weeks in the study of cyclosporine by Cattran et al). The nephrotoxic effects of calcineurin inhibitors are of concern, particularly if long-term treatment is required as a result of relapses. Managing the use of these agents in patients with reduced GFR can be difficult. Due to this issue, Ponticelli and Villa recommend alternative agents in patients with impaired renal function (creatinine clearance <60 ml/min), severe hypertension or severe interstitial fibrosis and tubular atrophy. Finally, the extent to which calcineurin inhibitors affect the underlying immune process rather than merely modifying disease expression is unclear. In view of the broad range of toxic effects and the high rates of relapse associated with the use of steroids, alkylating agents and

Mycophenolate is another agent used for MN treatment with varying results. Initial studies suggested that mycophenolate could reduce proteinuria in patients with MN resistant to other conventional therapies. However, a recent RCT detected no effect of mycophenolate monotherapy in patients with normal renal function and nephrotic levels of proteinuria, when compared to conservative antiproteinuric therapy. Corticosteroid treatment with mycophenolate therapy achieved a 1-year cumulative remission rate of 66% in a group of MN patients with moderate renal dysfunction, but was inferior to alkalating agents and steroids in a historically treated control group and demonstrated a relapse rate of nearly 40%. However, a small RCT revealed similar effects from 6 months of mycophenolate and steroids compared with chlorambucil and steroids at 15 months of follow-up. Given these small studies and lack of consistently demonstrated superior efficacy, mycophenolate is not a first-line agent for the treatment of MN, but may be considered with adjunctive

Clinical efficacy studies of mycophenolate mofetil (MMF) in IMN have produced mixed results in a multicenter study (Chan *et al.)* randomized 20 newly diagnosed patients with persistent proteinuria ≥3 g per day to undergo 6 months of treatment with either MMF plus prednisolone or with a regimen of chlorambucil alternating monthly with corticosteroids. The groups achieved similar remission rates (65%) and experienced few relapses, which suggests that MMF in conjunction with steroids has similar efficacy to a modified Ponticelli regimen. An open-label trial in the Netherlands evaluated the efficacy of MMF in patients considered to be at high risk of disease progression. The outcomes of 32 patients treated for 1 year with MMF 2 g per day and steroids were compared with those of historic matched controls treated with oral cyclophosphamide plus corticosteroids for 1 year. Patients in both groups had reduced GFR at baseline (median approximately 40 ml/min) and median proteinuria was >8 g/g creatinine. The two groups achieved similar remission rates (approximately 70%), but the relapse was higher in the MMF group such that by the end of follow-up, patients in the MMF arm were less likely to be in remission than those in the cyclophosphamide . Both treatments resulted in stabilization or improvement of renal function in the majority of patients, and infections and hospitalization occurred at a similar frequency in the two groups. Although the investigators concluded that MMF did not seem to be as effective as, or any better tolerated than cyclophosphamide, this study does suggest that a prolonged course of MMF might be of benefit even in patients with unfavorable

calcineurin inhibitors, alternative treatments have been investigated

corticosteroids, if standard therapies are not effective or cannot be tolerated.

**6. Antimetabolites** 

baseline characteristics.

**6.1 Mycophenolate mofetil** 

In contrast to the above-mentioned studies, responses to MMF in other multicenter randomized controlled trials the have been poor. Firm recommendations regarding the use of this agent as initial therapy are difficult to make. MMF might be a reasonable option when the toxic effects of alkylating agents and high-dose steroids are of particular concern or when severe azotemia prohibits use of calcineurin inhibitors. Studies in large numbers of patients with prolonged follow-up are needed to determine the long-term effectiveness of MMF for maintenance of remission and preservation of renal function. Additional information is also needed to fully evaluate the adverse effect profile of MMF. MMF is also associated with pregnancy loss and congenital malformations and it can also increase the risk of lymphoma and infection. Cases of JC-virus-associated progressive multifocal leukoencephalopathy in patients with systemic lupus erythematosus receiving MMF have elicited concern. All these considerations must be weighed in the decision to use MMF in IMN,

#### **6.2 Azathioprine**

Before the use of MMF became widespread, azathioprine was tested as a treatment for IMN in several small studies, with mixed results. A combination of azathioprine and corticosteroids was reported to be beneficial in high-risk patients with declining renal function. Some patients experienced reduction in proteinuria and stabilization or improvement of renal function. However, these studies were case series with no control groups and the combined number of patients analyzed was small. In contrast to these favorable findings, a recent retrospective review indicated that azathioprine had no longterm benefit in IMN. Due to the conflicting evidence regarding the efficacy of azathioprine in IMN and the popularity of MMF, azathioprine is unlikely to be tested in future randomized trials in this setting.

### **7. Alternative agents**

Due to the often severe adverse or nephrotoxic effects associated with cyclophosphamide and cyclosporine, several newer and potentially less toxic agents are under evaluation for the treatment of MN. Several small studies indicate the potential efficacy of rituximab, mycophenolate, or synthetic adrenocorticotrophic hormone (ACTH) in MN; unfortunately, none are large RCTs nor do they provide long-term follow-up data

#### **7.1 Rituximab**

Rituximab is a monoclonal anti-CD20 antibody that depletes B cells, Its rationale for use is provided by the suggested pathophysiological basis for MN of autoantibodies targeting a suspected glomerular antigen. Rituximab has been used in treatment of non-Hodgkin Lymphoma and others diseases. Although Rituximab appears to induce remission with an initial efficacy comparable to alkylating agents and corticosteroids, long-term data on dialysis-free survival have not been reported. In an open label trial of rituximab with a group of 15 high-risk idiopathic MN patients, there were 2 complete and 6 partial remissions at final follow-up. Others reported the effects of treatment with 4 weekly doses of rituximab on 50 consecutive patients with persistent nephrotic levels of proteinuria despite 6 months of conservative therapy," Ten patients achieved a full remission after treatment; however, they were more likely female and with lower baseline serum creatinine values, which is a population of high spontaneous remission. Recently, a small RCT study

Membranous Nephropathy 19

The role of sirolimus in IMN has been evaluated in two small pilot studies, with unfavorable results. No remissions occurred during therapy, but one patient achieved a partial remission after cessation of therapy. Severe adverse events, including pneumonitis, infection, persistent proteinuria and azotemia, necessitated discontinuation of the drug in the majority of cases. These trials were prematurely terminated owing to the unfavorable risk–benefit ratio. An open-label trial of sirolimus in 11 patients with a variety of chronic glomerulopathies and declining renal function, including three with membranous nephropathy, was associated with acute kidney injury in more than half of the patients; this event generally occurred within weeks of starting sirolimus. Thus, sirolimus does not seem

Eculizumab is a fully humanized monoclonal antibody directed against the complement protein C5, approved for the treatment of Paroxysmal Nocturnal Hematuria. Ecluzimab inhibit C5a and C5 b thus preventing complement activation. Treatment with eculizumab improves the quality of life and reduces the need of transfusions and the risk of thrombosis in patients with PNH. However, eculizumab can increase the risk of meningococcal infections perhaps due to the reduction in the levels of C5 activity. Patients should therefore be vaccinated or revaccinated with a meningococcal vaccine at least 2 weeks before receiving the first dose of eculizumab. Other side effects include headache, nasopharingitis, back pain and cough; nausea may occur in the period following injection. The mechanism of action of eculizumab renders this monoclonal antibody potentially attractive for treating patients with IMN, as the terminal components of the complement C5b--C9 play a prominent role in mediating the inflammation and the damage of podocytes and glomerular basement membrane. However, a RCT conducted in IMN failed to show any advantage over placebo of eculizumab 8 mg/kg every other week or every 4 weeks. Further trials are needed to establish whether a different dosage or more prolonged treatment may obtain

IVIG have been used in high dose in treatment of IMN. IVIG interferes with complementmediated immune damage by binding to C3b and C4b,by this mechanism preventing glomerular injury. This mechanism may be involved in IMN, as suggested by a study in passive Heymann nephritis, in which treatment with systemic immunoglobulin obtained a decrease in proteinuria, associated with a decreased glomerular deposition of C3c and C5b-- 9, without changes in the amount, size or distribution of the subepithelial immune complexes . A few anecdotal uncontrolled studies suggested a possible benefit of IVIG

Use of anticoagulation in nephrotic syndrome is controversial issue. Nephrotic syndrome (NS) is associated with high risk of for thromboembolic complications, including deep venous thrombosis, renal vein thrombosis, and pulmonary embolism; this risk seems to be greater for IMN especially in patient with low albumin and previous history of thromboembolic disease. Analyses showed that in patients with IMN the benefits of oral

**7.3 Sirolimus** 

**7.4 Eculizumab** 

therapeutic results in IMN

therapy in IMN.

**8.1 Anticoagulation** 

**8. Intravenous high-dose immunoglobulins [IVIG]** 

to have a role in the treatment of IMN

conducted in Spain demonstrated that rituximab was of benefit in 13 Spanish patients with idiopathic MN and CNI dependence, allowing successful weaning of the nephrotoxic CN. A review of the published literature about rituximab describing the use of rituximab in MN highlights that, while promising, the existing literature consists of too few patients, heterogeneous populations, and insufficient follow-up to recommend the use of rituximab outside the research setting.

There may be potentially fatal mucocutaneous reactions, such as stevens–Johnson syndrome and toxic epidermal necrolysis, can occur following rituximab exposure. Severe infections are infrequent, occurring in only 1–2% of patients. Of great concern, rare cases of progressive multifocal leukoencephalopathy have been reported with rituximab use, particularly as part of a multidrug immunosuppressive regimen. Physicians and patients need to be aware of the presenting features of this devastating demyelinating disease of the central nervous system, which include altered mental status, visual symptoms, motor deficits and ataxia. The preliminary results of rituximab treatment are encouraging, but concerns remain before this agent can be recommended for routine use in IMN. So far, no randomized, controlled trials have been conducted to clarify the role of rituximab in the treatment of IMN. Adequately powered, randomized, controlled trials with prolonged follow-up are needed to determine the long-term course of the disease following B-cell reconstitution; rates of relapse; subsequent redosing regimens; and effects on renal survival. Further studies must clarify whether rituximab should be used as monotherapy or in combination with other immunosuppressive drugs to achieve maximum anti proteinuric effect and durable remission. The preliminary small, uncontrolled study suggests that the addition of rituximab to tacrolimus can induce sustained remission of the nephrotic syndrome, allowing early tacrolimus withdrawal and thereby overcoming the issue of tacrolimus dependence.

#### **7.2 Adrenocorticotrophic hormone (ACTH)**

Several small, uncontrolled trials have reported beneficial effects of synthetic adrenocorticotropic hormone ACTH in patients with IMN. One small, randomized, controlled trial by Ponticelli *et al.* compared treatment with ACTH for 1 year to a 6-month regimen of methylprednisolone alternating monthly with a cytotoxic agent in 32 (mostly medium-risk) patients with IMN. The probability of complete or partial remission did not differ substantially between the groups (87% versus 93%), and the number of remissions, mean time to response and number of relapses were also comparable between the groups. The results suggest that prolonged ACTH treatment could be equivalent to the combined use of cytotoxic drugs and steroids. The side effects of ACTH include glucose intolerance, fluid retention, hypertension, diarrhea, bronze discoloration of the skin, dizziness and fatigue, all of which resolve after discontinuation of treatment. Extensive studies with long follow-up are needed to confirm the preliminary data on the use of ACTH in IMN. Further investigation is also required to find the mechanisms by which ACTH seems to decrease proteinuria and alter apolipoprotein metabolism. These effects are probably not entirely attributable to an increase in endogenous cortisol synthesis, since steroid monotherapy has not been shown to be effective in IMN. ACTH therapy can be effective in patients who are unresponsive to steroids. On the other hand, the endogenous cortisol liberated by the actions of exogenous ACTH might act differently and perhaps more effectively than orally administered steroid

### **7.3 Sirolimus**

18 An Update on Glomerulopathies – Clinical and Treatment Aspects

conducted in Spain demonstrated that rituximab was of benefit in 13 Spanish patients with idiopathic MN and CNI dependence, allowing successful weaning of the nephrotoxic CN. A review of the published literature about rituximab describing the use of rituximab in MN highlights that, while promising, the existing literature consists of too few patients, heterogeneous populations, and insufficient follow-up to recommend the use of rituximab

There may be potentially fatal mucocutaneous reactions, such as stevens–Johnson syndrome and toxic epidermal necrolysis, can occur following rituximab exposure. Severe infections are infrequent, occurring in only 1–2% of patients. Of great concern, rare cases of progressive multifocal leukoencephalopathy have been reported with rituximab use, particularly as part of a multidrug immunosuppressive regimen. Physicians and patients need to be aware of the presenting features of this devastating demyelinating disease of the central nervous system, which include altered mental status, visual symptoms, motor deficits and ataxia. The preliminary results of rituximab treatment are encouraging, but concerns remain before this agent can be recommended for routine use in IMN. So far, no randomized, controlled trials have been conducted to clarify the role of rituximab in the treatment of IMN. Adequately powered, randomized, controlled trials with prolonged follow-up are needed to determine the long-term course of the disease following B-cell reconstitution; rates of relapse; subsequent redosing regimens; and effects on renal survival. Further studies must clarify whether rituximab should be used as monotherapy or in combination with other immunosuppressive drugs to achieve maximum anti proteinuric effect and durable remission. The preliminary small, uncontrolled study suggests that the addition of rituximab to tacrolimus can induce sustained remission of the nephrotic syndrome, allowing early tacrolimus withdrawal and thereby overcoming the issue of

Several small, uncontrolled trials have reported beneficial effects of synthetic adrenocorticotropic hormone ACTH in patients with IMN. One small, randomized, controlled trial by Ponticelli *et al.* compared treatment with ACTH for 1 year to a 6-month regimen of methylprednisolone alternating monthly with a cytotoxic agent in 32 (mostly medium-risk) patients with IMN. The probability of complete or partial remission did not differ substantially between the groups (87% versus 93%), and the number of remissions, mean time to response and number of relapses were also comparable between the groups. The results suggest that prolonged ACTH treatment could be equivalent to the combined use of cytotoxic drugs and steroids. The side effects of ACTH include glucose intolerance, fluid retention, hypertension, diarrhea, bronze discoloration of the skin, dizziness and fatigue, all of which resolve after discontinuation of treatment. Extensive studies with long follow-up are needed to confirm the preliminary data on the use of ACTH in IMN. Further investigation is also required to find the mechanisms by which ACTH seems to decrease proteinuria and alter apolipoprotein metabolism. These effects are probably not entirely attributable to an increase in endogenous cortisol synthesis, since steroid monotherapy has not been shown to be effective in IMN. ACTH therapy can be effective in patients who are unresponsive to steroids. On the other hand, the endogenous cortisol liberated by the actions of exogenous ACTH might act differently and perhaps more effectively than orally

outside the research setting.

tacrolimus dependence.

administered steroid

**7.2 Adrenocorticotrophic hormone (ACTH)** 

The role of sirolimus in IMN has been evaluated in two small pilot studies, with unfavorable results. No remissions occurred during therapy, but one patient achieved a partial remission after cessation of therapy. Severe adverse events, including pneumonitis, infection, persistent proteinuria and azotemia, necessitated discontinuation of the drug in the majority of cases. These trials were prematurely terminated owing to the unfavorable risk–benefit ratio. An open-label trial of sirolimus in 11 patients with a variety of chronic glomerulopathies and declining renal function, including three with membranous nephropathy, was associated with acute kidney injury in more than half of the patients; this event generally occurred within weeks of starting sirolimus. Thus, sirolimus does not seem to have a role in the treatment of IMN

### **7.4 Eculizumab**

Eculizumab is a fully humanized monoclonal antibody directed against the complement protein C5, approved for the treatment of Paroxysmal Nocturnal Hematuria. Ecluzimab inhibit C5a and C5 b thus preventing complement activation. Treatment with eculizumab improves the quality of life and reduces the need of transfusions and the risk of thrombosis in patients with PNH. However, eculizumab can increase the risk of meningococcal infections perhaps due to the reduction in the levels of C5 activity. Patients should therefore be vaccinated or revaccinated with a meningococcal vaccine at least 2 weeks before receiving the first dose of eculizumab. Other side effects include headache, nasopharingitis, back pain and cough; nausea may occur in the period following injection. The mechanism of action of eculizumab renders this monoclonal antibody potentially attractive for treating patients with IMN, as the terminal components of the complement C5b--C9 play a prominent role in mediating the inflammation and the damage of podocytes and glomerular basement membrane. However, a RCT conducted in IMN failed to show any advantage over placebo of eculizumab 8 mg/kg every other week or every 4 weeks. Further trials are needed to establish whether a different dosage or more prolonged treatment may obtain therapeutic results in IMN

### **8. Intravenous high-dose immunoglobulins [IVIG]**

IVIG have been used in high dose in treatment of IMN. IVIG interferes with complementmediated immune damage by binding to C3b and C4b,by this mechanism preventing glomerular injury. This mechanism may be involved in IMN, as suggested by a study in passive Heymann nephritis, in which treatment with systemic immunoglobulin obtained a decrease in proteinuria, associated with a decreased glomerular deposition of C3c and C5b-- 9, without changes in the amount, size or distribution of the subepithelial immune complexes . A few anecdotal uncontrolled studies suggested a possible benefit of IVIG therapy in IMN.

#### **8.1 Anticoagulation**

Use of anticoagulation in nephrotic syndrome is controversial issue. Nephrotic syndrome (NS) is associated with high risk of for thromboembolic complications, including deep venous thrombosis, renal vein thrombosis, and pulmonary embolism; this risk seems to be greater for IMN especially in patient with low albumin and previous history of thromboembolic disease. Analyses showed that in patients with IMN the benefits of oral

Membranous Nephropathy 21

the disease course, and improved decision-making regarding necessity and duration of treatment. Treatment should be provided to those at high risk of progression to ESRD, including patients with persistent severe proteinuria or a documented loss of renal function. At present, alkylating agents and cyclosporine are the only clinically validated treatments with sufficient follow-up data; however, as the roles of tacrolimus, rituximab, mycophenolate, and ACTH grow, these agents may become the new treatments of choice for

We sincerely thank Dr. Irfan Warraich, Department of Pathology, Texas Tech University Medical Center for providing the photomicrographs from biopsies in subjects with

Ahmed M, Wong C .Rituximab and nephrotic syndrome: a new therapeutic hope? Nephrol

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idiopathic MN.

**12. References** 

**11. Acknowledgements** 

membranous nephropathy for inclusion in this chapter.

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anticoagulation outweigh the risks. However, before prescribing anticoagulants the physician should take into account the severity of the NS (as assessed by serum albumin concentration), pre-existing thrombotic states, and the overall likelihood of serious bleeding events consequent to oral anticoagulation The optimal duration of prophylactic anticoagulation is unknown but should probably last for as long as NS persists (Ponticelli C et al 2010).

## **9. Future directions**

If anti-PLA2R or other MN-specific autoantibodies can be demonstrated to be tightly associated with immunological disease activity in idiopathic MN, a serologic immunoassay would have several potential applications. it could use the anti- PLA2R as an initial assay for the diagnosis of idiopathic MN without kidney biopsy. Serial assays for the presence and titer of anti-PLA2R prior to therapeutic intervention in clinical trials could help reduce uncertainty as to whether rapid responders represent a true therapeutic effect or a spontaneous remission. Anti-PLA2R could also be followed during treatment to assess the efficacy of immunosuppressive therapy and to determine the length of treatment. It could also be useful in partial remission, when residual proteinuria could be caused either by ongoing but attenuated immune activity or by structural glomerular changes without immune activity.

### **10. Summary**

Membranous Nephropathy is a common cause of nephrotic syndrome in adults of all races and ethnicities. Its molecular pathogenesis is increasingly well understood, and identification of PLA2R as a target antigen may allow better diagnosis, better following of

anticoagulation outweigh the risks. However, before prescribing anticoagulants the physician should take into account the severity of the NS (as assessed by serum albumin concentration), pre-existing thrombotic states, and the overall likelihood of serious bleeding events consequent to oral anticoagulation The optimal duration of prophylactic anticoagulation is unknown but should probably last for as long as NS persists (Ponticelli C

If anti-PLA2R or other MN-specific autoantibodies can be demonstrated to be tightly associated with immunological disease activity in idiopathic MN, a serologic immunoassay would have several potential applications. it could use the anti- PLA2R as an initial assay for the diagnosis of idiopathic MN without kidney biopsy. Serial assays for the presence and titer of anti-PLA2R prior to therapeutic intervention in clinical trials could help reduce uncertainty as to whether rapid responders represent a true therapeutic effect or a spontaneous remission. Anti-PLA2R could also be followed during treatment to assess the efficacy of immunosuppressive therapy and to determine the length of treatment. It could also be useful in partial remission, when residual proteinuria could be caused either by ongoing but attenuated immune activity or by structural glomerular changes without

Membranous Nephropathy is a common cause of nephrotic syndrome in adults of all races and ethnicities. Its molecular pathogenesis is increasingly well understood, and identification of PLA2R as a target antigen may allow better diagnosis, better following of

et al 2010).

[Cattran et al 2010].

immune activity.

**10. Summary** 

**9. Future directions** 

the disease course, and improved decision-making regarding necessity and duration of treatment. Treatment should be provided to those at high risk of progression to ESRD, including patients with persistent severe proteinuria or a documented loss of renal function. At present, alkylating agents and cyclosporine are the only clinically validated treatments with sufficient follow-up data; however, as the roles of tacrolimus, rituximab, mycophenolate, and ACTH grow, these agents may become the new treatments of choice for idiopathic MN.

### **11. Acknowledgements**

We sincerely thank Dr. Irfan Warraich, Department of Pathology, Texas Tech University Medical Center for providing the photomicrographs from biopsies in subjects with membranous nephropathy for inclusion in this chapter.

### **12. References**


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Ehrenreich T, Churg J. Pathology of membranous nephropathy. Pathol Ann 1968;3:145. Ehrenreich T, Churg J. Pathology of membranous nephropathy. Pathol Ann 1968;3:145. Fervenza FC, Sethi S and Specks U, Idiopathic Membranous Nephropathy: Diagnosis and

Diagnosis and Management.Drugs Aging 2007; 24 (9): 717-732

Diseases, Vol 46, No 6 (December), 2005: pp 1012-1029.

Diseases, Vol 52, No 4 (October), 2008: pp 699-705

Treatment .Clin J Am Soc Nephrol 3: 905-919, 2008

High Doses of Human Immunoglobulins

Little, Brown and Company, 1974:393

Nephropathy-journal

Am Soc Nephrol 2: 932-937, 2007

Optimize Lymphocytolytic Therapy in Idiopathic Membranous Nephropathy.Clin J

Outline and Rationaleof a Treatment Strategy. American Journal of Kidney

Nephropathy:A 1-Year Randomized Controlled Trial.American Journal of Kidney

Protocol of Membranous Nephropathy Treatment with Endovenous Infusion of

RH, Idiopathic membranous, membranoproliferative, and lobular glomerulonephritis. In: Heptinstall RH, ed. The Pathology of the Kidney. Boston:

late start of immunosuppressive therapy in idiopathic membranous nephropathy:

Dose Chlorambucil in Nephrotic Patients with Idiopathic Membranous

in Adults with Nephrotic Syndrome Caused by IdiopathicMembranous

of immunosuppressive therapeutic regimens in patientswith nephrotic syndrome due to idiopathic membranous nephropathy. Renal Failure, 32, 566–571, 2010


**2** 

*USA* 

**Focal Segmental Glomerulosclerosis** 

 *Department of Medicine, Texas Tech University Health Sciences Center* 

Focal segmental glomerulosclerosis (FSGS) as the name implies is a histopathological pattern of lesions, where the "focal" refers to, involving minority of glomeruli and the "segmental" refers to, involving a portion of the glomerular capillary tuft caused by injury to podocytes (Fig 1A). Clinically it manifests proteinuria which can progress to nephrotic

Karl T. Fahr, a German pathologist, described "Progressive lipoid nephrosis" in 1925 which is currently recognized as FSGS. Arnold Rich (1957) was the first to report segmental sclerosis in juxtamedullary glomeruli in autopsy cases of children with nephrosis and uremia. In this report, he hypothesized that the progression to end stage renal disease (ESRD) in a subset of children with idiopathic nephrotic syndrome was because of the development of glomerular sclerosis. Churg et al published histopathological classification of nephrotic syndrome in children for the International Study of Kidney Disease in Children (ISKDC) in 1970, and the disease entity of FSGS was emphasized as a clinicopathological entity separate from minimal change disease (MCD) by its marked resistance to steroids and progression to ESRD. Habib (1973) described clinical and histopathological features of this

Brown et al (1978) reported the malignant form of FSGS, which is characterized by FSGS with rapid decline in renal function. Howie et al. (1984) described the glomerular tip lesion in FSGS, as glomeruli with segmental lesions at the outer 25% with adhesion or prominence of podocytes at the tubular neck. Patients with the glomerular tip lesion often develop nephrotic syndrome and have excellent response to steroids, favorable outcome, and their clinical course is similar to that of patients with MCD. Schwartz et al. (1985) reported another form of FSGS-related cellular lesion characterized by glomerular extracapillary epithelial hypercellularity and endocapillary hypercellularity with foam cells and infiltrating leukocytes, and this was considered to represent an early stage in the development of FSGS. Soon after a collapsing form of FSGS as a new clinic-pathological entity was described by Weiss et al. (1986), which is characterized by nephrotic syndrome with progressive irreversible ESRD and by glomerular collapse with epithelial hypercellularity. Subsequently, many cases of renal disease associated with HIV infection, (Rao TK et al 1984) termed as HIV-associated nephropathies, were reported with features

entity as a separate disease entity using the term 'focal glomerular scleroses'.

**1. Introduction** 

**1.1 Historical aspects** 

similar to the collapsing form of FSGS..

syndrome and eventually to end stage renal failure.

Dawinder S. Sohal and Sharma S. Prabhakar


## **Focal Segmental Glomerulosclerosis**

Dawinder S. Sohal and Sharma S. Prabhakar

 *Department of Medicine, Texas Tech University Health Sciences Center USA* 

#### **1. Introduction**

24 An Update on Glomerulopathies – Clinical and Treatment Aspects

Ruggenenti P,Cravedi P.Effects of Rituximab on Morphofunctional Abnormalities of Membranous Glomerulopathy, Clin J Am Soc Nephrol 3: 1652–1659, 2008. Salant D.In Search of the Elusive Membranous Nephropathy Antigen Nephron Physiol

Segara A ,Praga M,Ramos N.Successful treatment of membranous glomerulonephritis with

Sepe1 V, Libetta1C, GiulianoMG, Adamo G, Canton AD.Mycophenolate mofetil in primary

Stanescu HC, Arcos-Burgos M, Medlar A, et al. Risk HLADQA1 and PLA2R1 alleles in idiopathic membranous nephropathy. N Engl J Med 2011;364:616-26. Troyanov S,Wall CA, Miller JA. Scholey JW, Cattran DC, Idiopathic membranous

Waldman M,Austin HA.Controversies in the treatment of idiopathic membranous nephropathy Nat. Rev. Nephrol. 5, 469–479 (2009); published online 7 July 2009 Wehrmann M, Bohle A, Bogenschutz O, et al. Long-term prognosis of chronic idiopathic

glomerulopathies .Kidney International (2008) 73, 154–162

tubulointerstitial changes. Clin Nephrol 1989;3:67.

rituximab in calcineurin inhibitor-dependent patients Clin J Am Soc Nephrol. 2009

nephropathy: Definition and relevance of a partial remission. Kidney International,

membranous glomerulonephritis: An analysis of 334 cases with particular regard to

2009;112:p11–p12

Jun;4(6):1083-8. Epub 2009 May 28

Vol. 66 (2004), pp. 1199–1205

Focal segmental glomerulosclerosis (FSGS) as the name implies is a histopathological pattern of lesions, where the "focal" refers to, involving minority of glomeruli and the "segmental" refers to, involving a portion of the glomerular capillary tuft caused by injury to podocytes (Fig 1A). Clinically it manifests proteinuria which can progress to nephrotic syndrome and eventually to end stage renal failure.

#### **1.1 Historical aspects**

Karl T. Fahr, a German pathologist, described "Progressive lipoid nephrosis" in 1925 which is currently recognized as FSGS. Arnold Rich (1957) was the first to report segmental sclerosis in juxtamedullary glomeruli in autopsy cases of children with nephrosis and uremia. In this report, he hypothesized that the progression to end stage renal disease (ESRD) in a subset of children with idiopathic nephrotic syndrome was because of the development of glomerular sclerosis. Churg et al published histopathological classification of nephrotic syndrome in children for the International Study of Kidney Disease in Children (ISKDC) in 1970, and the disease entity of FSGS was emphasized as a clinicopathological entity separate from minimal change disease (MCD) by its marked resistance to steroids and progression to ESRD. Habib (1973) described clinical and histopathological features of this entity as a separate disease entity using the term 'focal glomerular scleroses'.

Brown et al (1978) reported the malignant form of FSGS, which is characterized by FSGS with rapid decline in renal function. Howie et al. (1984) described the glomerular tip lesion in FSGS, as glomeruli with segmental lesions at the outer 25% with adhesion or prominence of podocytes at the tubular neck. Patients with the glomerular tip lesion often develop nephrotic syndrome and have excellent response to steroids, favorable outcome, and their clinical course is similar to that of patients with MCD. Schwartz et al. (1985) reported another form of FSGS-related cellular lesion characterized by glomerular extracapillary epithelial hypercellularity and endocapillary hypercellularity with foam cells and infiltrating leukocytes, and this was considered to represent an early stage in the development of FSGS. Soon after a collapsing form of FSGS as a new clinic-pathological entity was described by Weiss et al. (1986), which is characterized by nephrotic syndrome with progressive irreversible ESRD and by glomerular collapse with epithelial hypercellularity. Subsequently, many cases of renal disease associated with HIV infection, (Rao TK et al 1984) termed as HIV-associated nephropathies, were reported with features similar to the collapsing form of FSGS..

Focal Segmental Glomerulosclerosis 27

pododcyte foot processes effacement and proteinuria: (1) interference with the slit diaphragm complex and its lipid rafts (2) direct interference with the actin cytoskeleton (3) interference with the GBM or with podocyte-GBM interaction, and (4) interference with negative surface charge of podocytes. Damage to podocytes triggers apoptosis and the detachment of podocytes from the glomerular basement membrane. The resulting reduction in podocyte number (podocytopenia) leaves the glomerular basement membrane to be exposed, (Fogo AB 2003) which leads to development of maladaptive interactions between the glomerular basement membrane and epithelial cells. This is followed by proliferation of epithelial, endothelial and mesangial cells. The combined reaction of cell proliferation and leakage of proteins into Bowman's space results in deposition of the collagen. Eventually the capillary loop collapses and endothelial cells are lost and the affected part of the glomerular tuft heals by scarring causing a characteristic lesion of FSGS. Thus the lesions initially are limited to a few segments in the glomerulus (segmental) and in a few regions of the kidney (focal) but the disease ultimately progresses to involve the entire kidney leading to end

The precise initial insult that leads to the above cascade of events is still unknown. A 'circulating permeability factor' which leads to glomerular basement membrane injury has been proposed in the pathogenesis of FSGS (Shalhoub RJ, 1974). The following information favors the hypothesis of 'circulating Permeability factor' (1) frequent recurrence of proteinuria after renal transplant [Ingulli E. et al. 1991] (2) the efficacy of extracorporeal techniques such as plasmapheresis in reducing the post renal transplant proteinuria [Artero M. et al 1992 and 1994]. (3) the results of *in vitro* bioassay which detects permeability changes induced by FSGS serum on isolated glomeruli [Savin VJ et al in 1992 and 1996]. (4)Rea et al (2001) demonstrated that the main clinical feature of FSGS i.e. proteinuria, disappeared within one year after transplantation in two recipients of kidneys from a patient with FSGS. Not taking into account ethical and legal implications, good outcome of the FSGS allograft kidneys into non FSGS recipients is another good evidence for humoral genesis/circulating permeability factor, as a cause of the FSGS. (5) Kemper M. et al (2001) demonstrated transmission of glomerular permeability factor from the mother affected by FSGS to her infant during gestation. After birth, proteinuria in the child decreased and then disappeared, suggesting a strong correlation with some circulating factor transmitted from

At the molecular level, cytokines and vasoactive factors are believed to play a major role in the progression of FSGS. The overexpression of transforming growth factor β (TGFβ) or its downstream proteins, the 'Smads' lead to glomerulosclerosis in animal models. Activation of the renin-angiotensin system upregulates TGFβ, which is considered to cause further progression of the disease (Harris RC et al. 2006). Angiogenic factors, like platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) seem to play a role in disease progression. This is based upon the rat remnant kidney model (RK model) experimental studies of progressive glomerulosclerosis. In this model, VEGF upregulation soon after the renal injury and later loss of VEGF expression correlates well with

Mechanical stress is also believed to play a role in the progression of FSGS. (Hostetter TH. 2003 & Kwoh C. et al 2006) The hyperfiltration due to the defects of the filtration barrier results in increased single nephron glomerular filtration rate (SNGFR), which results in hypertrophy of glomeruli. The hypertrophy exacerbates the mismatch between the glomerular basement membrane and the decreased numbers of podocytes, propagating the

progression of the glomerulosclerosis (Kang DH et al. 2001).

stage renal disease (ESRD).

the mother to the child.

Between 1980–2000, secondary FSGS (Rennke HG et al. 1989 & D'Agati V. 1994) was established as FSGS with recognized etiologic associations, including genetic mutations in podocyte-associated proteins, virus, drug toxicities, and structural–functional adaptations. Finally in 2004, the Columbia classification was proposed by D' Agati, Fogo, Bruijn, and Jenette, as a working classification for FSGS as given in table 2. Lately some celebrity figures like Sean Elliott and Alonzo Mourning (National Basketball Association) have been diagnosed with FSGS which has further enhanced awareness of this disease in public.

### **1.2 Epidemiology**

Focal segmental glomerulosclerosis (FSGS) currently is the leading cause of nephrotic syndrome in adults and children, particularly in the United States, Australia, Brazil, Canada, Kuwait, India and many other countries. (reference from: http://eng.hi138.com/?i295373\_The-epidemiology-of-focal-segmental-glomerulosclerosis). Renal biopsy survey for idiopathic nephrotic syndrome in adults in United States between 1995 to 1997, has revealed that FSGS is the most common cause of nephrotic syndrome, responsible for 35 percent of all cases and more than 50 percent of cases among black population, (67 percent of such cases in black adults were younger than 45 years of age). Idiopathic FSGS is now the most common cause of end stage renal disease (ESRD) caused by primary glomerular disease in the United States in both the black and white populations. The proportion of ESRD attributed to FSGS has increased 11-fold, from 0.2% in 1980 to 2.3% in 2000 (excluding patients with HIV). As per Kitiyakara C. et al (2004) the peak decade for FSGS ESRD incidence is 40 to 49 years among black patients as compared to, 70 to 79 years among white and Asian patients. Males have 1.5- to 2-fold greater risk than females. Recent incidence of end stage renal disease secondary to FSGS is five cases per million population in Caucasian US population and 30-40 cases per million population for African-American population (Hogg R. et al 2007).

### **2. Etio-pathogenesis**

While majority of FSGS cases are still considered idiopathic, the etiologies and mechanisms involved in FSGS development continue to be elucidated. FSGS can be divided into primary or idiopathic and secondary depending upon if the etiology is unknown vs. known respectively. For FSGS to produce nephrotic range or non-nephrotic range proteinuria, alterations of normal glomerular structure and function has to occur. Normal glomerular function requires, that the three major components of glomerular filter, namely *endothelial cells, podocytes, and glomerular basement membrane (GBM)*, be intact and are able to provide a permselective filtration barrier. Specialized tight junctions between podocyte foot processes create a slit diaphragm (SD) which is integral in preventing the loss of protein into Bowman's space (Kimberly et al. 2007 & Asanma et al 2003) . Even though the clinical presentation of FSGS is often heterogeneous, a cardinal feature of the disease is proteinuria, which implies loss of this permselective barrier (Schnaper HW, 2003 & Fogo AB, 2003). Electron microscopic picture clearly reveals distortion of normal architecture (or effacement) of the foot processes of podocytes in FSGS

(Fig 1B).

A key factor in the pathogenesis of FSGS is damage and loss of podocytes. Asanuma K. et al. (2003) described that based on recent insights into the molecular pathology of podocyte injury, at least four major causes have been identified that lead to the uniform reaction of

Between 1980–2000, secondary FSGS (Rennke HG et al. 1989 & D'Agati V. 1994) was established as FSGS with recognized etiologic associations, including genetic mutations in podocyte-associated proteins, virus, drug toxicities, and structural–functional adaptations. Finally in 2004, the Columbia classification was proposed by D' Agati, Fogo, Bruijn, and Jenette, as a working classification for FSGS as given in table 2. Lately some celebrity figures like Sean Elliott and Alonzo Mourning (National Basketball Association) have been diagnosed with FSGS which has further enhanced awareness of this disease in public.

Focal segmental glomerulosclerosis (FSGS) currently is the leading cause of nephrotic syndrome in adults and children, particularly in the United States, Australia, Brazil, Canada, Kuwait, India and many other countries. (reference from: http://eng.hi138.com/?i295373\_The-epidemiology-of-focal-segmental-glomerulosclerosis). Renal biopsy survey for idiopathic nephrotic syndrome in adults in United States between 1995 to 1997, has revealed that FSGS is the most common cause of nephrotic syndrome, responsible for 35 percent of all cases and more than 50 percent of cases among black population, (67 percent of such cases in black adults were younger than 45 years of age). Idiopathic FSGS is now the most common cause of end stage renal disease (ESRD) caused by primary glomerular disease in the United States in both the black and white populations. The proportion of ESRD attributed to FSGS has increased 11-fold, from 0.2% in 1980 to 2.3% in 2000 (excluding patients with HIV). As per Kitiyakara C. et al (2004) the peak decade for FSGS ESRD incidence is 40 to 49 years among black patients as compared to, 70 to 79 years among white and Asian patients. Males have 1.5- to 2-fold greater risk than females. Recent incidence of end stage renal disease secondary to FSGS is five cases per million population in Caucasian US population and 30-40 cases per million population for African-American

While majority of FSGS cases are still considered idiopathic, the etiologies and mechanisms involved in FSGS development continue to be elucidated. FSGS can be divided into primary or idiopathic and secondary depending upon if the etiology is unknown vs. known respectively. For FSGS to produce nephrotic range or non-nephrotic range proteinuria, alterations of normal glomerular structure and function has to occur. Normal glomerular function requires, that the three major components of glomerular filter, namely *endothelial cells, podocytes, and glomerular basement membrane (GBM)*, be intact and are able to provide a permselective filtration barrier. Specialized tight junctions between podocyte foot processes create a slit diaphragm (SD) which is integral in preventing the loss of protein into Bowman's space (Kimberly et al. 2007 & Asanma et al 2003) . Even though the clinical presentation of FSGS is often heterogeneous, a cardinal feature of the disease is proteinuria, which implies loss of this permselective barrier (Schnaper HW, 2003 & Fogo AB, 2003). Electron microscopic picture clearly reveals distortion of normal architecture (or effacement)

A key factor in the pathogenesis of FSGS is damage and loss of podocytes. Asanuma K. et al. (2003) described that based on recent insights into the molecular pathology of podocyte injury, at least four major causes have been identified that lead to the uniform reaction of

**1.2 Epidemiology** 

population (Hogg R. et al 2007).

of the foot processes of podocytes in FSGS

**2. Etio-pathogenesis** 

(Fig 1B).

pododcyte foot processes effacement and proteinuria: (1) interference with the slit diaphragm complex and its lipid rafts (2) direct interference with the actin cytoskeleton (3) interference with the GBM or with podocyte-GBM interaction, and (4) interference with negative surface charge of podocytes. Damage to podocytes triggers apoptosis and the detachment of podocytes from the glomerular basement membrane. The resulting reduction in podocyte number (podocytopenia) leaves the glomerular basement membrane to be exposed, (Fogo AB 2003) which leads to development of maladaptive interactions between the glomerular basement membrane and epithelial cells. This is followed by proliferation of epithelial, endothelial and mesangial cells. The combined reaction of cell proliferation and leakage of proteins into Bowman's space results in deposition of the collagen. Eventually the capillary loop collapses and endothelial cells are lost and the affected part of the glomerular tuft heals by scarring causing a characteristic lesion of FSGS. Thus the lesions initially are limited to a few segments in the glomerulus (segmental) and in a few regions of the kidney (focal) but the disease ultimately progresses to involve the entire kidney leading to end stage renal disease (ESRD).

The precise initial insult that leads to the above cascade of events is still unknown. A 'circulating permeability factor' which leads to glomerular basement membrane injury has been proposed in the pathogenesis of FSGS (Shalhoub RJ, 1974). The following information favors the hypothesis of 'circulating Permeability factor' (1) frequent recurrence of proteinuria after renal transplant [Ingulli E. et al. 1991] (2) the efficacy of extracorporeal techniques such as plasmapheresis in reducing the post renal transplant proteinuria [Artero M. et al 1992 and 1994]. (3) the results of *in vitro* bioassay which detects permeability changes induced by FSGS serum on isolated glomeruli [Savin VJ et al in 1992 and 1996].

(4)Rea et al (2001) demonstrated that the main clinical feature of FSGS i.e. proteinuria, disappeared within one year after transplantation in two recipients of kidneys from a patient with FSGS. Not taking into account ethical and legal implications, good outcome of the FSGS allograft kidneys into non FSGS recipients is another good evidence for humoral genesis/circulating permeability factor, as a cause of the FSGS. (5) Kemper M. et al (2001) demonstrated transmission of glomerular permeability factor from the mother affected by FSGS to her infant during gestation. After birth, proteinuria in the child decreased and then disappeared, suggesting a strong correlation with some circulating factor transmitted from the mother to the child.

At the molecular level, cytokines and vasoactive factors are believed to play a major role in the progression of FSGS. The overexpression of transforming growth factor β (TGFβ) or its downstream proteins, the 'Smads' lead to glomerulosclerosis in animal models. Activation of the renin-angiotensin system upregulates TGFβ, which is considered to cause further progression of the disease (Harris RC et al. 2006). Angiogenic factors, like platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) seem to play a role in disease progression. This is based upon the rat remnant kidney model (RK model) experimental studies of progressive glomerulosclerosis. In this model, VEGF upregulation soon after the renal injury and later loss of VEGF expression correlates well with progression of the glomerulosclerosis (Kang DH et al. 2001).

Mechanical stress is also believed to play a role in the progression of FSGS. (Hostetter TH. 2003 & Kwoh C. et al 2006) The hyperfiltration due to the defects of the filtration barrier results in increased single nephron glomerular filtration rate (SNGFR), which results in hypertrophy of glomeruli. The hypertrophy exacerbates the mismatch between the glomerular basement membrane and the decreased numbers of podocytes, propagating the

Focal Segmental Glomerulosclerosis 29

identified in association with FSGS like TRPC6 (Transient receptor potential cation chanel, subfamily C, member 6) which is a cation-selective, ion-channel protein that mediates calcium signals (Winn MP et al. 2005). The role of the other components of the slit

In summary, these data suggest that mutations in the cytoskeleton and membrane proteins specific to podocytes are responsible for most inherited forms of disease. The frequency of spontaneous mutations in the general population who develop nephrotic syndromes or FSGS still needs to be assessed as it has diagnostic, prognostic and therapeutic implications in case of FSGS. For example, patients who possess genetically defective podocytes should be unresponsive to conventional steroid treatment. Similarly, these patients should not have recurrent FSGS when transplanted with a structurally normal kidney allograft. Furthermore, family members who are potential living donors could be genetically screened for mutations associated with the development of kidney

Secondary FSGS can be seen in a variety of conditions such as renal agenesis, obesity, or sickle cell disease etc where hyperfiltration is a characteristic abnormality. Glomerulomegaly is common in situations with hyperfiltration. Obesity-associated FSGS needs a definitive diagnosis with a renal biopsy to exclude the presence of concurrent early diabetic nephropathy. Secondary FSGS may also result from intravenous drug abuse and reflux disease. It has been reported that toxins, including lithium and pamidronate, and sirolimus are associated with the development of FSGS lesions. Among the viral infections, HIV is the most common cause. Rarer viral causes of secondary FSGS include persistent parvovirus B19,

Primary FSGS more likely presents with sudden-onset nephrotic syndrome, whereas secondary FSGS presents more insidiously with subnephrotic range proteinuria and renal insufficiency. But the secondary FSGS from pamidronate toxicity though typically presents with full nephrotic syndrome and acute or subacute renal failure, with collapsing FSGS lesions on biopsy. Physical exam reveals elevated blood pressure and edema. Urine may be foamy and may have hematuria. The relative frequencies of these findings at presentation or biopsy based diagnosis in published series (Rydell JJ et al. 1995 & Chun MJ et al 2004) are as follows. Primary FSGS can present with nephrotic range proteinuria (60 to 75%), microscopic hematuria with variable degrees of proteinuria (30 to 50% ), hypertension (45 to 65 %), renal insufficiency (25 to 50 %) and with overlapping of these presentations. Children tend to present with more proteinuria whereas hypertension is more common in adults. A case of FSGS should be considered idiopathic only when other etiologies are thoroughly excluded. Uncommonly Muehrcke's lines (white banding on the nails due to hypoalbuminaemia), xanthelasma and xanthomata (cholesterol deposits in skin) are associated with FSGS as well. Following risk factors need to be considered when doing history and physical examination on such patients: male gender, black race, positive family history, heroin abuse, use of known causative medications, chronic viral

diaphragm in the pathophysiology of FSGS is not yet clear.

disease and excluded as candidate donors.

simian virus 40, and cytomegalovirus infection.

infection, a solitary kidney, and obesity.

**3. Secondary FSGS** 

**4. Clinical presentation** 

injury further. Another factor in the progression of FSGS is tubulointerstitial injury. Clinically, tubulointerstitial injury is a predictor of the loss of renal function in FSGS (D'Agati VD 2003 & Rodriguez-Iturbe B et al. 2005). The nonspecific entry of proteins into the tubular lumen is one potential source of damage to the tubulointerstitium. Indeed, persistence of nephrotic-range proteinuria is a negative prognostic factor for the progression of FSGS to ESRD (Walls J. 2001). Cytokines (such as TGFβ), when present in the tubules, will recruit monocytes, macrophage, and T-cells. This stimulates other cytokines, including interleukin-1, tumor necrosis factor alpha, and other chemokines. This inflammatory infiltrate leads to mesangial matrix deposition, promoting the collapse of glomeruli. The cellular infiltrate and cytokines also damage tubular epithelial cells, and some tubular epithelial cells may undergo transformation to mesenchymal cells. These mesenchymal cells, as well as recruited and stimulated fibroblasts, result in collagen matrix deposition and tubulointerstitial fibrosis

(Harris RC et al. 2006).

Regardless of the cause of podocyte injury, when the podocytes start dying, leakage of protein across the GBM leading to proteinuria and hypoalbuminemia ensues. At this time cholesterol levels start rising due to increased synthesis of cholesterol by the liver and loss of lipid regulating proteins in the urine, the underlying mechanism of these effects is not completely known yet. The beneficial effects of blocking the renin-angiotensin system may not be limited to their antiproteinuric or antihypertensive effects. As noted earlier, angiotensin stimulates TGFβ, in turn contributing to fibrosis. In addition, angiotensin affects intracellular calcium concentrations and the podocyte cytoskeleton (Harris RC et al. 2006). Inhibition of angiotensin may slow progression by these local mechanisms (Korbert SM, 2003).

With increasing incidence of FSGS, these pathways of podocyte injury and disease progression provide important targets for future intervention. Trials have already been initiated to antagonize cytokines, such as TGFβ (as discussed later in this chapter in treatment section).

Genetic mutations seen in congenital forms of nephrotic syndrome and FSGS enabled researchers to identify specific gene mutations involved in podocyte damage (Tryggvason K. et al. 2006). Mutations of the nephrin gene, a podocyte-specific transmembrane component of the slit diaphragm, are found in congenital Finnish-type nephrotic syndrome, and may lead to loss of normal caliber slit diaphragms. (Kestila M. et al. 1998, Tryggvason K. et al. 2006, Kwoh et al. 2006). In mouse models, mutations of nephrin-like transmembrane genes (NEPH-1) which also localize to the slit diaphragm result in proteinuria and early death. Other proteins which are part of the slit diaphragm complex include: podocin, CD2 associated protein (CD2AP), FAT, P-cadherin,ZO-1, LAP (leucine rich repeat and PDZ domain) protein. (Asanuma K et al 2003, Tryggvason K. et al. 2006). Mutations in podocin (a transmembrane protein that interacts with nephrin, NEPH-1 and CD2AP) have been identified in familial FSGS. Recently, mutations in CD2AP, an immunoglobulin-like protein that is involved in nephrin integration with podocyte cytoskeleton, have also been linked to genetic forms of FSGS (Shih NY. et al. 1999, Kim JM et al 2003, Tyggvason K. et al 2006). In mouse models, the loss of FAT1 and FAT2 (transmembrane proteins with cadherin-like repeats) results in the absence of slit diaphragms, proteinuria, and early death. Alphaactinin-4, an important structural component of the podocyte cytoskeleton, is mutated in some autosomal dominant forms of FSGS (Kaplan JM et al 2000, Yao J et al. 2004). In addition to the abnormal structural proteins mutations, some other mutations have been

injury further. Another factor in the progression of FSGS is tubulointerstitial injury. Clinically, tubulointerstitial injury is a predictor of the loss of renal function in FSGS (D'Agati VD 2003 & Rodriguez-Iturbe B et al. 2005). The nonspecific entry of proteins into the tubular lumen is one potential source of damage to the tubulointerstitium. Indeed, persistence of nephrotic-range proteinuria is a negative prognostic factor for the progression of FSGS to ESRD (Walls J. 2001). Cytokines (such as TGFβ), when present in the tubules, will recruit monocytes, macrophage, and T-cells. This stimulates other cytokines, including interleukin-1, tumor necrosis factor alpha, and other chemokines. This inflammatory infiltrate leads to mesangial matrix deposition, promoting the collapse of glomeruli. The cellular infiltrate and cytokines also damage tubular epithelial cells, and some tubular epithelial cells may undergo transformation to mesenchymal cells. These mesenchymal cells, as well as recruited and stimulated fibroblasts, result in collagen matrix deposition and

Regardless of the cause of podocyte injury, when the podocytes start dying, leakage of protein across the GBM leading to proteinuria and hypoalbuminemia ensues. At this time cholesterol levels start rising due to increased synthesis of cholesterol by the liver and loss of lipid regulating proteins in the urine, the underlying mechanism of these effects is not completely known yet. The beneficial effects of blocking the renin-angiotensin system may not be limited to their antiproteinuric or antihypertensive effects. As noted earlier, angiotensin stimulates TGFβ, in turn contributing to fibrosis. In addition, angiotensin affects intracellular calcium concentrations and the podocyte cytoskeleton (Harris RC et al. 2006). Inhibition of angiotensin may slow progression by these local mechanisms (Korbert SM,

With increasing incidence of FSGS, these pathways of podocyte injury and disease progression provide important targets for future intervention. Trials have already been initiated to antagonize cytokines, such as TGFβ (as discussed later in this chapter in

Genetic mutations seen in congenital forms of nephrotic syndrome and FSGS enabled researchers to identify specific gene mutations involved in podocyte damage (Tryggvason K. et al. 2006). Mutations of the nephrin gene, a podocyte-specific transmembrane component of the slit diaphragm, are found in congenital Finnish-type nephrotic syndrome, and may lead to loss of normal caliber slit diaphragms. (Kestila M. et al. 1998, Tryggvason K. et al. 2006, Kwoh et al. 2006). In mouse models, mutations of nephrin-like transmembrane genes (NEPH-1) which also localize to the slit diaphragm result in proteinuria and early death. Other proteins which are part of the slit diaphragm complex include: podocin, CD2 associated protein (CD2AP), FAT, P-cadherin,ZO-1, LAP (leucine rich repeat and PDZ domain) protein. (Asanuma K et al 2003, Tryggvason K. et al. 2006). Mutations in podocin (a transmembrane protein that interacts with nephrin, NEPH-1 and CD2AP) have been identified in familial FSGS. Recently, mutations in CD2AP, an immunoglobulin-like protein that is involved in nephrin integration with podocyte cytoskeleton, have also been linked to genetic forms of FSGS (Shih NY. et al. 1999, Kim JM et al 2003, Tyggvason K. et al 2006). In mouse models, the loss of FAT1 and FAT2 (transmembrane proteins with cadherin-like repeats) results in the absence of slit diaphragms, proteinuria, and early death. Alphaactinin-4, an important structural component of the podocyte cytoskeleton, is mutated in some autosomal dominant forms of FSGS (Kaplan JM et al 2000, Yao J et al. 2004). In addition to the abnormal structural proteins mutations, some other mutations have been

tubulointerstitial fibrosis (Harris RC et al. 2006).

2003).

treatment section).

identified in association with FSGS like TRPC6 (Transient receptor potential cation chanel, subfamily C, member 6) which is a cation-selective, ion-channel protein that mediates calcium signals (Winn MP et al. 2005). The role of the other components of the slit diaphragm in the pathophysiology of FSGS is not yet clear.

In summary, these data suggest that mutations in the cytoskeleton and membrane proteins specific to podocytes are responsible for most inherited forms of disease. The frequency of spontaneous mutations in the general population who develop nephrotic syndromes or FSGS still needs to be assessed as it has diagnostic, prognostic and therapeutic implications in case of FSGS. For example, patients who possess genetically defective podocytes should be unresponsive to conventional steroid treatment. Similarly, these patients should not have recurrent FSGS when transplanted with a structurally normal kidney allograft. Furthermore, family members who are potential living donors could be genetically screened for mutations associated with the development of kidney disease and excluded as candidate donors.

### **3. Secondary FSGS**

Secondary FSGS can be seen in a variety of conditions such as renal agenesis, obesity, or sickle cell disease etc where hyperfiltration is a characteristic abnormality. Glomerulomegaly is common in situations with hyperfiltration. Obesity-associated FSGS needs a definitive diagnosis with a renal biopsy to exclude the presence of concurrent early diabetic nephropathy. Secondary FSGS may also result from intravenous drug abuse and reflux disease. It has been reported that toxins, including lithium and pamidronate, and sirolimus are associated with the development of FSGS lesions. Among the viral infections, HIV is the most common cause. Rarer viral causes of secondary FSGS include persistent parvovirus B19, simian virus 40, and cytomegalovirus infection.

### **4. Clinical presentation**

Primary FSGS more likely presents with sudden-onset nephrotic syndrome, whereas secondary FSGS presents more insidiously with subnephrotic range proteinuria and renal insufficiency. But the secondary FSGS from pamidronate toxicity though typically presents with full nephrotic syndrome and acute or subacute renal failure, with collapsing FSGS lesions on biopsy. Physical exam reveals elevated blood pressure and edema. Urine may be foamy and may have hematuria. The relative frequencies of these findings at presentation or biopsy based diagnosis in published series (Rydell JJ et al. 1995 & Chun MJ et al 2004) are as follows. Primary FSGS can present with nephrotic range proteinuria (60 to 75%), microscopic hematuria with variable degrees of proteinuria (30 to 50% ), hypertension (45 to 65 %), renal insufficiency (25 to 50 %) and with overlapping of these presentations. Children tend to present with more proteinuria whereas hypertension is more common in adults. A case of FSGS should be considered idiopathic only when other etiologies are thoroughly excluded. Uncommonly Muehrcke's lines (white banding on the nails due to hypoalbuminaemia), xanthelasma and xanthomata (cholesterol deposits in skin) are associated with FSGS as well. Following risk factors need to be considered when doing history and physical examination on such patients: male gender, black race, positive family history, heroin abuse, use of known causative medications, chronic viral infection, a solitary kidney, and obesity.

Focal Segmental Glomerulosclerosis 31

Differences in clinical manifestations correlate with differences in pathologic phenotypes. Collapsing variant of FSGS often has more severe proteinuria and renal insufficiency but less hypertension than typical variant. Patients with collapsing FSGS frequently have extrarenal manifestations of the disease, a few weeks before the onset of the nephrosis e.g. episodes of upper respiratory infections, diarrhea that are usually ascribed to the viral or other infectious processes. However, the symptoms of fever, anorexia, aches and pains are present only in 20% of patients at the time of onset of nephrosis. Glomerular tip lesion variant (Fig 4) often presents with rapid onset of edema similar to minimal change disease. Glomerular tip lesion patients may develop reversible acute renal failure, at the times of initial presentation when the degree of proteinuria, edema, hypoalbuminemia are at their peak. This behaves like minimal change disease and this rarely happens in any other forms of FSGS. Patients with glomerular tip lesion FSGS tend to be older white males in

Distinguishing between primary and secondary FSGS is important, since secondary FSGS should not respond to immunosuppressive therapy. Instead, the inciting condition or toxin should be alleviated or stopped in secondary form FSGS if possible. Unfortunately, distinguishing between primary vs. secondary FSGS can be challenging, since focal sclerosis lesions may be present in a diverse assortment of glomerular, vascular, and tubular injuries, just as global sclerosis represents a common endpoint lesion for the end-stage renal disease.

Urine analysis may have wide range of findings starting from fatty casts to dysmorphic red blood cells and red blood cell casts. Proteinuria varies from less than 1 gm to 30g/day. Hypoproteinemia is common in patients with FSGS, with total serum protein reduced to varying extents. Hypoalbuminemia may drop as low as below 2 g/dL, especially in patients with the collapsing and glomerular tip variants of FSGS. Cholesterol levels are increased. Serum complement components are typically in normal range in FSGS. CD4 cell count and HIV test is essential in all patients with FSGS, especially those with the collapsing pattern. DNA/PCR for Parvo B19 and CMV test is an essential part of work up to rule out the rare forms of secondary FSGS. Finally renal biopsy is the final step to make the diagnosis of FSGS.

The diagnosis of FSGS is not an easy one to make, because the morphologic features of FSGS are nonspecific and can occur in a variety of other conditions or superimposed on other glomerular disease processes. Additionally, because the defining glomerular lesion is focal, it may not be adequately sampled in small needle biopsies. The diagnosis of FSGS is further complicated by the existence of a primary (or idiopathic) form and many secondary forms (Table 1). Before a diagnosis of primary FSGS can be made, secondary forms must be excluded. Idiopathic FSGS must be distinguished from human immunodeficiency virus (HIV) -associated nephropathy, heroin nephropathy and other large group of secondary FSGS caused by structural-functional adaptations mediated by intrarenal vasodilatation, and increased glomerular capillary pressures, (as listed in Table 1). The morphological types are not used to guide treatment but to provide useful prognostic information. Electron microscopy can be used to distinguish primary and secondary FSGS. In primary FSGS, foot process fusion is diffuse and occurs throughout the glomeruli. In secondary FSGS, foot

comparison to younger black males predominance in collapsing variant FSGS.

**5. Laboratory findings** 

**6. Histopathology** 

process fusion is mostly limited to the sclerotic areas.

Primary or idiopathic FSGS: Refer to Table 2

Secondary FSGS:

	- a. HIV associated nephropathy (HIVAN)
	- b. Parvovirus B19
	- c. SV40
	- d. CMV
	- a. Pamidronate
	- b. Lithium
	- c. Interferon –alpha
	- d. Heroin
	- e. Sirolimus

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_


\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Table 1. Etiologic Classification of Focal Segmental Glomerulosclerosis

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Primary or idiopathic FSGS: Refer to Table 2

a. HIV associated nephropathy (HIVAN)

3. Secondary FSGS mediated by adaptive structural-functional responses

Any advanced renal disease with reduction in functioning nephrons

mutations in genes NPHS1 (coding for nephrin) ,NPHS2 (coding for podocin) and

Mutations in genes ACTN1 (coding for alpha actinin4), TRPC6 (coding for TRPC6),

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Atheroembolic or other acute vaso-occlusive processes

Table 1. Etiologic Classification of Focal Segmental Glomerulosclerosis

Secondary FSGS: 1. Virus assoiated

c. SV40 d. CMV 2. Drug toxicity

> d. Heroin e. Sirolimus

 Obesity Hypertension

4. Familial FSGS

 Sickle cell anemia Anabolic steroids

b. Parvovirus B19

a. Pamidronate b. Lithium

c. Interferon –alpha

a. Reduced renal mass Unilateral renal agenesis Oligomeganephronia Renal dysplasia Reflux nephropathy Sequela to cortical necrosis Surgical renal ablation

Chronic allograft nephropathy

b. Initially normal renal mass

Cyanotic congenital heart disease

PLCE1 (coding for PLC epsilon1)

LAMB2 (coding for Laminin beta 2 chain)

a. Autosomal recessive :

b. Autosomal dominant

 INF2 (coding for INF2) WT1 (coding for WT1)

Differences in clinical manifestations correlate with differences in pathologic phenotypes. Collapsing variant of FSGS often has more severe proteinuria and renal insufficiency but less hypertension than typical variant. Patients with collapsing FSGS frequently have extrarenal manifestations of the disease, a few weeks before the onset of the nephrosis e.g. episodes of upper respiratory infections, diarrhea that are usually ascribed to the viral or other infectious processes. However, the symptoms of fever, anorexia, aches and pains are present only in 20% of patients at the time of onset of nephrosis. Glomerular tip lesion variant (Fig 4) often presents with rapid onset of edema similar to minimal change disease. Glomerular tip lesion patients may develop reversible acute renal failure, at the times of initial presentation when the degree of proteinuria, edema, hypoalbuminemia are at their peak. This behaves like minimal change disease and this rarely happens in any other forms of FSGS. Patients with glomerular tip lesion FSGS tend to be older white males in comparison to younger black males predominance in collapsing variant FSGS.

Distinguishing between primary and secondary FSGS is important, since secondary FSGS should not respond to immunosuppressive therapy. Instead, the inciting condition or toxin should be alleviated or stopped in secondary form FSGS if possible. Unfortunately, distinguishing between primary vs. secondary FSGS can be challenging, since focal sclerosis lesions may be present in a diverse assortment of glomerular, vascular, and tubular injuries, just as global sclerosis represents a common endpoint lesion for the end-stage renal disease.

#### **5. Laboratory findings**

Urine analysis may have wide range of findings starting from fatty casts to dysmorphic red blood cells and red blood cell casts. Proteinuria varies from less than 1 gm to 30g/day. Hypoproteinemia is common in patients with FSGS, with total serum protein reduced to varying extents. Hypoalbuminemia may drop as low as below 2 g/dL, especially in patients with the collapsing and glomerular tip variants of FSGS. Cholesterol levels are increased. Serum complement components are typically in normal range in FSGS. CD4 cell count and HIV test is essential in all patients with FSGS, especially those with the collapsing pattern. DNA/PCR for Parvo B19 and CMV test is an essential part of work up to rule out the rare forms of secondary FSGS. Finally renal biopsy is the final step to make the diagnosis of FSGS.

#### **6. Histopathology**

The diagnosis of FSGS is not an easy one to make, because the morphologic features of FSGS are nonspecific and can occur in a variety of other conditions or superimposed on other glomerular disease processes. Additionally, because the defining glomerular lesion is focal, it may not be adequately sampled in small needle biopsies. The diagnosis of FSGS is further complicated by the existence of a primary (or idiopathic) form and many secondary forms (Table 1). Before a diagnosis of primary FSGS can be made, secondary forms must be excluded. Idiopathic FSGS must be distinguished from human immunodeficiency virus (HIV) -associated nephropathy, heroin nephropathy and other large group of secondary FSGS caused by structural-functional adaptations mediated by intrarenal vasodilatation, and increased glomerular capillary pressures, (as listed in Table 1). The morphological types are not used to guide treatment but to provide useful prognostic information. Electron microscopy can be used to distinguish primary and secondary FSGS. In primary FSGS, foot process fusion is diffuse and occurs throughout the glomeruli. In secondary FSGS, foot process fusion is mostly limited to the sclerotic areas.

Focal Segmental Glomerulosclerosis 33

effacement of foot processes (Fig 1.B)and podocyte hypertrophy. The adjacent nonsclerotic

 Fig. 1. (A-left) Focal segmental distribution of glomerular lesions. The glomerulus at the top right of the picture is normal. The glomerulus at the bottom shows dense segmental scars with adhesion to Bowman's capsule. (Periodic acid methenamine silver, 20X). (B-Right) Patchy effacement of foot processes is present. No immune complex-type deposits are seen

**Perihilar Variant of Focal Segmental Glomerulosclerosis** : This variant is defined as perihilar hyalinosis and sclerosis (Fig 2. A & B) which involves more than 50% of glomeruli with segmental lesions. This category requires that the cellular, tip, and collapsing variants

Fig. 2. (A-left): Perihillar variant FSGS (B): Glomerulus with "Hyalinosis": homogeneous, eosinophilic material may be present at the periphery of sclerotic foci, or within the capillary

Immunofluorescence (IF) reveals segmental deposits of IgM and C3 in areas of sclerosis and

Electron microscopy (EM) demonstrates variable foot process effacement. The perihilar variant may occur in primary or secondary FSGS. It is more common in secondary forms of FSGS mediated by adaptive structural-functional responses, in which it is typically accompanied by glomerular hypertrophy. In this setting, the greater filtration pressures at

http://www.unckidneycenter.org/kidneyhealthlibrary/fsgs.html#causes)

glomerular capillaries show only foot process effacement.

along the GBM. (Electron Microscopy).

be excluded. Podocyte hyperplasia is uncommon.

subendothelial space (PAS, 40X). (Fig 2A-obtained from

hyalinosis.


#### Five main light microscopic patterns of FSGS have been defined, as given below:

Classified based upon the 'Working Columbia Classification 2004'.

Table 2. Morphological variants of FSGS

Although appearance of the glomerular tuft differs in these forms, all share the common feature of podocyte alterations at the ultrastructural level. At present, it is unclear if these morphologic variants reflect pathogenetic differences or they are the consequence of different severities of podocyte injury or histopathologic evolution. Future studies are needed to address these questions.

**Classic Focal Segmental Glomerulosclerosis** (Focal Segmental Glomerulosclerosis Not Otherwise Specified) : also called FSGS NOS, or typical FSGS. FSGS NOS requires exclusion of the other more specific subtypes described in the table 2. Light Micrscopic examination reveals accumulation of extracellular matrix which occlude glomerular capillaries, forming discrete segmental solidifications involving affected portion of the glomerular tuft. Also seen is plasmatic insudation of amorphous glassy material beneath the GBM, endocapillary foam cells, and wrinkling of the GBM. Adhesions to Bowman's capsule are common, and overlying visceral epithelial cells often appear swollen over the sclerosing segment. Non sclerotic glomerular lobules appear normal by light microscopy except for mild podocyte swelling.

Immunofluorescence: focal and segmental granular deposition of IgM, and C3 is seen often, but C1 in the distribution of segmental glomerular sclerosis may also be seen. Nonsclerotic glomeruli may have weak mesangial staining for IgM and C3. By electron microscopic examination, segmental sclerotic lesions exhibit increased matrix, wrinkling and retraction of GBM. Accumulation of inframembranous hyaline material but *no immune complex* electron-dense deposits are seen. Overlying the segmental sclerosis, there is usually

Although appearance of the glomerular tuft differs in these forms, all share the common feature of podocyte alterations at the ultrastructural level. At present, it is unclear if these morphologic variants reflect pathogenetic differences or they are the consequence of different severities of podocyte injury or histopathologic evolution. Future studies are

**Classic Focal Segmental Glomerulosclerosis** (Focal Segmental Glomerulosclerosis Not Otherwise Specified) : also called FSGS NOS, or typical FSGS. FSGS NOS requires exclusion of the other more specific subtypes described in the table 2. Light Micrscopic examination reveals accumulation of extracellular matrix which occlude glomerular capillaries, forming discrete segmental solidifications involving affected portion of the glomerular tuft. Also seen is plasmatic insudation of amorphous glassy material beneath the GBM, endocapillary foam cells, and wrinkling of the GBM. Adhesions to Bowman's capsule are common, and overlying visceral epithelial cells often appear swollen over the sclerosing segment. Non sclerotic glomerular lobules appear normal by light microscopy except for mild podocyte swelling. Immunofluorescence: focal and segmental granular deposition of IgM, and C3 is seen often, but C1 in the distribution of segmental glomerular sclerosis may also be seen. Nonsclerotic glomeruli may have weak mesangial staining for IgM and C3. By electron microscopic examination, segmental sclerotic lesions exhibit increased matrix, wrinkling and retraction of GBM. Accumulation of inframembranous hyaline material but *no immune complex* electron-dense deposits are seen. Overlying the segmental sclerosis, there is usually

Five main light microscopic patterns of FSGS have been defined, as given below:

Classified based upon the 'Working Columbia Classification 2004'.

Table 2. Morphological variants of FSGS

needed to address these questions.

effacement of foot processes (Fig 1.B)and podocyte hypertrophy. The adjacent nonsclerotic glomerular capillaries show only foot process effacement.

Fig. 1. (A-left) Focal segmental distribution of glomerular lesions. The glomerulus at the top right of the picture is normal. The glomerulus at the bottom shows dense segmental scars with adhesion to Bowman's capsule. (Periodic acid methenamine silver, 20X). (B-Right) Patchy effacement of foot processes is present. No immune complex-type deposits are seen along the GBM. (Electron Microscopy).

**Perihilar Variant of Focal Segmental Glomerulosclerosis** : This variant is defined as perihilar hyalinosis and sclerosis (Fig 2. A & B) which involves more than 50% of glomeruli with segmental lesions. This category requires that the cellular, tip, and collapsing variants be excluded. Podocyte hyperplasia is uncommon.

Fig. 2. (A-left): Perihillar variant FSGS (B): Glomerulus with "Hyalinosis": homogeneous, eosinophilic material may be present at the periphery of sclerotic foci, or within the capillary subendothelial space (PAS, 40X). (Fig 2A-obtained from http://www.unckidneycenter.org/kidneyhealthlibrary/fsgs.html#causes)

Immunofluorescence (IF) reveals segmental deposits of IgM and C3 in areas of sclerosis and hyalinosis.

Electron microscopy (EM) demonstrates variable foot process effacement. The perihilar variant may occur in primary or secondary FSGS. It is more common in secondary forms of FSGS mediated by adaptive structural-functional responses, in which it is typically accompanied by glomerular hypertrophy. In this setting, the greater filtration pressures at

Focal Segmental Glomerulosclerosis 35

IF microscopy of collapsing lesions often is positive for IgM and C3. EM reveals severe foot

This variant is defined by the presence of at least one segmental lesion involving the tip domain (i.e., the outer 25% of the tuft next to the origin of the proximal tubule). There is either adhesion (Fig 4) between the tuft and Bowman's capsule or confluence of swollen podocytes with parietal or tubular epithelial cells at the tubular lumen or neck. In some cases, the affected segment appears to herniate into the tubular lumen. The segmental lesions may be cellular or sclerosing type. Although initially peripherally located, these lesions may progress more centrally. The presence of perihilar sclerosis or collapsing

IF microscopy reveals involved tip area positive for IgM and C3 and EM findings

Fig. 4. Sclerotic glomerular segment with a small adhesion; "Glomerular tip lesion" (Periodic

Typically the goals of treatment in FSGS include-(1) to reduce, eliminate or at least suppress the proteinuria and (2) to avoid or at least retard the progression to ESRD. Treatment of the FSGS can be arbitrarily divided into (1) **Nonspecific; nutritional management** (2)**Non immunosuppressive therapy**: diuretics and RAS interference. (3) **Immunosuppressive treatment: which includes the use of s**teroids, steroid sparing medications like cyclophosphamide, cyclosporine A, tacrolimus and mycophenolic mofetil. **(4)-Antifibrotic drugs:Pirfenidone, Rosiglitazone (FONT Phase 1 trial completed and now FONT phase II trial is ongoing) (5)-Monoclonal/polyclonal Antibodies: Adalimumab, Rituximab,** 

Nonspecific treatment: Nonspecific treatment goals in FSGS are like any other glomerulopathy associated with nephrotic syndrome, which include maintenance of adequate nutrition, minimization or elimination of proteinuria, and prevention of complications resulting from edema. The mainstay of treatment is reduction in daily salt intake to 2 g of sodium. A high level of protein intake may further aggravate proteinuria, adversely affecting renal function. Current recommendations call for an intake of 1to1.3 g of

process effacement affecting both collapsed and noncollapsed glomeruli

**Tip Variant of Focal Segmental Glomerulosclerosis :** 

sclerosis rules out the tip variant.

acid methenamine silver, 40X)

**Fresolmumab (6)-Plasmapheresis** 

**7. Treatment** 

demonstrate foot process effacement.

the proximal end of the glomerular capillary bed may favor the development of lesions at the vascular pole.

**Cellular Variant of Focal Segmental Glomerulosclerosis** : This variant is characterized by focal and segmental endocapillary hypercellularity that may mimic a form of focal proliferative glomerulonephritis. Glomerular capillaries are segmentally occluded by endocapillary hypercellularity, including foam cells, infiltrating leukocytes, karyorrhectic debris, and hyaline. There is often hyperplasia of the visceral epithelial cells, which may appear swollen and crowded, sometimes forming pseudocrescents. This variant requires that tip lesions and collapsing lesions be excluded.

IF shows focal and segmental glomerular positivity for IgM and C3 and EM reveals severe foot process effacement.

**Collapsing Variant of Focal Segmental Glomerulosclerosis:** This variant is defined by at least one glomerulus with segmental or global collapse (Fig 3) and overlying hypertrophy and hyperplasia of visceral epithelial cells. There is occlusion of glomerular capillary lumina by implosive wrinkling and collapse of the GBMs. This lesion is more often global than segmental. Overlying podocytes display striking hypertrophy, hyperplasia and express proliferation markers. Podocytes often contain prominent intracytoplasmic protein resorption droplets and may fill Bowman's space, forming pseudocrescents . Although podocyte hyperplasia is found in both the collapsing and cellular variants of FSGS, collapsing glomerulopathy is distinguished by the absence of endocapillary hypercellularity. In collapsing FSGS, there is prominent tubulointerstitial disease, including tubular atrophy, interstitial fibrosis, interstitial edema, and inflammation. A distinctive feature is the presence of dilated tubules forming microcysts that contain loose proteinaceous casts. This pattern can occur both in primary FSGS and also in secondary FSGS due to HIV,parvovirus B12,pamidronate toxicity and interferon therapy. Endothelial tubuloreticular inclusions are identified in over 90% of patients with HIVinfection collapsing glomerulopathy and interferon therapy, whereas only in 10% cases with idiopathic collapsing glomerulopathy. Besides these conditions endothelial tubuloreticular inclusions are seen commonly in systemic lupus erythematosus nephritis.

Fig. 3. A glomerulus displays collapsing variant of FSGS , glomerular capillaries show collapse with absent lumen (H&E, 40X).

the proximal end of the glomerular capillary bed may favor the development of lesions at

**Cellular Variant of Focal Segmental Glomerulosclerosis** : This variant is characterized by focal and segmental endocapillary hypercellularity that may mimic a form of focal proliferative glomerulonephritis. Glomerular capillaries are segmentally occluded by endocapillary hypercellularity, including foam cells, infiltrating leukocytes, karyorrhectic debris, and hyaline. There is often hyperplasia of the visceral epithelial cells, which may appear swollen and crowded, sometimes forming pseudocrescents. This variant requires

IF shows focal and segmental glomerular positivity for IgM and C3 and EM reveals severe

**Collapsing Variant of Focal Segmental Glomerulosclerosis:** This variant is defined by at least one glomerulus with segmental or global collapse (Fig 3) and overlying hypertrophy and hyperplasia of visceral epithelial cells. There is occlusion of glomerular capillary lumina by implosive wrinkling and collapse of the GBMs. This lesion is more often global than segmental. Overlying podocytes display striking hypertrophy, hyperplasia and express proliferation markers. Podocytes often contain prominent intracytoplasmic protein resorption droplets and may fill Bowman's space, forming pseudocrescents . Although podocyte hyperplasia is found in both the collapsing and cellular variants of FSGS, collapsing glomerulopathy is distinguished by the absence of endocapillary hypercellularity. In collapsing FSGS, there is prominent tubulointerstitial disease, including tubular atrophy, interstitial fibrosis, interstitial edema, and inflammation. A distinctive feature is the presence of dilated tubules forming microcysts that contain loose proteinaceous casts. This pattern can occur both in primary FSGS and also in secondary FSGS due to HIV,parvovirus B12,pamidronate toxicity and interferon therapy. Endothelial tubuloreticular inclusions are identified in over 90% of patients with HIVinfection collapsing glomerulopathy and interferon therapy, whereas only in 10% cases with idiopathic collapsing glomerulopathy. Besides these conditions endothelial tubuloreticular

inclusions are seen commonly in systemic lupus erythematosus nephritis.

Fig. 3. A glomerulus displays collapsing variant of FSGS , glomerular capillaries show

collapse with absent lumen (H&E, 40X).

the vascular pole.

foot process effacement.

that tip lesions and collapsing lesions be excluded.

IF microscopy of collapsing lesions often is positive for IgM and C3. EM reveals severe foot process effacement affecting both collapsed and noncollapsed glomeruli

**Tip Variant of Focal Segmental Glomerulosclerosis :** 

This variant is defined by the presence of at least one segmental lesion involving the tip domain (i.e., the outer 25% of the tuft next to the origin of the proximal tubule). There is either adhesion (Fig 4) between the tuft and Bowman's capsule or confluence of swollen podocytes with parietal or tubular epithelial cells at the tubular lumen or neck. In some cases, the affected segment appears to herniate into the tubular lumen. The segmental lesions may be cellular or sclerosing type. Although initially peripherally located, these lesions may progress more centrally. The presence of perihilar sclerosis or collapsing sclerosis rules out the tip variant.

IF microscopy reveals involved tip area positive for IgM and C3 and EM findings demonstrate foot process effacement.

Fig. 4. Sclerotic glomerular segment with a small adhesion; "Glomerular tip lesion" (Periodic acid methenamine silver, 40X)

### **7. Treatment**

Typically the goals of treatment in FSGS include-(1) to reduce, eliminate or at least suppress the proteinuria and (2) to avoid or at least retard the progression to ESRD. Treatment of the FSGS can be arbitrarily divided into (1) **Nonspecific; nutritional management** (2)**Non immunosuppressive therapy**: diuretics and RAS interference. (3) **Immunosuppressive treatment: which includes the use of s**teroids, steroid sparing medications like cyclophosphamide, cyclosporine A, tacrolimus and mycophenolic mofetil. **(4)-Antifibrotic drugs:Pirfenidone, Rosiglitazone (FONT Phase 1 trial completed and now FONT phase II trial is ongoing) (5)-Monoclonal/polyclonal Antibodies: Adalimumab, Rituximab, Fresolmumab (6)-Plasmapheresis** 

Nonspecific treatment: Nonspecific treatment goals in FSGS are like any other glomerulopathy associated with nephrotic syndrome, which include maintenance of adequate nutrition, minimization or elimination of proteinuria, and prevention of complications resulting from edema. The mainstay of treatment is reduction in daily salt intake to 2 g of sodium. A high level of protein intake may further aggravate proteinuria, adversely affecting renal function. Current recommendations call for an intake of 1to1.3 g of

Focal Segmental Glomerulosclerosis 37

this treatment regimen, and relapses are frequent when steroids are discontinued. Blacks and patients with collapsing FSGS are generally refractory to treatment and progress to renal failure. In steroid responsive patients, the goal is to titrate prednisone to the lowest dose needed to stop proteinuria and to prevent relapses. Use of steroids on alternate days can also reduce toxicity. The optimal duration of treatment is uncertain; some authorities recommend use of steroids indefinitely. If no remission after 4 months of corticosteroid

In patients who are refractory to 2-3 months of prednisone therapy, the recommendation is to reduce the steroid dose and to add cyclophosphamide (2.5 mg/kg [150-200 mg/day]), monitor patients for bone marrow suppression, and encourage adequate fluid intake to prevent hemorrhagic cystitis. Prolonged use of cyclophosphamide may lead to gonadal toxicity; therefore, persisting with cyclophosphamide beyond 3 months in patients who do not respond is unwise. Cyclosporine (3.5mg/kg/day) can induce remission and preserve renal function, although relapse occurs in 60% of patients when cyclosporine alone is used (Ponticelli C. et al. 1993 & Cattran DC et al. 1999). Continuing treatment with cyclosporine for 1 year after remission followed by a slow tapering of the dose results in a longer remission (Meyrier A 1994). Low-dose cyclosporine in combination with low-dose prednisone (0.15mg/kg/day) in corticosteroid-resistant patients is more effective than cyclosporine alone (Cattaran DC. Et al 1999 & Meyrier A 2009). Rates of complete (Niaudet P. 1994 & Braun N et al 2008) and partial remission achieved with cyclosporine are greater if low-dose prednisone is given at the same time. Continuous use of cyclosporine for >12 months is associated with a significant increase in tubulointerstitial fibrosis. Its use should,

treatment, disease is defined as being corticosteroid-resistant (Meyrier A.,2009).

therefore, be limited to patients with creatinine clearance >60 ml/min/1.73 m2

were uncontrolled investigations that were limited to a few patients.

partial remission (4 patients). [Segarra A et al 2002].

**7.3 Tacrolimus** 

**7.4 Mycophenolate mofetil** 

Isolated reports have suggested that patients who are refractory to steroids and cyclophosphamide, treatment with other immunosuppressive agents, such as tacrolimus and sirolimus, may be beneficial in inducing remission. However, studies using these agents

Few studies have reported the use of tacrolimus for Idiopathic FSGS. In the largest study, 25 patients with nephrotic syndrome due to primary FSGS and known resistance to or dependence on cyclosporine (cyclosporine) were given tacrolimus plus prednisolone (prednisone) for 6 months. Seventeen patients had a reduced proteinuria to <3 g/day, and 12 had complete or partial remission. Thirteen patients relapsed after discontinuing tacrolimus; reinstitution of therapy for 1 year resulted in complete remission (5 patients) and

Mycophenolate mofetil (MMF) is a reversible inhibitor of inosine monophosphate dehydrogenase (IMPDH) in purine biosynthesis. MMF is selective for the de novo pathway critical to lymphocytic proliferation and activation. Because of favorable results in other glomerular diseases, mycophenolate mofetil has also been evaluated in FSGS. Although the experience is limited, the suggested dose is 750-1000 mg twice daily in patients who are refractory to corticosteroids and in whom calcineurin inhibitors may not be appropriate. However, adequate randomized studies supporting this approach are lacking (Cattran DC et al. 2004). If mycophenolate is being considered, cyclosporine should not be used concurrently.

Mycophenolate can be either used alone or in combination with corticosteroids.

high biologic value protein per kilogram of body weight and a reduction of fat intake. Lipid lowering is necessary to reduce cardiovascular risk and to possibly delay the progression of renal disease.

#### **7.1 Non-immunosuppressive drug treatment**

Symptomatic relief of edema helps the patient feel better. In most patients, loop diuretics are needed to promote diuresis. Patients with massive edema with impaired oral absorption may require intravenous administration of loop diuretics. In patients with refractory conditions, addition of other diuretics (eg, metolazone) and potassium-sparing agents (eg, spironolactone, triamterene) facilitates diuresis and prevents hypokalemia. Rarely, some patients (especially children) with intractable edema may need intravenous albumin and diuretics in a hospital setting to initiate diuresis. Protracted use of intravenous albumin should be discouraged; the regimen is expensive and ineffective, because most of the infused albumin is lost in the urine. Continuous IV drip of furosemide is preferred over large boluses of IV push to avoid side effects*.* 

Control of hypertension is one of the most important aspects of overall management in FSGS, like any other glomerular disease. Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) are nonspecific agents that reduce proteinuria because of their antihypertensive and intrarenal hemodynamic effects of reducing glomerular capillary pressure and resistance. ACEIs and ARBs are effective in reducing protein loss even in normotensive patients. These agents do have an effect in slowing down of the disease progression by downregulating the TGFβ by interfering with renin angiotensin system, regardless of any antihypertensive effects.These agents do not abolish proteinuria completely or reverse the primary glomerular disease process per se.

As hypertension develops in most patients with FSGS which further causes deterioration of renal function, the control of blood pressure is an important part of the treatment of FSGS. Many patients, may require combination antihypertensive therapy to maintain blood pressure in the normal range. Lipid lowering therapy like statins is warranted to correct hyperlipidemia to reduce the risk of cardiovascular disease in this subset of patient population.

#### **7.2 Immunosuppressive treatment**

Idiopathic FSGS is a difficult disease to treat because of its highly variable clinical course. The specific treatment approach is still empirical, and no consensus has evolved because of a lack of prospective controlled trials. Current evidence is, mostly derived from retrospective analyses, and favors prolonged steroid therapy (6 months or longer) to induce remission in patients with idiopathic FSGS.

Criteria for remission: (1) complete remission is considered when urinary protein excretion of less than 200-300 mg/day, and (2) partial response is considered when urinary protein excretion of 200-3500 mg/day, or a greater than 50% reduction in baseline proteinuria.

Since long-term steroid therapy is not without serious toxicity, the patient counselling regarding the goals of the therapy, possible potential side affects and expected outcomes, is essential before starting such treatment. The current consensus is to initiate therapy with prednisone in a dose of 1 mg/kg (60-80 mg/d) for 2-6 months or longer, depending on patient's response as assessed by presence or absence of edema, 24-hour urine protein excretions, creatinine clearance, serum creatinine, serum albumin, and lipid levels. Literature reveals that 30-60% of patients may undergo complete or partial remission with

high biologic value protein per kilogram of body weight and a reduction of fat intake. Lipid lowering is necessary to reduce cardiovascular risk and to possibly delay the progression of

Symptomatic relief of edema helps the patient feel better. In most patients, loop diuretics are needed to promote diuresis. Patients with massive edema with impaired oral absorption may require intravenous administration of loop diuretics. In patients with refractory conditions, addition of other diuretics (eg, metolazone) and potassium-sparing agents (eg, spironolactone, triamterene) facilitates diuresis and prevents hypokalemia. Rarely, some patients (especially children) with intractable edema may need intravenous albumin and diuretics in a hospital setting to initiate diuresis. Protracted use of intravenous albumin should be discouraged; the regimen is expensive and ineffective, because most of the infused albumin is lost in the urine. Continuous IV drip of furosemide is preferred over

Control of hypertension is one of the most important aspects of overall management in FSGS, like any other glomerular disease. Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) are nonspecific agents that reduce proteinuria because of their antihypertensive and intrarenal hemodynamic effects of reducing glomerular capillary pressure and resistance. ACEIs and ARBs are effective in reducing protein loss even in normotensive patients. These agents do have an effect in slowing down of the disease progression by downregulating the TGFβ by interfering with renin angiotensin system, regardless of any antihypertensive effects.These agents do not abolish

As hypertension develops in most patients with FSGS which further causes deterioration of renal function, the control of blood pressure is an important part of the treatment of FSGS. Many patients, may require combination antihypertensive therapy to maintain blood pressure in the normal range. Lipid lowering therapy like statins is warranted to correct hyperlipidemia to reduce the risk of cardiovascular disease in this subset of patient

Idiopathic FSGS is a difficult disease to treat because of its highly variable clinical course. The specific treatment approach is still empirical, and no consensus has evolved because of a lack of prospective controlled trials. Current evidence is, mostly derived from retrospective analyses, and favors prolonged steroid therapy (6 months or longer) to induce remission in

Criteria for remission: (1) complete remission is considered when urinary protein excretion of less than 200-300 mg/day, and (2) partial response is considered when urinary protein excretion of 200-3500 mg/day, or a greater than 50% reduction in baseline proteinuria. Since long-term steroid therapy is not without serious toxicity, the patient counselling regarding the goals of the therapy, possible potential side affects and expected outcomes, is essential before starting such treatment. The current consensus is to initiate therapy with prednisone in a dose of 1 mg/kg (60-80 mg/d) for 2-6 months or longer, depending on patient's response as assessed by presence or absence of edema, 24-hour urine protein excretions, creatinine clearance, serum creatinine, serum albumin, and lipid levels. Literature reveals that 30-60% of patients may undergo complete or partial remission with

proteinuria completely or reverse the primary glomerular disease process per se.

renal disease.

population.

**7.2 Immunosuppressive treatment** 

patients with idiopathic FSGS.

**7.1 Non-immunosuppressive drug treatment** 

large boluses of IV push to avoid side effects*.* 

this treatment regimen, and relapses are frequent when steroids are discontinued. Blacks and patients with collapsing FSGS are generally refractory to treatment and progress to renal failure. In steroid responsive patients, the goal is to titrate prednisone to the lowest dose needed to stop proteinuria and to prevent relapses. Use of steroids on alternate days can also reduce toxicity. The optimal duration of treatment is uncertain; some authorities recommend use of steroids indefinitely. If no remission after 4 months of corticosteroid treatment, disease is defined as being corticosteroid-resistant (Meyrier A.,2009).

In patients who are refractory to 2-3 months of prednisone therapy, the recommendation is to reduce the steroid dose and to add cyclophosphamide (2.5 mg/kg [150-200 mg/day]), monitor patients for bone marrow suppression, and encourage adequate fluid intake to prevent hemorrhagic cystitis. Prolonged use of cyclophosphamide may lead to gonadal toxicity; therefore, persisting with cyclophosphamide beyond 3 months in patients who do not respond is unwise. Cyclosporine (3.5mg/kg/day) can induce remission and preserve renal function, although relapse occurs in 60% of patients when cyclosporine alone is used (Ponticelli C. et al. 1993 & Cattran DC et al. 1999). Continuing treatment with cyclosporine for 1 year after remission followed by a slow tapering of the dose results in a longer remission (Meyrier A 1994). Low-dose cyclosporine in combination with low-dose prednisone (0.15mg/kg/day) in corticosteroid-resistant patients is more effective than cyclosporine alone (Cattaran DC. Et al 1999 & Meyrier A 2009). Rates of complete (Niaudet P. 1994 & Braun N et al 2008) and partial remission achieved with cyclosporine are greater if low-dose prednisone is given at the same time. Continuous use of cyclosporine for >12 months is associated with a significant increase in tubulointerstitial fibrosis. Its use should, therefore, be limited to patients with creatinine clearance >60 ml/min/1.73 m2

Isolated reports have suggested that patients who are refractory to steroids and cyclophosphamide, treatment with other immunosuppressive agents, such as tacrolimus and sirolimus, may be beneficial in inducing remission. However, studies using these agents were uncontrolled investigations that were limited to a few patients.

#### **7.3 Tacrolimus**

Few studies have reported the use of tacrolimus for Idiopathic FSGS. In the largest study, 25 patients with nephrotic syndrome due to primary FSGS and known resistance to or dependence on cyclosporine (cyclosporine) were given tacrolimus plus prednisolone (prednisone) for 6 months. Seventeen patients had a reduced proteinuria to <3 g/day, and 12 had complete or partial remission. Thirteen patients relapsed after discontinuing tacrolimus; reinstitution of therapy for 1 year resulted in complete remission (5 patients) and partial remission (4 patients). [Segarra A et al 2002].

#### **7.4 Mycophenolate mofetil**

Mycophenolate mofetil (MMF) is a reversible inhibitor of inosine monophosphate dehydrogenase (IMPDH) in purine biosynthesis. MMF is selective for the de novo pathway critical to lymphocytic proliferation and activation. Because of favorable results in other glomerular diseases, mycophenolate mofetil has also been evaluated in FSGS. Although the experience is limited, the suggested dose is 750-1000 mg twice daily in patients who are refractory to corticosteroids and in whom calcineurin inhibitors may not be appropriate. However, adequate randomized studies supporting this approach are lacking (Cattran DC et al. 2004). If mycophenolate is being considered, cyclosporine should not be used concurrently. Mycophenolate can be either used alone or in combination with corticosteroids.

Focal Segmental Glomerulosclerosis 39

Secondary FSGS treatment management is directed toward the etiology or associated disorder. For example, discontinuing pamidronate in pamidronate induced FSGS, and in HIV-associated FSGS, HAART is associated with remission of proteinuria and preservation of renal function. In heroin-associated FSGS, discontinuation of the drug may result in

Prognosis of idiopathic FSGS is variable. Important prognostic factors are, the amount of proteinuria, the level of plasma creatinine, the morphological subtype, and the response to therapy as listed in table 3. Korbet SM (1999) described that nephrotic patients with FSGS, particularly those with massive proteinuria, have a significantly poorer prognosis than nonnephrotic patients, with 50% progressing to end-stage renal disease (ESRD) over 3-8 years as compared with a 10-year survival of >80%, respectively. In addition, the recurrence rate of this lesion is high in transplanted patients with primary FSGS. When clinical and histological features at presentation have been evaluated by multivariate analysis, the significant positive predictors of progression to ESRD have consistently been the serum creatinine (>1.3 mg/dl), amount of proteinuria and the presence of interstitial fibrosis (> or =20%). The one factor which is a significant negative predictor of progression to ESRD is the achievement of a remission in proteinuria. Unfortunately, spontaneous remissions are rare

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Thomas DB et al. (2006) described that the morphological subtype identified on renal biopsy also provided useful prognostic information. The collapsing variant, the main variant seen in HIV-induced FSGS, is associated with a worse prognosis than the other forms. The tip

Focal segmental glomerulosclerosis is still largely an idiopathic disease but in the recent past, more genetic mutations and secondary causes have been described. As more and more pathogenetic mechanisms involved in idiopathic FSGS are coming into light and more secondary causes of FSGS are described, the occurrence of true idiopathic FSGS diagnosis is

remission of proteinuria and improvement in renal function.

in FSGS, occurring in < or =6% of patients only.

2. Histopathologic features at the time of biopsy

3. Clinical features during the course of the FSGS

variant has a better prognosis than the other forms.

a. Nephrotic range proteinuria or massive proteinuria

a. Failure to achieve partial or complete remission.

Table 3. Risk factors for progressive loss of renal function in FSGS

1. Clinical features at the time of biopsy

b. Elevated serum creatinine

c. Black race

**9. Summary** 

a. Collapsing variant b. Tubulointerstitial fibrosis

**8. Prognosis** 

### **7.5 Monoclonal antibody treatment**

**Rituximab**: Rituximab is an anti CD20 chimeric monoclonal antibody. Case reports have suggested rituximab may be effective in treating patients with minimal change nephropathy and FSGS. (Peters HP et al 2008). . Four patients with nephrotic syndrome due to minimal change nephropathy or FSGS were treated with rituximab because of failure of or intolerance to the standard immunosuppressive therapy. Other cases of FSGS reported in the literature (6 pediatric patients) that were successfully treated with rituximab were also included. Complete remission was reported within 1 month for a 7-year-old boy with FSGS during treatment with rituximab and concurrent treatment with mycophenolate, low-dose prednisolone (prednisone), and tacrolimus. Controlled trials are needed to further evaluate the efficacy of rituximab in FSGS.

**Fresolimumab**: Recently a phase one trial (Trachtman H. et al 2011) has been completed which evaluated the safety and pharmacokinetics of single-dose infusions of fresolimumab, a human monoclonal antibody that inactivates all forms of transforming growth factor-beta (TGF-β), in a phase I open-label, dose-ranging study. Patients with biopsy-confirmed, treatment-resistant, primary FSGS with a minimum estimated glomerular filtration rate (eGFR) of 25 ml/min per 1.73m2, and a urine protein to creatinine ratio over 1.8mg/mg were enrolled. All 16 patients completed the study in which each received one of four single-dose levels of fresolimumab (up to 4mg/kg) and was followed for 112 days. Fresolimumab was well tolerated with pustular rash, the only adverse event, developing in two patients. Single-dose fresolimumab was well tolerated in patients with primary resistant FSGS. Additional evaluation in a larger dose-ranging study is necessary.

#### **7.6 Antifibrotic agents**

**Pirfenidone** has been shown to have therapeutic potential in fibrotic diseases, although the mechanism of action is not well understood. It has been shown to reduce transforming growth factor-β 1 production, antagonize TNF- signaling, and scavenge reactive oxygen species. It also reduces fibrosis and prevents loss of glomerular filtration in animal models of renal disease. An open-label trial evaluated the safety and efficacy of pirfenidone in patients with idiopathic and post adaptive FSGS. Pirfenidone had no effect on BP or proteinuria but it did preserve renal function. Controlled trials are needed to further evaluate the efficacy of pirfenidone in FSGS (Cho ME. et al 2007).

**Rosiglitazone**: Renal mesangial cells express peroxisome proliferator-activated receptor (PPAR –gamma). PPAR-gamma activation, can exhibit anti-inflammatory effects. Weissgarten et al (2006) demonstrated that PPAR-gamma activation by rosiglitazone resulted in decreased manifestation of inflammatory hallmarks, including inhibition of mesangial cell proliferation, downregulation of apoptosis and blunted responsiveness to angiotensin II in animal models. FONT-I (a phase 1 clinical trial) showed that this agent was safe and well tolerated in 11 patients with biopsy-demonstrated FSGS. The results of further studies are awaited.

Despite all attempts, some patients continue to deteriorate and progress to ESRD. Patients and their families should be counselled in detail regarding the treatment options for ESRD so that they can choose appropriate treatment tailored to their life style among maintenance hemodialysis, continuous ambulatory peritoneal dialysis, or renal transplantation. FSGS may recur in the transplanted kidney, but most centers do not consider this a contraindication for renal transplantation.

Secondary FSGS treatment management is directed toward the etiology or associated disorder. For example, discontinuing pamidronate in pamidronate induced FSGS, and in HIV-associated FSGS, HAART is associated with remission of proteinuria and preservation of renal function. In heroin-associated FSGS, discontinuation of the drug may result in remission of proteinuria and improvement in renal function.

### **8. Prognosis**

38 An Update on Glomerulopathies – Clinical and Treatment Aspects

**Rituximab**: Rituximab is an anti CD20 chimeric monoclonal antibody. Case reports have suggested rituximab may be effective in treating patients with minimal change nephropathy

change nephropathy or FSGS were treated with rituximab because of failure of or intolerance to the standard immunosuppressive therapy. Other cases of FSGS reported in the literature (6 pediatric patients) that were successfully treated with rituximab were also included. Complete remission was reported within 1 month for a 7-year-old boy with FSGS during treatment with rituximab and concurrent treatment with mycophenolate, low-dose prednisolone (prednisone), and tacrolimus. Controlled trials are needed to further evaluate

**Fresolimumab**: Recently a phase one trial (Trachtman H. et al 2011) has been completed which evaluated the safety and pharmacokinetics of single-dose infusions of fresolimumab, a human monoclonal antibody that inactivates all forms of transforming growth factor-beta (TGF-β), in a phase I open-label, dose-ranging study. Patients with biopsy-confirmed, treatment-resistant, primary FSGS with a minimum estimated glomerular filtration rate (eGFR) of 25 ml/min per 1.73m2, and a urine protein to creatinine ratio over 1.8mg/mg were enrolled. All 16 patients completed the study in which each received one of four single-dose levels of fresolimumab (up to 4mg/kg) and was followed for 112 days. Fresolimumab was well tolerated with pustular rash, the only adverse event, developing in two patients. Single-dose fresolimumab was well tolerated in patients with primary resistant

**Pirfenidone** has been shown to have therapeutic potential in fibrotic diseases, although the mechanism of action is not well understood. It has been shown to reduce transforming growth factor-β 1 production, antagonize TNF- signaling, and scavenge reactive oxygen species. It also reduces fibrosis and prevents loss of glomerular filtration in animal models of renal disease. An open-label trial evaluated the safety and efficacy of pirfenidone in patients with idiopathic and post adaptive FSGS. Pirfenidone had no effect on BP or proteinuria but it did preserve renal function. Controlled trials are needed to further

**Rosiglitazone**: Renal mesangial cells express peroxisome proliferator-activated receptor (PPAR –gamma). PPAR-gamma activation, can exhibit anti-inflammatory effects. Weissgarten et al (2006) demonstrated that PPAR-gamma activation by rosiglitazone resulted in decreased manifestation of inflammatory hallmarks, including inhibition of mesangial cell proliferation, downregulation of apoptosis and blunted responsiveness to angiotensin II in animal models. FONT-I (a phase 1 clinical trial) showed that this agent was safe and well tolerated in 11 patients with biopsy-demonstrated FSGS. The results of further

Despite all attempts, some patients continue to deteriorate and progress to ESRD. Patients and their families should be counselled in detail regarding the treatment options for ESRD so that they can choose appropriate treatment tailored to their life style among maintenance hemodialysis, continuous ambulatory peritoneal dialysis, or renal transplantation. FSGS may recur in the transplanted kidney, but most centers do not consider this a

FSGS. Additional evaluation in a larger dose-ranging study is necessary.

evaluate the efficacy of pirfenidone in FSGS (Cho ME. et al 2007).

Four patients with nephrotic syndrome due to minimal

**7.5 Monoclonal antibody treatment** 

and FSGS. (Peters HP et al 2008). .

the efficacy of rituximab in FSGS.

**7.6 Antifibrotic agents** 

studies are awaited.

contraindication for renal transplantation.

Prognosis of idiopathic FSGS is variable. Important prognostic factors are, the amount of proteinuria, the level of plasma creatinine, the morphological subtype, and the response to therapy as listed in table 3. Korbet SM (1999) described that nephrotic patients with FSGS, particularly those with massive proteinuria, have a significantly poorer prognosis than nonnephrotic patients, with 50% progressing to end-stage renal disease (ESRD) over 3-8 years as compared with a 10-year survival of >80%, respectively. In addition, the recurrence rate of this lesion is high in transplanted patients with primary FSGS. When clinical and histological features at presentation have been evaluated by multivariate analysis, the significant positive predictors of progression to ESRD have consistently been the serum creatinine (>1.3 mg/dl), amount of proteinuria and the presence of interstitial fibrosis (> or =20%). The one factor which is a significant negative predictor of progression to ESRD is the achievement of a remission in proteinuria. Unfortunately, spontaneous remissions are rare in FSGS, occurring in < or =6% of patients only.

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

	- a. Nephrotic range proteinuria or massive proteinuria
	- b. Elevated serum creatinine
	- c. Black race
	- a. Collapsing variant
	- b. Tubulointerstitial fibrosis
	- a. Failure to achieve partial or complete remission.

Table 3. Risk factors for progressive loss of renal function in FSGS

Thomas DB et al. (2006) described that the morphological subtype identified on renal biopsy also provided useful prognostic information. The collapsing variant, the main variant seen in HIV-induced FSGS, is associated with a worse prognosis than the other forms. The tip variant has a better prognosis than the other forms.

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

### **9. Summary**

Focal segmental glomerulosclerosis is still largely an idiopathic disease but in the recent past, more genetic mutations and secondary causes have been described. As more and more pathogenetic mechanisms involved in idiopathic FSGS are coming into light and more secondary causes of FSGS are described, the occurrence of true idiopathic FSGS diagnosis is

Focal Segmental Glomerulosclerosis 41

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decreasing and thus the frequency of the latter is often overstated. Other than the genetic and secondary causes of FSGS, the treatment strategies are still not based upon multiple large randomized controlled trials, and in fact are based upon predominantly anecdotal experiences. Still there are more questions than the answers for the pathogenesis, classification, treatment and prognosis of this an almost a century old disease. This clincopathologic entity still remains a challenge for the nephrologists and transplant physicians.

### **10. Acknowledgements**

We acknowledge and thank Irfan Warraich, MD (Dept. of pathology, Texas Tech University Health Science Center Lubbock, Texas) for his generous contribution by providing us with histopathology figures used in this chapter.

### **11. References**


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Rodriguez-Iturbe B, Johnson RJ, Herrera-Acosta J (2005) Tubulointerstitial damage and

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adults: presentation, course, and response to treatment. Am J Kidney Dis 1995;

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segmental glomerulosclerosis: report of four cases and review of the literature.

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CD2-associated protein haploinsufficiency is linked to glomerular disease

Morita T, Nissinen M, Herva R,

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Neth J Med. 2008;66:408-415.

25:534.

glomerulopathies. Annu Rev Pathol Mech Dis 1:349–374


**3** 

*1United Kingdom* 

*2United States of America* 

**Membranoproliferative Glomerulonephritis** 

*1Centre for Complement & Inflammation Research (CCIR), Imperial College, London,* 

Membranoproliferative glomerulonephritis (MPGN) refers to glomerular pathology in which there is thickening of the capillary wall together with mesangial expansion. In this article we firstly review the pathological features of MPGN and discuss how advances in our understanding of the association between abnormalities in the regulation of complement and MPGN have revealed limitations in the historical pathological sub-division of MPGN. Secondly we review the clinical presentation of MPGN, its prognosis and

The name 'membranoproliferative glomerulonephritis' derives from the light microscopic glomerular histologic pattern. MPGN is synonymous with 'mesangiocapillary glomerulonephritis'. The glomeruli are large and hypercellular. The hypercellularity is typically uniform though-out the glomeruli. Mesangial hypercellularity and expansion of the mesangial matrix can accentuate the appearance of discrete lobules within the glomeruli. In some cases the hypercellularity includes infiltration of the glomerulus with neutrophils [1] and in severe cases monocytes have been detected in the glomerulus [2]. The degree of leukocyte infiltration shows some correlation with the degree of C3d deposition, possibly due to the chemotactic effects of complement split products. In patients who have undergone two biopsies, for example, when the abundance of C3d decreased in the second biopsy fewer leukocytes were observed [3]. The basement membranes of glomerular capillaries in MPGN are thickened. A characteristic change in the capillary wall is splitting of the glomerular basement membrane (GBM), termed 'tram-tracking' or 'double-contours'. This is due to the inter-position of the proliferated mesangial cells between the endothelial cells and the GBM. The inter-positioned mesangial cells generate new basement membrane material between the endothelial and mesangial cells, a process that is readily identifiable on

Immunofluorescence studies of glomerular immunoglobulin and C3 in MPGN typically demonstrated granular deposition of these immune factors along the capillary loops. Staining for IgG is often fainter than it is for complement C3 and is sometimes absent [4 5].

**1. Introduction** 

therapeutic considerations.

electron microscopy.

**2. Pathological features of MPGN** 

Matthew C. Pickering1 and Joshua M. Thurman2

*University of Colorado Denver School of Medicine, Colorado* 

*2Division of Nephrology and Hypertension,* 

Yao J, Le TC, Kos CH, Henderson JM, Allen PG, Denker BM, Pollak MR (2004) Alphaactinin-4-mediated FSGS: an inherited kidney disease caused by an aggregated and rapidly degraded cytoskeletal protein. PLoS Biol 2(6):787–794

## **Membranoproliferative Glomerulonephritis**

Matthew C. Pickering1 and Joshua M. Thurman2

*1Centre for Complement & Inflammation Research (CCIR), Imperial College, London, 2Division of Nephrology and Hypertension, University of Colorado Denver School of Medicine, Colorado 1United Kingdom 2United States of America* 

### **1. Introduction**

44 An Update on Glomerulopathies – Clinical and Treatment Aspects

Yao J, Le TC, Kos CH, Henderson JM, Allen PG, Denker BM, Pollak MR (2004) Alpha-

rapidly degraded cytoskeletal protein. PLoS Biol 2(6):787–794

actinin-4-mediated FSGS: an inherited kidney disease caused by an aggregated and

Membranoproliferative glomerulonephritis (MPGN) refers to glomerular pathology in which there is thickening of the capillary wall together with mesangial expansion. In this article we firstly review the pathological features of MPGN and discuss how advances in our understanding of the association between abnormalities in the regulation of complement and MPGN have revealed limitations in the historical pathological sub-division of MPGN. Secondly we review the clinical presentation of MPGN, its prognosis and therapeutic considerations.

### **2. Pathological features of MPGN**

The name 'membranoproliferative glomerulonephritis' derives from the light microscopic glomerular histologic pattern. MPGN is synonymous with 'mesangiocapillary glomerulonephritis'. The glomeruli are large and hypercellular. The hypercellularity is typically uniform though-out the glomeruli. Mesangial hypercellularity and expansion of the mesangial matrix can accentuate the appearance of discrete lobules within the glomeruli. In some cases the hypercellularity includes infiltration of the glomerulus with neutrophils [1] and in severe cases monocytes have been detected in the glomerulus [2]. The degree of leukocyte infiltration shows some correlation with the degree of C3d deposition, possibly due to the chemotactic effects of complement split products. In patients who have undergone two biopsies, for example, when the abundance of C3d decreased in the second biopsy fewer leukocytes were observed [3]. The basement membranes of glomerular capillaries in MPGN are thickened. A characteristic change in the capillary wall is splitting of the glomerular basement membrane (GBM), termed 'tram-tracking' or 'double-contours'. This is due to the inter-position of the proliferated mesangial cells between the endothelial cells and the GBM. The inter-positioned mesangial cells generate new basement membrane material between the endothelial and mesangial cells, a process that is readily identifiable on electron microscopy.

Immunofluorescence studies of glomerular immunoglobulin and C3 in MPGN typically demonstrated granular deposition of these immune factors along the capillary loops. Staining for IgG is often fainter than it is for complement C3 and is sometimes absent [4 5].

Membranoproliferative Glomerulonephritis 47

include mesangial proliferative and crescentic lesions [10]. In fact more than half of the dense deposit disease biopsies did not show MPGN. The emerging consensus is that dense deposit disease is a distinct pathologic entity and should not be thought of as an MPGN variant [10 14]. Secondly, we now know that defects in complement regulation are strongly associated with glomerular inflammation in which there is isolated glomerular C3 deposition irrespective of whether the glomerular lesion is MPGN. We discuss the intimate

**type II** 

MEMBRANOPROLIFERATIVE GLOMERULONEPHRITIS

**type III** 

complement C3 variable IgG

sub-endothelial & sub-epithelial

complement C3

intra-membranous

association between complement and MPGN next.

Immunofluorescence:

GBM deposits:

**3. Complement and MPGN** 

**type I** 

complement C3 and IgG

sub-endothelial

complement C3 and IgG. GBM – glomerular basement membrane.

Fig. 1. The traditional classification of MPGN. Dense deposit disease was renamed MPGN

The complement system is an integral component of immunity. Its principal role is concerned with host defence against pathogens and it forms an important component of innate immunity. Complement also acts as natural adjuvant enhancing the B cell response to antigen and more recent data implicates an important role for complement in T cell responses [15]. To understand the relationship between MPGN and complement it is important to understand how complement is activated. Binding of antibody to antigen forms an immune complex and immune complexes are the triggers of the complement classical pathway. In immune-complex associated MPGN the classical pathway is activated and contributes to glomerular inflammation. The complement alternative pathway is continuously activated i.e. requires no specific activating trigger. Unlike the classical pathway, the alternative pathway is antibody-independent and, through this pathway, the key effector molecules of the pathway (C3 and C5) can be deposited on surfaces in the absence of immunoglobulin. The key negative regulator of the alternative pathway is an abundant plasma protein called complement factor H (CFH). Early investigators studying MPGN hypothesized that MPGN lesions in which complement components such as C3 were present in the absence of immunoglobulin, were mediated by alternative pathway activation. We now know that both inherited and acquired causes of alternative pathway regulation are associated with this type of MPGN. Dense deposit disease, in which

type II in this classification. Immunofluorescence refers to glomerular staining for

An early study described three immunofluorescence patterns in MPGN: glomerular deposition of both immunoglobulin and C3 (66%), predominant deposition of C3 (21%) and deposition of C3 only (13%) [4]. The finding of immunoglobulin and complement is characteristic of immune complex-mediated glomerular inflammation and suggests that the MPGN is secondary to systemic disorders in which there is a propensity for immunecomplexes to deposit or form within the kidney. The known conditions include diseases such as autoimmune disorders (e.g. systemic lupus erythematosus), malignancies and chronic infections. Hepatitis C, for example, is now recognized as a major cause of mixed cryoglobulinemia and MPGN [6]. Where a systemic disorder is identifiable the MPGN is referred to as secondary MPGN. In the absence of a clear aetiology MPGN is appropriately termed 'idiopathic or primary MPGN'.

The finding of glomerular complement deposition alone suggests activation of the complement system in the absence of immunoglobulin. This most commonly is a consequence of activation of the complement alternative pathway. Perhaps not surprisingly we now know that inherited and acquired disorders of the alternative pathway are associated with MPGN in which the histological features are characterized by predominant or isolated glomerular C3 deposition. The prototypic example of this is dense deposit disease. In the 1960s it was recognized that ribbon-like electron dense deposits are detectable within the lamina densa of the glomerular basement membrane in some patients with glomerulonephritis [7]. These intra-membranous deposits are the histological defining feature of dense deposit disease and this, rather than mesangial inter-positioning, produces thickening of the GBM. Dense deposit disease is rare: of children whose biopsies demonstrate an MPGN pattern by light microscopy, less than 20% have dense deposit disease [8 9]. Prominent C3 deposits are virtually always present in the glomeruli of patients with dense deposit disease. Granular C3 deposits are almost always present within the mesangium, although different patterns have also been observed [8], e.g. "ring-like" pattern of mesangial C3 staining. Glomerular C1q and/or immunoglobulins may be seen [10]. Our current understanding of dense deposit disease has recently been reviewed [11].

Since the MPGN in dense deposit disease was associated with distinct immunofluorescence studies (predominant or isolated glomerular C3 deposition) and GBM ultrastructural appearances (striking linear electron dense transformation of the lamina densa) an MPGN classification emerged which sub-divided MPGN initially into two groups: MPGN type I and MPGN type II. MPGN type I was characterized by immunoglobulin and C3 deposition and sub-endothelial electron dense GBM deposits [5]. MPGN type I contained both primary and secondary types. MPGN type II was used to describe dense deposit disease. Hence in the literature dense deposit disease was renamed MPGN type II. A further group, MPGN type III was subsequently added to describe MPGN where there were prominent subepithelial GBM deposits, possibly caused by immune-complexes similar to those found in membranous disease [12]. As in membranous disease, the deposits are associated with spikes along the GBM that can be detected by silver stain. A further MPGN type III variant was characterized by the presence of basement membrane ruptures on electron microscopy [13]. C3 deposition is invariably present in the glomeruli of patients with MPGN type III whilst immunoglobulin deposition is variable [13]. The traditional classification of MPGN is depicted in figure 1.

Subsequent studies have revealed limitations in this MPGN classification. Firstly, it is now recognized that patients with the dense deposit disease may present with many different patterns of glomerular injury by light microscopy. These patterns, in addition to MPGN,

An early study described three immunofluorescence patterns in MPGN: glomerular deposition of both immunoglobulin and C3 (66%), predominant deposition of C3 (21%) and deposition of C3 only (13%) [4]. The finding of immunoglobulin and complement is characteristic of immune complex-mediated glomerular inflammation and suggests that the MPGN is secondary to systemic disorders in which there is a propensity for immunecomplexes to deposit or form within the kidney. The known conditions include diseases such as autoimmune disorders (e.g. systemic lupus erythematosus), malignancies and chronic infections. Hepatitis C, for example, is now recognized as a major cause of mixed cryoglobulinemia and MPGN [6]. Where a systemic disorder is identifiable the MPGN is referred to as secondary MPGN. In the absence of a clear aetiology MPGN is appropriately

The finding of glomerular complement deposition alone suggests activation of the complement system in the absence of immunoglobulin. This most commonly is a consequence of activation of the complement alternative pathway. Perhaps not surprisingly we now know that inherited and acquired disorders of the alternative pathway are associated with MPGN in which the histological features are characterized by predominant or isolated glomerular C3 deposition. The prototypic example of this is dense deposit disease. In the 1960s it was recognized that ribbon-like electron dense deposits are detectable within the lamina densa of the glomerular basement membrane in some patients with glomerulonephritis [7]. These intra-membranous deposits are the histological defining feature of dense deposit disease and this, rather than mesangial inter-positioning, produces thickening of the GBM. Dense deposit disease is rare: of children whose biopsies demonstrate an MPGN pattern by light microscopy, less than 20% have dense deposit disease [8 9]. Prominent C3 deposits are virtually always present in the glomeruli of patients with dense deposit disease. Granular C3 deposits are almost always present within the mesangium, although different patterns have also been observed [8], e.g. "ring-like" pattern of mesangial C3 staining. Glomerular C1q and/or immunoglobulins may be seen [10]. Our

current understanding of dense deposit disease has recently been reviewed [11].

Since the MPGN in dense deposit disease was associated with distinct immunofluorescence studies (predominant or isolated glomerular C3 deposition) and GBM ultrastructural appearances (striking linear electron dense transformation of the lamina densa) an MPGN classification emerged which sub-divided MPGN initially into two groups: MPGN type I and MPGN type II. MPGN type I was characterized by immunoglobulin and C3 deposition and sub-endothelial electron dense GBM deposits [5]. MPGN type I contained both primary and secondary types. MPGN type II was used to describe dense deposit disease. Hence in the literature dense deposit disease was renamed MPGN type II. A further group, MPGN type III was subsequently added to describe MPGN where there were prominent subepithelial GBM deposits, possibly caused by immune-complexes similar to those found in membranous disease [12]. As in membranous disease, the deposits are associated with spikes along the GBM that can be detected by silver stain. A further MPGN type III variant was characterized by the presence of basement membrane ruptures on electron microscopy [13]. C3 deposition is invariably present in the glomeruli of patients with MPGN type III whilst immunoglobulin deposition is variable [13]. The traditional classification of MPGN is

Subsequent studies have revealed limitations in this MPGN classification. Firstly, it is now recognized that patients with the dense deposit disease may present with many different patterns of glomerular injury by light microscopy. These patterns, in addition to MPGN,

termed 'idiopathic or primary MPGN'.

depicted in figure 1.

include mesangial proliferative and crescentic lesions [10]. In fact more than half of the dense deposit disease biopsies did not show MPGN. The emerging consensus is that dense deposit disease is a distinct pathologic entity and should not be thought of as an MPGN variant [10 14]. Secondly, we now know that defects in complement regulation are strongly associated with glomerular inflammation in which there is isolated glomerular C3 deposition irrespective of whether the glomerular lesion is MPGN. We discuss the intimate association between complement and MPGN next.


Fig. 1. The traditional classification of MPGN. Dense deposit disease was renamed MPGN type II in this classification. Immunofluorescence refers to glomerular staining for complement C3 and IgG. GBM – glomerular basement membrane.

### **3. Complement and MPGN**

The complement system is an integral component of immunity. Its principal role is concerned with host defence against pathogens and it forms an important component of innate immunity. Complement also acts as natural adjuvant enhancing the B cell response to antigen and more recent data implicates an important role for complement in T cell responses [15]. To understand the relationship between MPGN and complement it is important to understand how complement is activated. Binding of antibody to antigen forms an immune complex and immune complexes are the triggers of the complement classical pathway. In immune-complex associated MPGN the classical pathway is activated and contributes to glomerular inflammation. The complement alternative pathway is continuously activated i.e. requires no specific activating trigger. Unlike the classical pathway, the alternative pathway is antibody-independent and, through this pathway, the key effector molecules of the pathway (C3 and C5) can be deposited on surfaces in the absence of immunoglobulin. The key negative regulator of the alternative pathway is an abundant plasma protein called complement factor H (CFH). Early investigators studying MPGN hypothesized that MPGN lesions in which complement components such as C3 were present in the absence of immunoglobulin, were mediated by alternative pathway activation. We now know that both inherited and acquired causes of alternative pathway regulation are associated with this type of MPGN. Dense deposit disease, in which

Membranoproliferative Glomerulonephritis 49

INCLUSION CRITERIA:

EXAMPLES:

glomerulonephritis and CFHR5 nephropathy are discussed separately.

Fig. 2. Definition and examples of C3 glomerulopathy

**5. Clinical features of MPGN** 

**MPGN type I** 

some of the patients.

Glomerular deposition of complement C3 with scanty or no immunoglobulin

C3 glomerulonephritis

Dense deposit disease

CFHR5 nephropathy

In summary the traditional MPGN classification has become outdated due to advances in our understanding of complement-mediated glomerular inflammation and our knowledge of the histopathological spectrum of dense deposit disease. Nevertheless, in reviewing historical studies the use of the traditional MPGN sub-groups is unavoidable. In the next sections we have used the traditional sub-groups acknowledging that the reader will now be aware of the limitations of this classification. The recent descriptions of C3

MPGN commonly presents with the nephritic syndrome (microscopic hematuria, nonnephrotic proteinuria, and renal insufficiency). However, up to one third of the patients present with relatively preserved renal function and the nephrotic syndrome [27]. The spectrum of clinical findings is generally the same for all of the subgroups considered in this review, but some differences exist and are discussed under individual headings below.

MPGN type I may be more common in children than in adults [28]. It typically presents as a renal limited disease, although many patients have hypertension at the time of diagnosis [29]. Hypertension may be less common in children [29], particularly when the disease is detected early through screening of asymptomatic individuals. The majority of patients have microscopic hematuria, and some have macroscopic haematuria [29]. Cameron reported that 75% of their patients with MPGN type I had normal C3 levels at the time of disease onset [29]. In an analysis of 9 patients with MPGN type I, however, Ooi and colleagues reported depressed C3 levels for all of the patients on at least one occasion [30]. Approximately 30% of patients have a C3 nephritic factor [29 30]. C4 levels were low for

C3 glomerulopathy

glomerular C3 deposition is typically seen with little or no immunoglobulin, is associated with genetic and acquired factors that enhance alternative pathway activation (reviewed in [11]). C3 nephritic factor was associated with dense deposit disease decades ago [14] and the 'factor' is now known to be an immunoglobulin which targets an enzyme complex within the alternative pathway. This autoantibody stabilizes the enzyme complex and enhances alternative pathway activation. Hence C3 nephritic factor is associated with over-activation of the alternative pathway. Consequently, through consumption, plasma C3 levels are typically low in individuals with C3 nephritic factor. Genetic factors include genetic deficiency of the alternative pathway regulatory protein, CFH (reviewed in [16]) and 'gain of function' mutations in the alternative pathway activation protein, complement C3 [17]. Genetic deficiency of CFH in pigs and gene-targeted CFH-deficient mice also results in spontaneous MPGN [18 19]. Acquired dysregulation of the alternative pathway due to neutralizing autoantibodies against CFH [20] has also been described. Recently, an autoantibody to factor B, an activation protein within the alternative pathway, has also been associated with MPGN [21]. In summary factors that increase alternative pathway activation have been associated with MPGN in which there is glomerular C3 with little or no immunoglobulin. It is clearly important to distinguish MPGN driven by these factors from MPGN associated with systemic immune complex disease or MPGN due to other aetiologies. Consequently, there has been much discussion on how to develop our current classification of MPGN.

#### **4. C3 glomerulopathy – moving away from the traditional classification of MPGN**

In order to identify individuals with inherited or acquired defects in complement regulation we proposed a classification called C3 glomerulopathy (Figure 2) [22]. C3 glomerulopathy defines glomerular pathology characterized by isolated or predominant glomerular C3 deposition in the absence of immunoglobulin irrespective of both the glomerular light microscopic appearances and the ultrastructural appearance of the GBM [22]. Whilst many patients with isolated glomerular C3 deposition and complement abnormalities develop MPGN some do not [23 24]. For example, Servais and colleagues described 19 cases of primary glomerulonephritis cases with isolated deposition of C3 in the absence of morphological GBM changes of dense deposit disease [24]. Thirteen cases had an MPGN pattern by light microscopy whilst the remaining 6 did not [24]. They used the term 'C3 glomerulonephritis'. Recently a familial C3 glomerulopathy associated with a mutation in a complement protein called complement factor H-related protein 5 (CFHR5) was characterised [23]. Affected individuals have renal biopsies consistent with C3 glomerulonephritis. Biopsies show mesangial C3 deposition and variable degrees of mesangial hypercellularity. Mesangial and sub-endothelial GBM electron dense deposits are typical and some develop MPGN. These patients were identified and specifically investigated for complement disorders because renal biopsies demonstrated glomerular C3 deposition in the absence of immunoglobulin. The discovery of CFHR5 nephropathy is fascinating and we direct the interested reader to [25]. The impetus to propose the term C3 glomerulopathy was to enable the rapid identification of patients with glomerular disease who ought to be investigated for complement abnormalities and who may benefit from complement modulating therapeutic strategies. The relationship between C3 glomerulopathy and MPGN is discussed in detail in reference [26].

glomerular C3 deposition is typically seen with little or no immunoglobulin, is associated with genetic and acquired factors that enhance alternative pathway activation (reviewed in [11]). C3 nephritic factor was associated with dense deposit disease decades ago [14] and the 'factor' is now known to be an immunoglobulin which targets an enzyme complex within the alternative pathway. This autoantibody stabilizes the enzyme complex and enhances alternative pathway activation. Hence C3 nephritic factor is associated with over-activation of the alternative pathway. Consequently, through consumption, plasma C3 levels are typically low in individuals with C3 nephritic factor. Genetic factors include genetic deficiency of the alternative pathway regulatory protein, CFH (reviewed in [16]) and 'gain of function' mutations in the alternative pathway activation protein, complement C3 [17]. Genetic deficiency of CFH in pigs and gene-targeted CFH-deficient mice also results in spontaneous MPGN [18 19]. Acquired dysregulation of the alternative pathway due to neutralizing autoantibodies against CFH [20] has also been described. Recently, an autoantibody to factor B, an activation protein within the alternative pathway, has also been associated with MPGN [21]. In summary factors that increase alternative pathway activation have been associated with MPGN in which there is glomerular C3 with little or no immunoglobulin. It is clearly important to distinguish MPGN driven by these factors from MPGN associated with systemic immune complex disease or MPGN due to other aetiologies. Consequently, there has been much discussion on how to develop our current

**4. C3 glomerulopathy – moving away from the traditional classification of** 

glomerulopathy and MPGN is discussed in detail in reference [26].

In order to identify individuals with inherited or acquired defects in complement regulation we proposed a classification called C3 glomerulopathy (Figure 2) [22]. C3 glomerulopathy defines glomerular pathology characterized by isolated or predominant glomerular C3 deposition in the absence of immunoglobulin irrespective of both the glomerular light microscopic appearances and the ultrastructural appearance of the GBM [22]. Whilst many patients with isolated glomerular C3 deposition and complement abnormalities develop MPGN some do not [23 24]. For example, Servais and colleagues described 19 cases of primary glomerulonephritis cases with isolated deposition of C3 in the absence of morphological GBM changes of dense deposit disease [24]. Thirteen cases had an MPGN pattern by light microscopy whilst the remaining 6 did not [24]. They used the term 'C3 glomerulonephritis'. Recently a familial C3 glomerulopathy associated with a mutation in a complement protein called complement factor H-related protein 5 (CFHR5) was characterised [23]. Affected individuals have renal biopsies consistent with C3 glomerulonephritis. Biopsies show mesangial C3 deposition and variable degrees of mesangial hypercellularity. Mesangial and sub-endothelial GBM electron dense deposits are typical and some develop MPGN. These patients were identified and specifically investigated for complement disorders because renal biopsies demonstrated glomerular C3 deposition in the absence of immunoglobulin. The discovery of CFHR5 nephropathy is fascinating and we direct the interested reader to [25]. The impetus to propose the term C3 glomerulopathy was to enable the rapid identification of patients with glomerular disease who ought to be investigated for complement abnormalities and who may benefit from complement modulating therapeutic strategies. The relationship between C3

classification of MPGN.

**MPGN** 

Fig. 2. Definition and examples of C3 glomerulopathy

In summary the traditional MPGN classification has become outdated due to advances in our understanding of complement-mediated glomerular inflammation and our knowledge of the histopathological spectrum of dense deposit disease. Nevertheless, in reviewing historical studies the use of the traditional MPGN sub-groups is unavoidable. In the next sections we have used the traditional sub-groups acknowledging that the reader will now be aware of the limitations of this classification. The recent descriptions of C3 glomerulonephritis and CFHR5 nephropathy are discussed separately.

### **5. Clinical features of MPGN**

MPGN commonly presents with the nephritic syndrome (microscopic hematuria, nonnephrotic proteinuria, and renal insufficiency). However, up to one third of the patients present with relatively preserved renal function and the nephrotic syndrome [27]. The spectrum of clinical findings is generally the same for all of the subgroups considered in this review, but some differences exist and are discussed under individual headings below.

#### **MPGN type I**

MPGN type I may be more common in children than in adults [28]. It typically presents as a renal limited disease, although many patients have hypertension at the time of diagnosis [29]. Hypertension may be less common in children [29], particularly when the disease is detected early through screening of asymptomatic individuals. The majority of patients have microscopic hematuria, and some have macroscopic haematuria [29]. Cameron reported that 75% of their patients with MPGN type I had normal C3 levels at the time of disease onset [29]. In an analysis of 9 patients with MPGN type I, however, Ooi and colleagues reported depressed C3 levels for all of the patients on at least one occasion [30]. Approximately 30% of patients have a C3 nephritic factor [29 30]. C4 levels were low for some of the patients.

Membranoproliferative Glomerulonephritis 51

internal duplication in exons 2 and 3 of the *CFHR5* gene. This results in a secreted abnormally large CFHR5 protein. The clinical course of CFHR5 nephropathy has been described in a comprehensive review of 91 patients from 16 pedigrees [39]. Affected patients have continuous microscopic haematuria and often develop macroscopic haematuria during periods of infection. Hypertension, proteinuria and end-stage renal failure are more common in men. In this report of affected individuals aged over 50 years, 80% of affected men developed chronic renal failure whilst 21% of affected women developed chronic renal

As highlighted in the above discussion, many of the patients who were previously identified simply as having MPGN are now recognized as having distinct disease processes. Consequently, older data regarding the prognosis of MPGN may have combined patients who would now be categorized differently. For example, early studies of MPGN may well have included patients with dense deposit disease due to defective complement regulation or patients with secondary MPGN caused by hepatitis C associated cryoglobulinemia.

Studies of patients with MPGN type I have reported fairly wide variation in the long-term prognosis of the disease. The 10-year renal survival for children has been reported to be 60-80% [41 42]. The patients included in these studies were treated with corticosteroids and other immunosuppression. The prognosis may be improved by early detection, such as that afforded by screening of school children [43]. This improvement could be due to lead-time bias, however. Adverse prognostic features include nephrotic syndrome [32 44], an elevated creatinine at presentation or within the first year, and structural injury on the renal

The prognosis of dense deposit disease may be worse than that for MPGN type I [8 9 29], although the small numbers of patients with each disease make it difficult to control for other variables. Spontaneous remissions of dense deposit disease are rare, and approximately 50% of patients will reach end-stage renal disease within 10 years [29]. Of the 27 patients in the report by Nasr *et al.* for whom follow-up was known, 25.9% had a complete response to therapy. There was no response in the remaining patients and 25.9% progressed to end-stage renal disease (duration of follow-up 2 months to 24 years) [31]. Age and the serum creatinine at biopsy were predictive of progression to end-stage renal disease. Only 7.1% of the adults had a complete response to therapy. Although uncontrolled complement activation is believed to be pathogenic in this disease, perturbations in C3 levels do not appear to correlate with clinical outcomes [29 31]. Recurrence in renal

During the period of follow-up (ranging from 0.4 – 34 years) in the series reported by Servais *et al.* [24], most of the patients had a decline in renal function. Three patients reached end stage renal disease, and another two patients had creatinine clearances below 15 mL/min. The patients described by Sethi et al. did not show a decline in renal function

during the short period of follow-up (6 months to 3 years) [36].

failure [39]. The condition recurs in the transplanted kidney [40].

**6. Prognostic considerations in MPGN** 

**MPGN type II/Dense deposit disease** 

allografts is common [31 45]. **C3 glomerulonephritis** 

**MPGN type I** 

biopsy [44].

### **MPGN type II/Dense deposit disease**

Dense deposit disease usually presents in children between the ages of 5 and 15 [14], although a recent series included more patients diagnosed in adulthood than in childhood (12). There appears to be a slight female to male preponderance [31]. The clinical presentation is similar to that of MPGN type I. MPGN type II usually presents with the nephritic syndrome [28], but proteinuria is usually present and is in the nephrotic range in approximately 50% of patients [31]. Interestingly, the renal disease is often preceded by an infection [31]. More than 80% of patients with dense deposit disease have hypocomplementemia at some point [29], and 100% of children with the disease had depressed C3 levels in the report of Nasr et al. [31]. Approximately 80% of patients with dense deposit disease have detectable C3 nephritic factor [32]. As described above, dense deposit disease is strongly associated with defects in regulation of the alternative pathway of complement. This underlying defect probably explains the association of dense deposit disease with acquired partial lipodystrophy [33] and with retinal drusen (deposits within Bruch's membrane of the retina) [34]. In acquired partial lipodystrophy, uncontrolled complement activation causes the loss of subcutaneous fat. The drusen resemble those seen in patients with age related macular degeneration and presumably also form as a result of defective complement regulation. However, only one patient with each of these co-morbidities was described in a recent report of 32 patients with dense deposit disease [31].

### **MPGN type III**

MPGN type III usually presents with similar clinical findings to those seen in MPGN type I. The proportion of patients with hypocomplementaemia was comparable to that of MPGN type I [35].

### **C3 glomerulonephritis**

C3 glomerulonephritis is a recently described entity so the full spectrum of its clinical manifestations is not yet known. Servais *et al.* reported a series of 19 patients [24]. Men and women were represented nearly equally, and the patients ranged in age from 7 years old to 70. Sethi *et al.* recently reported three more cases of C3 glomerulopathy [36], all of whom were adult males (aged 38-73). Approximately 30% of the patients in the two reports had a creatinine clearance below 60 ml/min. Most of the patients had proteinuria (eight of the 22 patients described had nephrotic range proteinuria) and 15 patients had hematuria [24 36]. A comprehensive evaluation of the complement system was performed in both reports. Nine of the 22 total patients had depressed C3 levels. Six patients in the first series had C3 nephritic factor, and one of the patients reported by Sethi *et al.* had C3 nephritic factor. Mutations in complement regulatory proteins were identified in six of the patients reported by Servais *et al.* [24]. The three patients reported by *Sethi et al.* all carried the Tyr402His allele of CFH which has been identified as a risk allele for dense deposit disease [36].

#### **CFHR5 nephropathy**

CFHR5 nephropathy is a familial form of C3 glomerulopathy that has to date only been described among individuals with Cypriot ancestry. It was characterized only recently [23 25]. Affected individuals all carry a mutation in CFHR5 and to date only heterozygous affected individuals have been identified. The biological role of CFHR5 is unknown although there is evidence that it interacts with complement deposited within the glomeruli in many different glomerular pathologies [37 38]. The mutation in CFHR5 nephropathy is an internal duplication in exons 2 and 3 of the *CFHR5* gene. This results in a secreted abnormally large CFHR5 protein. The clinical course of CFHR5 nephropathy has been described in a comprehensive review of 91 patients from 16 pedigrees [39]. Affected patients have continuous microscopic haematuria and often develop macroscopic haematuria during periods of infection. Hypertension, proteinuria and end-stage renal failure are more common in men. In this report of affected individuals aged over 50 years, 80% of affected men developed chronic renal failure whilst 21% of affected women developed chronic renal failure [39]. The condition recurs in the transplanted kidney [40].

### **6. Prognostic considerations in MPGN**

As highlighted in the above discussion, many of the patients who were previously identified simply as having MPGN are now recognized as having distinct disease processes. Consequently, older data regarding the prognosis of MPGN may have combined patients who would now be categorized differently. For example, early studies of MPGN may well have included patients with dense deposit disease due to defective complement regulation or patients with secondary MPGN caused by hepatitis C associated cryoglobulinemia.

#### **MPGN type I**

50 An Update on Glomerulopathies – Clinical and Treatment Aspects

Dense deposit disease usually presents in children between the ages of 5 and 15 [14], although a recent series included more patients diagnosed in adulthood than in childhood (12). There appears to be a slight female to male preponderance [31]. The clinical presentation is similar to that of MPGN type I. MPGN type II usually presents with the nephritic syndrome [28], but proteinuria is usually present and is in the nephrotic range in approximately 50% of patients [31]. Interestingly, the renal disease is often preceded by an infection [31]. More than 80% of patients with dense deposit disease have hypocomplementemia at some point [29], and 100% of children with the disease had depressed C3 levels in the report of Nasr et al. [31]. Approximately 80% of patients with dense deposit disease have detectable C3 nephritic factor [32]. As described above, dense deposit disease is strongly associated with defects in regulation of the alternative pathway of complement. This underlying defect probably explains the association of dense deposit disease with acquired partial lipodystrophy [33] and with retinal drusen (deposits within Bruch's membrane of the retina) [34]. In acquired partial lipodystrophy, uncontrolled complement activation causes the loss of subcutaneous fat. The drusen resemble those seen in patients with age related macular degeneration and presumably also form as a result of defective complement regulation. However, only one patient with each of these co-morbidities was described in a recent report of 32 patients with dense deposit disease

MPGN type III usually presents with similar clinical findings to those seen in MPGN type I. The proportion of patients with hypocomplementaemia was comparable to that of MPGN

C3 glomerulonephritis is a recently described entity so the full spectrum of its clinical manifestations is not yet known. Servais *et al.* reported a series of 19 patients [24]. Men and women were represented nearly equally, and the patients ranged in age from 7 years old to 70. Sethi *et al.* recently reported three more cases of C3 glomerulopathy [36], all of whom were adult males (aged 38-73). Approximately 30% of the patients in the two reports had a creatinine clearance below 60 ml/min. Most of the patients had proteinuria (eight of the 22 patients described had nephrotic range proteinuria) and 15 patients had hematuria [24 36]. A comprehensive evaluation of the complement system was performed in both reports. Nine of the 22 total patients had depressed C3 levels. Six patients in the first series had C3 nephritic factor, and one of the patients reported by Sethi *et al.* had C3 nephritic factor. Mutations in complement regulatory proteins were identified in six of the patients reported by Servais *et al.* [24]. The three patients reported by *Sethi et al.* all carried the Tyr402His allele

CFHR5 nephropathy is a familial form of C3 glomerulopathy that has to date only been described among individuals with Cypriot ancestry. It was characterized only recently [23 25]. Affected individuals all carry a mutation in CFHR5 and to date only heterozygous affected individuals have been identified. The biological role of CFHR5 is unknown although there is evidence that it interacts with complement deposited within the glomeruli in many different glomerular pathologies [37 38]. The mutation in CFHR5 nephropathy is an

of CFH which has been identified as a risk allele for dense deposit disease [36].

**MPGN type II/Dense deposit disease** 

[31].

**MPGN type III** 

**C3 glomerulonephritis**

**CFHR5 nephropathy** 

type I [35].

Studies of patients with MPGN type I have reported fairly wide variation in the long-term prognosis of the disease. The 10-year renal survival for children has been reported to be 60-80% [41 42]. The patients included in these studies were treated with corticosteroids and other immunosuppression. The prognosis may be improved by early detection, such as that afforded by screening of school children [43]. This improvement could be due to lead-time bias, however. Adverse prognostic features include nephrotic syndrome [32 44], an elevated creatinine at presentation or within the first year, and structural injury on the renal biopsy [44].

#### **MPGN type II/Dense deposit disease**

The prognosis of dense deposit disease may be worse than that for MPGN type I [8 9 29], although the small numbers of patients with each disease make it difficult to control for other variables. Spontaneous remissions of dense deposit disease are rare, and approximately 50% of patients will reach end-stage renal disease within 10 years [29]. Of the 27 patients in the report by Nasr *et al.* for whom follow-up was known, 25.9% had a complete response to therapy. There was no response in the remaining patients and 25.9% progressed to end-stage renal disease (duration of follow-up 2 months to 24 years) [31]. Age and the serum creatinine at biopsy were predictive of progression to end-stage renal disease. Only 7.1% of the adults had a complete response to therapy. Although uncontrolled complement activation is believed to be pathogenic in this disease, perturbations in C3 levels do not appear to correlate with clinical outcomes [29 31]. Recurrence in renal allografts is common [31 45].

#### **C3 glomerulonephritis**

During the period of follow-up (ranging from 0.4 – 34 years) in the series reported by Servais *et al.* [24], most of the patients had a decline in renal function. Three patients reached end stage renal disease, and another two patients had creatinine clearances below 15 mL/min. The patients described by Sethi et al. did not show a decline in renal function during the short period of follow-up (6 months to 3 years) [36].

Membranoproliferative Glomerulonephritis 53

The patients treated with these agents are probably selected because they have concerning prognostic factors. Thus, it is difficult to determine the efficacy of these agents. Case reports have also described patients with steroid-resistant MPGN type I who responded to mycophenolate mofetil [57 58]. Certainly more data is needed, but given the relative safety of

Approximately 30-60% of patients with MPGN type I who undergo renal transplantation have a recurrence of the disease, and disease recurrence adversely affects graft survival [59 60]. Although there are anecdotal reports that increasing immunosuppression may be

No clinical trials have been conducted in patients with dense deposit disease. Based upon what is known about the pathophysiology of the disease, the complement inhibitor eculizumab may be beneficial, and a clinical trial of this agent in dense deposit disease is currently underway. Eculizumab is a monoclonal antibody that blocks C5 activation and is currently licensed for treatment of anemia in paroxysmal nocturnal haemoglobinuria. It has been used successfully to treat atypical haemolytic uraemic syndrome and is likely to be

Plasma exchange may be effective at removing autoantibodies or dysfunctional complement components, while also enhancing CFH function through the infusion of plasma. Plasma exchange was reported to be effective in two affected sisters who had a factor H mutation

The role of immunosuppressive agents in dense deposit disease is uncertain. Theoretically, immunosuppressive drugs may be beneficial in patients with evidence of autoantibodies, yet corticosteroids are not of clear benefit in this disease [14]. The patients reported by Nasr et al. included 18 patients who received immunosuppression [31]. The immunosuppression regimens included steroids in all patients. Two patients were also treated with mycophenolate mofetil and three received calcineurin inhibitors. A trend towards a benefit was seen in patients treated with immunosuppression but this did not reach significance. The greatest benefit was seen in those who received immunosuppression and a reninangiotensin system inhibitor. Recently, a patient with fulminant disease was treated with high-dose corticosteroids, plasma exchange, and cyclophosphamide, and apparently responded to treatment [62]. This patient had a low C3 level, but did not have C3 nephritic factor or a complement mutation, so the mechanism by which the treatment benefited the

Based on existing data, the optimal treatment of patients with dense deposit disease is uncertain. A treatment algorithm incorporating complement testing and the above treatment options has been proposed [63]. Treatment may need to be initiated before genetic testing can be performed, however, and the presence or absence of C3 nephritic factor is probably not sufficiently accurate to guide therapy. Thus, the decision to use plasma exchange, standard immunosuppressive drugs, and/or eculizumab must ultimately be

Recurrence of MPGN type II is very common in patients who receive renal transplants, and some estimate the recurrence rate is 100% [60]. Graft survival at 5 years is approximately 50%, and the most common cause of graft loss is recurrent disease [64]. The impact of more aggressive (e.g. peri-transplant plasma exchange) or newer therapies (e.g eculizumab)

this medication it is a reasonable choice for patients who do not respond to steroids.

beneficial, there is no well established therapy for recurrent disease [60].

**MPGN type II/Dense deposit disease** 

licensed for this indication soon.

and C3 nephritic factor [61].

patient is difficult to infer.

remain unknown.

made based on the clinical severity of the disease.

#### **CFHR5 nephropathy**

As mentioned above the course of this condition is more severe in males. In affected individuals aged over 50 years the incidence of end-stage renal failure was 78% in men and 22% in women [39].

### **7. Therapeutic approaches to MPGN**

Given the distinct mechanisms of glomerular injury between immune-complex-mediated MPGN and the primary complement-mediated MPGN groups, evidence of treatment efficacy in one group may not be applicable to the other. However, for both groups nonspecific therapies may be beneficial at slowing the progression of renal disease. The blood pressure should be rigorously controlled, and ACE inhibitors or angiotensin receptor blockers are probably agents of choice [46]. Complications of the nephrotic syndrome, such as hyperlipidemia, should be treated.

#### **MPGN type I**

As this is a disease of immune-complex deposition, there is a rationale for treating this disease with immunosuppression. Unfortunately, there is not conclusive evidence that any of the common treatments are effective. Perhaps the best study to date was a randomized controlled trial of alternate day prednisone that included 41 children with MPGN type I [47]. The patients had high-grade proteinuria or renal impairment, and renal survival was better in the group that received steroids. Although this difference did not reach significance (P = 0.07), the authors concluded that this was due to the small number of patients. Other uncontrolled studies further support the finding that long-term treatment with corticosteroids may be effective at inducing disease remission [9 48 49]. One of these studies included patients with diffuse lesions on their biopsies, but who were detected early through school-based screening [49]. These patients were treated with alternate day steroids, and all of the patients but one was treated for at least four years. Of 19 patients evaluated, four patients had persistent mild proteinuria but only one patient had a disease relapse (successfully treated with a second course of steroids). Other case series have not shown improved outcomes in patients who received steroids. In one such study, however, the authors determined that patients who received steroids were more likely to have had the nephrotic syndrome [44]. They concluded, therefore, that steroids may, in fact, have been beneficial. After analyzing the available data, Levin concluded that corticosteroids are indicated for children with nephrotic syndrome or with renal insufficiency [50], but the optimal criteria by which patients should be stratified for treatment are still under debate [44].

Similarly, some studies have suggested a benefit of treatment with anti-platelet agents [51 52]. A randomized, controlled trial of aspirin and dipyridamole, for example, indicated that treatment with these agents was effective at preserving renal function [51]. However, a longterm follow-up study that examined renal survival in these patients from the time of diagnosis (not from the start of treatment) did not see a sustained benefit [53]. Another randomized trial of patients demonstrated that aspirin plus dipyridamole was effective at reducing proteinuria at 36 months [54]. The serum creatinines in both groups were unchanged, however, so the effect of this treatment on the progression of renal disease remains uncertain.

Several case series and case reports have described patients treated with other immunosuppressive agents, such as cyclophosphamide or calcineurin inhibitors [29 44 55 56].

As mentioned above the course of this condition is more severe in males. In affected individuals aged over 50 years the incidence of end-stage renal failure was 78% in men and

Given the distinct mechanisms of glomerular injury between immune-complex-mediated MPGN and the primary complement-mediated MPGN groups, evidence of treatment efficacy in one group may not be applicable to the other. However, for both groups nonspecific therapies may be beneficial at slowing the progression of renal disease. The blood pressure should be rigorously controlled, and ACE inhibitors or angiotensin receptor blockers are probably agents of choice [46]. Complications of the nephrotic syndrome, such

As this is a disease of immune-complex deposition, there is a rationale for treating this disease with immunosuppression. Unfortunately, there is not conclusive evidence that any of the common treatments are effective. Perhaps the best study to date was a randomized controlled trial of alternate day prednisone that included 41 children with MPGN type I [47]. The patients had high-grade proteinuria or renal impairment, and renal survival was better in the group that received steroids. Although this difference did not reach significance (P = 0.07), the authors concluded that this was due to the small number of patients. Other uncontrolled studies further support the finding that long-term treatment with corticosteroids may be effective at inducing disease remission [9 48 49]. One of these studies included patients with diffuse lesions on their biopsies, but who were detected early through school-based screening [49]. These patients were treated with alternate day steroids, and all of the patients but one was treated for at least four years. Of 19 patients evaluated, four patients had persistent mild proteinuria but only one patient had a disease relapse (successfully treated with a second course of steroids). Other case series have not shown improved outcomes in patients who received steroids. In one such study, however, the authors determined that patients who received steroids were more likely to have had the nephrotic syndrome [44]. They concluded, therefore, that steroids may, in fact, have been beneficial. After analyzing the available data, Levin concluded that corticosteroids are indicated for children with nephrotic syndrome or with renal insufficiency [50], but the optimal criteria by which patients should be stratified for

Similarly, some studies have suggested a benefit of treatment with anti-platelet agents [51 52]. A randomized, controlled trial of aspirin and dipyridamole, for example, indicated that treatment with these agents was effective at preserving renal function [51]. However, a longterm follow-up study that examined renal survival in these patients from the time of diagnosis (not from the start of treatment) did not see a sustained benefit [53]. Another randomized trial of patients demonstrated that aspirin plus dipyridamole was effective at reducing proteinuria at 36 months [54]. The serum creatinines in both groups were unchanged, however, so the effect of this treatment on the progression of renal disease

Several case series and case reports have described patients treated with other immunosuppressive agents, such as cyclophosphamide or calcineurin inhibitors [29 44 55 56].

**CFHR5 nephropathy** 

22% in women [39].

**MPGN type I** 

**7. Therapeutic approaches to MPGN** 

as hyperlipidemia, should be treated.

treatment are still under debate [44].

remains uncertain.

The patients treated with these agents are probably selected because they have concerning prognostic factors. Thus, it is difficult to determine the efficacy of these agents. Case reports have also described patients with steroid-resistant MPGN type I who responded to mycophenolate mofetil [57 58]. Certainly more data is needed, but given the relative safety of this medication it is a reasonable choice for patients who do not respond to steroids.

Approximately 30-60% of patients with MPGN type I who undergo renal transplantation have a recurrence of the disease, and disease recurrence adversely affects graft survival [59 60]. Although there are anecdotal reports that increasing immunosuppression may be beneficial, there is no well established therapy for recurrent disease [60].

#### **MPGN type II/Dense deposit disease**

No clinical trials have been conducted in patients with dense deposit disease. Based upon what is known about the pathophysiology of the disease, the complement inhibitor eculizumab may be beneficial, and a clinical trial of this agent in dense deposit disease is currently underway. Eculizumab is a monoclonal antibody that blocks C5 activation and is currently licensed for treatment of anemia in paroxysmal nocturnal haemoglobinuria. It has been used successfully to treat atypical haemolytic uraemic syndrome and is likely to be licensed for this indication soon.

Plasma exchange may be effective at removing autoantibodies or dysfunctional complement components, while also enhancing CFH function through the infusion of plasma. Plasma exchange was reported to be effective in two affected sisters who had a factor H mutation and C3 nephritic factor [61].

The role of immunosuppressive agents in dense deposit disease is uncertain. Theoretically, immunosuppressive drugs may be beneficial in patients with evidence of autoantibodies, yet corticosteroids are not of clear benefit in this disease [14]. The patients reported by Nasr et al. included 18 patients who received immunosuppression [31]. The immunosuppression regimens included steroids in all patients. Two patients were also treated with mycophenolate mofetil and three received calcineurin inhibitors. A trend towards a benefit was seen in patients treated with immunosuppression but this did not reach significance. The greatest benefit was seen in those who received immunosuppression and a reninangiotensin system inhibitor. Recently, a patient with fulminant disease was treated with high-dose corticosteroids, plasma exchange, and cyclophosphamide, and apparently responded to treatment [62]. This patient had a low C3 level, but did not have C3 nephritic factor or a complement mutation, so the mechanism by which the treatment benefited the patient is difficult to infer.

Based on existing data, the optimal treatment of patients with dense deposit disease is uncertain. A treatment algorithm incorporating complement testing and the above treatment options has been proposed [63]. Treatment may need to be initiated before genetic testing can be performed, however, and the presence or absence of C3 nephritic factor is probably not sufficiently accurate to guide therapy. Thus, the decision to use plasma exchange, standard immunosuppressive drugs, and/or eculizumab must ultimately be made based on the clinical severity of the disease.

Recurrence of MPGN type II is very common in patients who receive renal transplants, and some estimate the recurrence rate is 100% [60]. Graft survival at 5 years is approximately 50%, and the most common cause of graft loss is recurrent disease [64]. The impact of more aggressive (e.g. peri-transplant plasma exchange) or newer therapies (e.g eculizumab) remain unknown.

Membranoproliferative Glomerulonephritis 55

[6] Alpers CE, Smith KD. Cryoglobulinemia and renal disease. Curr Opin Nephrol

[7] Berger J, Galle P. [Dense Deposits within the Basal Membranes of the Kidney. Optical

[8] Habib R, Gubler MC, Loirat C*, et al.* Dense deposit disease: a variant of membranoproliferative glomerulonephritis. Kidney Int 1975; 7:204-215. [9] Habib R, Kleinknecht C, Gubler MC*, et al.* Idiopathic membranoproliferative glomerulonephritis in children. Report of 105 cases. Clin Nephrol 1973; 1:194-214. [10] Walker PD, Ferrario F, Joh K*, et al.* Dense deposit disease is not a membranoproliferative glomerulonephritis. Mod Pathol 2007; 20:605-616.

[12] Burkholder PM, Marchand A, Krueger RP. Mixed membranous and proliferative

[13] Strife CF, McEnery PT, McAdams AJ*, et al.* Membranoproliferative glomerulonephritis

[14] Appel GB, Cook HT, Hageman G*, et al.* Membranoproliferative glomerulonephritis type II (dense deposit disease): an update. J Am Soc Nephrol 2005; 16:1392-1403. [15] Kemper C, Atkinson JP. T-cell regulation: with complements from innate immunity. Nat

[16] Pickering MC, Cook HT. Translational mini-review series on complement factor H:

[17] Martinez-Barricarte R, Heurich M, Valdes-Canedo F*, et al.* Human C3 mutation reveals a

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[20] Meri S, Koistinen V, Miettinen A*, et al.* Activation of the alternative pathway of

[21] Strobel S, Zimmering M, Papp K*, et al.* Anti-factor B autoantibody in dense deposit

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[23] Gale DP, de Jorge EG, Cook HT*, et al.* Identification of a mutation in complement factor

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### **MPGN type III**

Patients with MPGN type III do not seem to respond as well to corticosteroids as do those with type I disease as assessed by disease relapse and estimated GFR [35]. Thus, other than non-specfic therapies there is scant evidence to guide the treatment of these patients.

#### **C3 glomerulonephritis**

In the series by Servais et al., five of the patients were treated with steroids [24]. The authors reported that there was no clear effect of treatment on the disease outcomes. The patients reported by Sethi et al. were treated conservatively (no patients received immunosuppression), and no deterioration in renal function was seen during the period of follow-up [36]. Thus, based upon the available data there is little evidence to support immunosuppression in these patients.

#### **CFHR5 nephropathy**

The optimum treatment for CFHR5 nephropathy presently remains unknown. There are theoretical grounds to investigate the utility of eculizumab in this condition e.g. during disease flares. The relationship between renal decline and infective episodes in CFHR5 nephropathy implies that immunosuppressive strategies may be a potentially harmful approach.

### **8. Conclusions**

MPGN is a fascinating glomerular pathology. We have made significant progress in understanding the role of complement in MPGN. There are limitations to the traditional histological classification. Dense deposit disease should not be referred to as MPGN type II since many patients with dense deposit disease do not have MPGN. C3 glomerulopathy is a new term which encompasses glomerular pathologies in which there is isolated or predominant deposition of glomerular C3. C3 glomerulopathy includes dense deposit disease and C3 glomerulonephritis. The most recent addition is CFHR5 nephropathy. Individuals with C3 glomerulopathy should be investigated for complement dysregulation and represent logical patient populations in which to explore the efficacy of complement modulating therapies.

### **9. References**


Patients with MPGN type III do not seem to respond as well to corticosteroids as do those with type I disease as assessed by disease relapse and estimated GFR [35]. Thus, other than

In the series by Servais et al., five of the patients were treated with steroids [24]. The authors reported that there was no clear effect of treatment on the disease outcomes. The patients reported by Sethi et al. were treated conservatively (no patients received immunosuppression), and no deterioration in renal function was seen during the period of follow-up [36]. Thus, based upon the available data there is little evidence to support

The optimum treatment for CFHR5 nephropathy presently remains unknown. There are theoretical grounds to investigate the utility of eculizumab in this condition e.g. during disease flares. The relationship between renal decline and infective episodes in CFHR5 nephropathy implies that immunosuppressive strategies may be a potentially harmful

MPGN is a fascinating glomerular pathology. We have made significant progress in understanding the role of complement in MPGN. There are limitations to the traditional histological classification. Dense deposit disease should not be referred to as MPGN type II since many patients with dense deposit disease do not have MPGN. C3 glomerulopathy is a new term which encompasses glomerular pathologies in which there is isolated or predominant deposition of glomerular C3. C3 glomerulopathy includes dense deposit disease and C3 glomerulonephritis. The most recent addition is CFHR5 nephropathy. Individuals with C3 glomerulopathy should be investigated for complement dysregulation and represent logical patient populations in which to explore the efficacy of complement

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**8. Conclusions** 

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[27] Watson AR, Poucell S, Thorner P*, et al.* Membranoproliferative glomerulonephritis type

[28] Magil AB, Price JD, Bower G*, et al.* Membranoproliferative glomerulonephritis type 1:

[29] Cameron JS, Turner DR, Heaton J*, et al.* Idiopathic mesangiocapillary

[30] Ooi YM, Vallota EH, West CD. Classical complement pathway activation in membranoproliferative glomerulonephritis. Kidney Int 1976; 9:46-53. [31] Nasr SH, Valeri AM, Appel GB*, et al.* Dense deposit disease: clinicopathologic study of 32 pediatric and adult patients. Clin J Am Soc Nephrol 2009; 4:22-32. [32] Schwertz R, Rother U, Anders D*, et al.* Complement analysis in children with idiopathic

[33] Eisinger AJ, Shortland JR, Moorhead PJ. Renal disease in partial lipodystrophy. Q J Med

[34] Duvall-Young J, Short CD, Raines MF*, et al.* Fundus changes in mesangiocapillary

[35] Braun MC, West CD, Strife CF. Differences between membranoproliferative

[36] Sethi S, Fervenza FC, Zhang Y*, et al.* Proliferative glomerulonephritis secondary to

[37] McRae JL, Cowan PJ, Power DA*, et al.* Human factor H-related protein 5 (FHR-5). A new complement-associated protein. J Biol Chem 2001; 276:6747-6754. [38] McRae JL, Duthy TG, Griggs KM*, et al.* Human factor H-related protein 5 has cofactor

[39] Athanasiou Y, Voskarides K, Gale DP*, et al.* Familial C3 Glomerulopathy Associated

[40] Vernon KA, Gale DP, de Jorge EG*, et al.* Recurrence of complement factor H-related protein 5 nephropathy in a renal transplant. Am J Transplant 2011; 11:152-155. [41] Arslan S, Saatci U, Ozen S*, et al.* Membranoproliferative glomerulonephritis in childhood: factors affecting prognosis. Int Urol Nephrol 1997; 29:711-716. [42] McEnery PT. Membranoproliferative glomerulonephritis: the Cincinnati experience- cumulative renal survival from 1957 to 1989. J Pediatr 1990; 116:S109-114. [43] Kawasaki Y, Suzuki J, Nozawa R*, et al.* Efficacy of school urinary screening for membranoproliferative glomerulonephritis type 1. Arch Dis Child 2002; 86:21-25.

prednisone regimen. Am J Kidney Dis 1999; 34:1022-1032.

associates with lipoprotein. J Immunol 2005; 174:6250-6256.

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comparison of natural history in children and adults. Clin Nephrol 1979; 11:239-

glomerulonephritis. Comparison of types I and II in children and adults and long-

membranoproliferative glomerulonephritis: a long-term follow-up. Pediatr Allergy

glomerulonephritis type II: clinical and fluorescein angiographic findings. Br J

glomerulonephritis types I and III in long-term response to an alternate-day

dysfunction of the alternative pathway of complement. Clin J Am Soc Nephrol

activity, inhibits C3 convertase activity, binds heparin and C-reactive protein, and

with CFHR5 Mutations: Clinical Characteristics of 91 Patients in 16 Pedigrees. Clin

Nephrol Hypertens 2011; 20:271-277.

term prognosis. Am J Med 1983; 74:175-192.

Dis 1984; 4:141-146.

Immunol 2001; 12:166-172.

Ophthalmol 1989; 73:900-906.

J Am Soc Nephrol 2011; 6:1436-1446.

1972; 41:343-354.

2011; 6:1009-1017.

244.


**4** 

*France* 

**Primary IgA Nephropathy: An Update in 2011** 

*1Dialysis and Renal Transplantation Department, University Hospital of Saint-Etienne,* 

IgA Nephropathy (IGAN) is also called mesangial IgA glomerulonephritis and was first described by Jean Berger, a French general pathologist, in 1968 in the journal d'Urologie-

The definition is histopathologic with the characteristical **deposition** of the **immunoglobulin A** in the renal **mesangium**: these deposits are **predominant** (or codominant with other immunoglobulins, IgG and/or IgM), **granular**, **coarse** (with the "en mottes" aspect), **generalized** in the glomerulus and **diffuse** to all glomeruli. These deposits are evidenced usually by the technique of direct immunofluorescence on a semi-quantitative scale: traces (+/-), 1+, 2+, and ≥ 3+. There is a general agreement to accept **at least 1+ IgA** 

By contrast, the lesions observed by light microscopy are often segmental and focal. The main lesions concern the mesangium with increased matrix and hypercellularity with in addition endocapillary proliferation. The most severe lesions are represented by obsolescent glomeruli ("pains à cacheter"), focal and segmental glomerulosclerosis (FSGS) with capsular adhesions,

The deposition of mesangial IgA has been observed in different types of glomerulonephritis leading to the classification of **Primary IgA nephropathy** (Berger's disease) versus the **Secondary IgA nephropathies**: associated to Schönlein-Henoch Purpura, to patent Alcoholic Liver Cirrhosis, to Systemic lupus Erythematosus, or to Ankylosing

Primary IGAN is worldwide the most frequent glomerulonephritis; it accounts for about a quarter of the percutaneous renal biopsies performed on native kidneys. In France, the **incidence** is about 30 new cases per million inhabitants (pmp) and its **prevalence** is closed

and extracapillary proliferation with the formation of cellular/hyalinized crescents.

Spondylarthritis. Primary disease represents at least 80 % of the cases.

Néphrologie [1, 2]. Many reviews have been written on the subject [3, 4, 5].

**1. Introduction** 

**2. Definition [3, 4, 5]** 

**deposits** for the definition.

**3. The IgA nephritides [3, 4, 5]** 

**4. Epidemiology [3, 4, 5]** 

Francois Berthoux1 and Amir Kamal Aziz2

*Medical Faculty of Saint-Etienne, Saint-Etienne Cedex 2* 

*2Nephrology and Dialysis Department, Louis Pasteur Hospital, Dole* 


## **Primary IgA Nephropathy: An Update in 2011**

Francois Berthoux1 and Amir Kamal Aziz2

*1Dialysis and Renal Transplantation Department, University Hospital of Saint-Etienne, Medical Faculty of Saint-Etienne, Saint-Etienne Cedex 2 2Nephrology and Dialysis Department, Louis Pasteur Hospital, Dole France* 

### **1. Introduction**

58 An Update on Glomerulopathies – Clinical and Treatment Aspects

[60] Ponticelli C, Glassock RJ. Posttransplant recurrence of primary glomerulonephritis. Clin

[61] Licht C, Heinen S, Jozsi M*, et al.* Deletion of Lys224 in regulatory domain 4 of Factor H

[62] Krmar RT, Holtback U, Linne T*, et al.* Acute renal failure in dense deposit disease:

[63] Smith RJ, Alexander J, Barlow PN*, et al.* New approaches to the treatment of dense

[64] Braun MC, Stablein DM, Hamiwka LA*, et al.* Recurrence of membranoproliferative

reveals a novel pathomechanism for dense deposit disease (MPGN II). Kidney Int

complete recovery after combination therapy with immunosuppressant and plasma

glomerulonephritis type II in renal allografts: The North American Pediatric Renal Transplant Cooperative Study experience. J Am Soc Nephrol 2005; 16:2225-2233.

J Am Soc Nephrol 2010; 5:2363-2372.

exchange. Clin Nephrol 2011; 75 Suppl 1:4-10.

deposit disease. J Am Soc Nephrol 2007; 18:2447-2456.

2006; 70:42-50.

IgA Nephropathy (IGAN) is also called mesangial IgA glomerulonephritis and was first described by Jean Berger, a French general pathologist, in 1968 in the journal d'Urologie-Néphrologie [1, 2]. Many reviews have been written on the subject [3, 4, 5].

### **2. Definition [3, 4, 5]**

The definition is histopathologic with the characteristical **deposition** of the **immunoglobulin A** in the renal **mesangium**: these deposits are **predominant** (or codominant with other immunoglobulins, IgG and/or IgM), **granular**, **coarse** (with the "en mottes" aspect), **generalized** in the glomerulus and **diffuse** to all glomeruli. These deposits are evidenced usually by the technique of direct immunofluorescence on a semi-quantitative scale: traces (+/-), 1+, 2+, and ≥ 3+. There is a general agreement to accept **at least 1+ IgA deposits** for the definition.

By contrast, the lesions observed by light microscopy are often segmental and focal. The main lesions concern the mesangium with increased matrix and hypercellularity with in addition endocapillary proliferation. The most severe lesions are represented by obsolescent glomeruli ("pains à cacheter"), focal and segmental glomerulosclerosis (FSGS) with capsular adhesions, and extracapillary proliferation with the formation of cellular/hyalinized crescents.

### **3. The IgA nephritides [3, 4, 5]**

The deposition of mesangial IgA has been observed in different types of glomerulonephritis leading to the classification of **Primary IgA nephropathy** (Berger's disease) versus the **Secondary IgA nephropathies**: associated to Schönlein-Henoch Purpura, to patent Alcoholic Liver Cirrhosis, to Systemic lupus Erythematosus, or to Ankylosing Spondylarthritis. Primary disease represents at least 80 % of the cases.

### **4. Epidemiology [3, 4, 5]**

Primary IGAN is worldwide the most frequent glomerulonephritis; it accounts for about a quarter of the percutaneous renal biopsies performed on native kidneys. In France, the **incidence** is about 30 new cases per million inhabitants (pmp) and its **prevalence** is closed

Primary IgA Nephropathy: An Update in 2011 61

few days; there is no clot and the characteristics of this gross haematuria is nephrological with the presence on urine cytology of typical red blood cell casts. After such an episode, the patient is usually presenting microscopic haematuria or could be in total remission

The discovery can be **chance proteinuria** or **chance microscopic haematuria** at time of systemic urine control for medical check-up at school, at different institutions or at work; the time of onset is therefore imprecise and we should refer to the last negative control if

The disease can be diagnosed later on with arterial hypertension (HT) and/or oedema

Overall, IGAN is a **progressive disease** both clinically and pathologically. The **clinical progression** starts with urine abnormalities, followed by occurrence of HT, sometimes oedema related to massive proteinuria or nephrotic syndrome, and later on occurrence of CKD-3 through CKD-5 and ultimately renal death necessitating chronic dialysis. The progression is also **pathological** and this was demonstrated by repeated renal biopsies 5 years later [6, 13]: it was shown that the global optical score (GOS) progressed in the majority of the patients with increased glomerular but also vascular and tubular/interstitial

We are taking as example our prospective cohort of primary IGAN patients [12] whose diagnostic biopsy was performed between January 1st 1990 and December 31st 1999 at our institution with loco-regional patients coming from the Saint-Etienne area, the IGAN-STET-CO. This cohort is composed of 332 patients (237 men, 71.4 %) with a mean age of 35.9 at onset, 41.4 at diagnosis and 48.8 years at dialysis/death or at last follow-up visit. The total exposure time was about 13 years. Overall, 32 patients needed dialysis, 13 died before reaching dialysis, and 45 (13.6 %) reached the primary composite outcome while 99 (29.8 %)

**7. Predictive risk factors and the absolute renal risk of dialysis/death: A new** 

One very important goal was to sort out the major and independent risk factors (RF), present at diagnosis, able to predict accurately the ultimate final prognosis (dialysis or death as primary end-point). From the literature, we already know that the amount of proteinuria (g per day), the occurrence/presence of HT, and the severity of renal lesions on the initial biopsy were associated with progression [6-11]. Many other risk factors were also described such as gender, overweight/obesity [14], metabolic syndrome, age at onset [15], hypertriglyceridemia/hyperuricemia [16], and also different immunogenetic markers (HLA antigens, different cytokines polymorphisms [17, 18],…) but they were not consistent or controversial in different published cohorts. We recently confirmed [12] that these 3 major risk factors were sufficient to cover the whole prediction in our cohort. These risk factors were simplified, dichotomized for easier use and shown to be independent predictors in a multivariate model of Cox regression: - the most important is the **presence of HT (Yes or No)** defined according to WHO (≥ 140/90); - the next is the **presence of Proteinuria ≥ 1g/d (Yes or No);** and - the last is the scoring of the renal lesions; we have used our own **global** 

and/or chronic kidney disease stage 3 or up (CKD-3+).

**6. Progression of the disease [6-12]** 

presented the secondary outcome (CKD-3+).

until the next episode.

any.

indices.

**concept [12]** 

to 1000 pmp according to the disease duration from onset to dialysis or to last follow-up (from few months to more than 5O years). All these numbers are of course dependent of the "politic" of renal biopsy: liberal indication or restricted to the most severe cases with already proteinuria over 1g/day or already some degree of renal insufficiency (Glomerular Filtration Rate <60 ml/mn/1.73 m² body surface area).

The disease is more frequent in men than in women, about 70% males, in most continents except in Asia. Age at onset ranges from 5 years to 75 with a peak frequency in adolescents and young adults.

Fig. 1. HistoPathology of IgA Nephropathy:

(a) Light microscopy showing one glomerulus with segmental increased in mesangial matrix and mild hypercellularity.

(b) Immunofluorescent microscopy showing diffuse IgA deposits in all glomeruli.

(c) Immunofluorescent microscopy showing generalized mesangial IgA deposits within all the glomerulus surface/volume.

### **5. Mode of onset/initial presentation [3, 4, 5]**

The onset of the disease can be **acute/subacute** in about 30 to 35 % of the patients with the classical **intra-infectious gross haematuria**: at time of an infectious episode of various origin (pharyngitis, bronchitis, or even intestinal or urinary infection), the patient is urinating blood with colored urine, more brown than red with a color of coffee or cocacola; this is a total haematuria (sometimes with loin pain) which lasts from few hours to

to 1000 pmp according to the disease duration from onset to dialysis or to last follow-up (from few months to more than 5O years). All these numbers are of course dependent of the "politic" of renal biopsy: liberal indication or restricted to the most severe cases with already proteinuria over 1g/day or already some degree of renal insufficiency (Glomerular

The disease is more frequent in men than in women, about 70% males, in most continents except in Asia. Age at onset ranges from 5 years to 75 with a peak frequency in adolescents

(a) Light microscopy showing one glomerulus with segmental increased in mesangial

(c) Immunofluorescent microscopy showing generalized mesangial IgA deposits within all

The onset of the disease can be **acute/subacute** in about 30 to 35 % of the patients with the classical **intra-infectious gross haematuria**: at time of an infectious episode of various origin (pharyngitis, bronchitis, or even intestinal or urinary infection), the patient is urinating blood with colored urine, more brown than red with a color of coffee or cocacola; this is a total haematuria (sometimes with loin pain) which lasts from few hours to

(b) Immunofluorescent microscopy showing diffuse IgA deposits in all glomeruli.

Filtration Rate <60 ml/mn/1.73 m² body surface area).

Fig. 1. HistoPathology of IgA Nephropathy:

**5. Mode of onset/initial presentation [3, 4, 5]** 

matrix and mild hypercellularity.

the glomerulus surface/volume.

and young adults.

few days; there is no clot and the characteristics of this gross haematuria is nephrological with the presence on urine cytology of typical red blood cell casts. After such an episode, the patient is usually presenting microscopic haematuria or could be in total remission until the next episode.

The discovery can be **chance proteinuria** or **chance microscopic haematuria** at time of systemic urine control for medical check-up at school, at different institutions or at work; the time of onset is therefore imprecise and we should refer to the last negative control if any.

The disease can be diagnosed later on with arterial hypertension (HT) and/or oedema and/or chronic kidney disease stage 3 or up (CKD-3+).

### **6. Progression of the disease [6-12]**

Overall, IGAN is a **progressive disease** both clinically and pathologically. The **clinical progression** starts with urine abnormalities, followed by occurrence of HT, sometimes oedema related to massive proteinuria or nephrotic syndrome, and later on occurrence of CKD-3 through CKD-5 and ultimately renal death necessitating chronic dialysis. The progression is also **pathological** and this was demonstrated by repeated renal biopsies 5 years later [6, 13]: it was shown that the global optical score (GOS) progressed in the majority of the patients with increased glomerular but also vascular and tubular/interstitial indices.

We are taking as example our prospective cohort of primary IGAN patients [12] whose diagnostic biopsy was performed between January 1st 1990 and December 31st 1999 at our institution with loco-regional patients coming from the Saint-Etienne area, the IGAN-STET-CO. This cohort is composed of 332 patients (237 men, 71.4 %) with a mean age of 35.9 at onset, 41.4 at diagnosis and 48.8 years at dialysis/death or at last follow-up visit. The total exposure time was about 13 years. Overall, 32 patients needed dialysis, 13 died before reaching dialysis, and 45 (13.6 %) reached the primary composite outcome while 99 (29.8 %) presented the secondary outcome (CKD-3+).

### **7. Predictive risk factors and the absolute renal risk of dialysis/death: A new concept [12]**

One very important goal was to sort out the major and independent risk factors (RF), present at diagnosis, able to predict accurately the ultimate final prognosis (dialysis or death as primary end-point). From the literature, we already know that the amount of proteinuria (g per day), the occurrence/presence of HT, and the severity of renal lesions on the initial biopsy were associated with progression [6-11]. Many other risk factors were also described such as gender, overweight/obesity [14], metabolic syndrome, age at onset [15], hypertriglyceridemia/hyperuricemia [16], and also different immunogenetic markers (HLA antigens, different cytokines polymorphisms [17, 18],…) but they were not consistent or controversial in different published cohorts. We recently confirmed [12] that these 3 major risk factors were sufficient to cover the whole prediction in our cohort. These risk factors were simplified, dichotomized for easier use and shown to be independent predictors in a multivariate model of Cox regression: - the most important is the **presence of HT (Yes or No)** defined according to WHO (≥ 140/90); - the next is the **presence of Proteinuria ≥ 1g/d (Yes or No);** and - the last is the scoring of the renal lesions; we have used our own **global** 

Primary IgA Nephropathy: An Update in 2011 63

**The permanent control of HT** is a major step; the goal is to lower BP ≤ 130/80. Sodium chloride restriction is recommended with 24 h urinary sodium below 100 mmol/d corresponding to a maximum of 6 g daily sodium chloride. All antihypertensive agents can be used: diuretics, beta blockers, calcium blockers, central-acting, ACE inhibitors, angiotensin-2-receptor blockers (ARBs), and more recently renin inhibitors. However two classes have demonstrated a better protection and should be used alone or in association: ACEI and ARBs [25, 26]. In our prospective cohort [12], we have demonstrated that survival without dialysis/death improved in patients with adequately controlled BP on long term

**The significant reduction of proteinuria** is another major step [28]; the reduction can be obtained with the use of ACEI and ARBs which have a significant antiproteinuric effect. It is recommended to start with either one of these drugs, to titrate the dose to the effect, and to use the association in case of resistant proteinuria; the goal is to bring proteinuria ≤ 1g/d or ideally < 0.30 g/d. We have also demonstrated [12] that the permanent reduction of

The **treatment of severe renal lesions** on the biopsy; this could be achieved by Prednisone or Prednisolone treatment [29-31] which should be in theory able to reduce hypercellularity and cellular infiltration within the glomeruli; however in the trial, there was no repeated biopsies at the end of the steroid therapy. For the most severe cases with extracapillary GN (>50 % crescents), the association of high dose steroids and immunosuppressive agents as

We are still in the need of large randomized controlled trials with a long duration (5 years

Very significant progress has been made during the last decades. The key protein in this disease is the immunoglobulin A and more precisely the subgroup 1; in fact IgA1 is deposited in the mesangium but not IgA2 [34]. The major difference between IgA1 and IgA2 is the presence of an hinge region in IgA1 composed of 23 aminoacids with usually five sugar side chains linked to threonin or serin. There is now a consensus [35-39] about the fact that the main difference between IgA1 in Controls and in IGAN patients is the hypogalactosylation of these glycosylated side chains. The normal complete sugar chain is O-linked to threonine or serin and composed of one molecule of N-acetyl galactosamine (GalNac) and one molecule of Galactose; in addition a molecule of sialic acid can be bound in terminal to Galactose or in lateral to GalNac. In IGAN, more side chains are truncated with loss of terminal Galactose with its terminal sialic acid and is referred to the Galactose-deficient IgA1 (deGal-IgA1). This deGal-IgA1 represents the specific autoantigen in this disease and is present in the serum, within the circulating immune complexes and in the mesangial deposits [40]. This loss in terminal galactose is associated with a downregulation of the gene controlling the linkage of galactose to GalNac, C1GALT1 [41] and the description of specific polymorphism [42] raising the possibility of genetic predisposition

The loss of terminal galactose unusually exposed the GalNac molecules, which become antigenic with elicitation of a specific antibody response [44, 45]: IgG and/or IgA anti O-GalNac, the specific auto-antibodies. It is now possible to measure the amount of circulating

proteinuria is associated with a better survival on long-term.

The use of Fish Oil was limited with controversial results [32, 33].

seems optimal) to draw definite conclusions on the treatment of IGAN.

**10. Pathogenesis and patho-physiology of primary IgA nephropathy** 

already proposed might be a good option.

[27].

[43].

**optical score** (GOS) developed 20 years ago and integrating all elementary lesions (glomerular from 0 to 6, vascular from 0 to 5, tubular from 0 to 4, and interstitial from 0 to 5) with a GOS up to 20. We set up by ROC analysis that the best cut-off for predicting dialysis was the **presence of GOS ≥ 8 (Yes or No).** These 3 RF turned out to have a similar weight in the prediction of dialysis/death by the different accuracy parameters and also by the Cox regression (β/SE ratio of the same magnitude).

In analogy to the absolute cardio-vascular risk (ACVR) of death or major CV events at 10 years [19, 20], we proposed the **Absolute Renal Risk of Dialysis/Death**; this ARR is calculated at diagnosis and is very simply the number of these RF present: 0, 1, 2 or 3. By Kaplan-Meier survival curves, we could calculate the cumulative rate of primary event at 10 and 20 years after onset (time zero); it was respectively 2 and 4 % for ARR=0; 2 and 9 % for ARR=1; 7 and 18 % for ARR=2; and 29 and 64% for ARR=3.

In addition, this ARR integrates gender (less RF for women), age at diagnosis (more RF for older patients), and also body mass index (more RF in overweight/obese patients). We could also use it as prospective with time zero set-up at diagnosis and not at onset; the cumulative incidence of dialysis/death 10 years after diagnosis is 4% for ARR=0, 8% for ARR=1, 18% for ARR=2, and 68% for ARR=3. It is remarkable to underline the similarity of the values obtained 20 y after onset and 10y after diagnosis.

**Distribution of these Risk Factors at time of Diagnosis and at last follow-up (LFU):** HT was present in 120 patients (36.1%) at diagnosis and in 164 (49.4%) at LFU; Proteinuria ≥1g/d was present in 100 patients (30.1%) at diagnosis and in only 61 (18.4%) at LFU; and GOS ≥ 8 was present in 120 patients (36.1%) at time of diagnosis. The distribution of the ARR was as follows at time of diagnosis: 151 patients (45.5%) with ARR=0, 69 (20.8%) with ARR=1, 65 (19.6%) with ARR=2, and 47 patients (14.1%) with ARR=3.

It should be stressed that this cohort is an adequately treated cohort with all RF targeted as soon as they were identified: perfect control of blood pressure (target <130/80) with all antihypertensive agents; persistent reduction of proteinuria with ACEI and ARBs; and prednisolone for severe renal lesions.

### **8. Pathological classification of IgA nephropathy**

We have developed our own classification in 1990 [6, 13] with the Global Optical Score already described. During the past decades, the classifications of Haas [21] or Hass modified by Lee [22] were frequently used. The international Oxford classification was published in 2009 [23, 24] and retained only 4 parameters with significant clinical prediction: - mesangial hypercellularity (M score= 0 or 1); - endocapillary hypercellurarity (E score= 0 or 1); - segmental glomerulosclerosis (S score= 0 or 1); and - tubular atrophy/interstitial fibrosis (T score= 0 or 1 or 2). Overall, the MEST score ranges from 0 to 5. One limitation is that patients were only included if proteinuria was ≥ 1g/d in adults and there was no patients with ARR=0 who are in fact the majority of the patients; in addition patients with extracapillary GN (≥ 50% crescents) were also excluded; the two tails of the IGAN cohorts were therefore lacking!

### **9. Principles of treatment in IGAN**

The treatment should in fact target all major risk factors when present: hypertension, proteinuria, and severe renal lesions.

**optical score** (GOS) developed 20 years ago and integrating all elementary lesions (glomerular from 0 to 6, vascular from 0 to 5, tubular from 0 to 4, and interstitial from 0 to 5) with a GOS up to 20. We set up by ROC analysis that the best cut-off for predicting dialysis was the **presence of GOS ≥ 8 (Yes or No).** These 3 RF turned out to have a similar weight in the prediction of dialysis/death by the different accuracy parameters and also by the Cox

In analogy to the absolute cardio-vascular risk (ACVR) of death or major CV events at 10 years [19, 20], we proposed the **Absolute Renal Risk of Dialysis/Death**; this ARR is calculated at diagnosis and is very simply the number of these RF present: 0, 1, 2 or 3. By Kaplan-Meier survival curves, we could calculate the cumulative rate of primary event at 10 and 20 years after onset (time zero); it was respectively 2 and 4 % for ARR=0; 2 and 9 % for

In addition, this ARR integrates gender (less RF for women), age at diagnosis (more RF for older patients), and also body mass index (more RF in overweight/obese patients). We could also use it as prospective with time zero set-up at diagnosis and not at onset; the cumulative incidence of dialysis/death 10 years after diagnosis is 4% for ARR=0, 8% for ARR=1, 18% for ARR=2, and 68% for ARR=3. It is remarkable to underline the similarity of

**Distribution of these Risk Factors at time of Diagnosis and at last follow-up (LFU):** HT was present in 120 patients (36.1%) at diagnosis and in 164 (49.4%) at LFU; Proteinuria ≥1g/d was present in 100 patients (30.1%) at diagnosis and in only 61 (18.4%) at LFU; and GOS ≥ 8 was present in 120 patients (36.1%) at time of diagnosis. The distribution of the ARR was as follows at time of diagnosis: 151 patients (45.5%) with ARR=0, 69 (20.8%) with

It should be stressed that this cohort is an adequately treated cohort with all RF targeted as soon as they were identified: perfect control of blood pressure (target <130/80) with all antihypertensive agents; persistent reduction of proteinuria with ACEI and ARBs; and

We have developed our own classification in 1990 [6, 13] with the Global Optical Score already described. During the past decades, the classifications of Haas [21] or Hass modified by Lee [22] were frequently used. The international Oxford classification was published in 2009 [23, 24] and retained only 4 parameters with significant clinical prediction: - mesangial hypercellularity (M score= 0 or 1); - endocapillary hypercellurarity (E score= 0 or 1); - segmental glomerulosclerosis (S score= 0 or 1); and - tubular atrophy/interstitial fibrosis (T score= 0 or 1 or 2). Overall, the MEST score ranges from 0 to 5. One limitation is that patients were only included if proteinuria was ≥ 1g/d in adults and there was no patients with ARR=0 who are in fact the majority of the patients; in addition patients with extracapillary GN (≥ 50% crescents) were also excluded; the two tails of the IGAN cohorts

The treatment should in fact target all major risk factors when present: hypertension,

regression (β/SE ratio of the same magnitude).

ARR=1; 7 and 18 % for ARR=2; and 29 and 64% for ARR=3.

the values obtained 20 y after onset and 10y after diagnosis.

**8. Pathological classification of IgA nephropathy** 

prednisolone for severe renal lesions.

were therefore lacking!

**9. Principles of treatment in IGAN** 

proteinuria, and severe renal lesions.

ARR=1, 65 (19.6%) with ARR=2, and 47 patients (14.1%) with ARR=3.

**The permanent control of HT** is a major step; the goal is to lower BP ≤ 130/80. Sodium chloride restriction is recommended with 24 h urinary sodium below 100 mmol/d corresponding to a maximum of 6 g daily sodium chloride. All antihypertensive agents can be used: diuretics, beta blockers, calcium blockers, central-acting, ACE inhibitors, angiotensin-2-receptor blockers (ARBs), and more recently renin inhibitors. However two classes have demonstrated a better protection and should be used alone or in association: ACEI and ARBs [25, 26]. In our prospective cohort [12], we have demonstrated that survival without dialysis/death improved in patients with adequately controlled BP on long term [27].

**The significant reduction of proteinuria** is another major step [28]; the reduction can be obtained with the use of ACEI and ARBs which have a significant antiproteinuric effect. It is recommended to start with either one of these drugs, to titrate the dose to the effect, and to use the association in case of resistant proteinuria; the goal is to bring proteinuria ≤ 1g/d or ideally < 0.30 g/d. We have also demonstrated [12] that the permanent reduction of proteinuria is associated with a better survival on long-term.

The **treatment of severe renal lesions** on the biopsy; this could be achieved by Prednisone or Prednisolone treatment [29-31] which should be in theory able to reduce hypercellularity and cellular infiltration within the glomeruli; however in the trial, there was no repeated biopsies at the end of the steroid therapy. For the most severe cases with extracapillary GN (>50 % crescents), the association of high dose steroids and immunosuppressive agents as already proposed might be a good option.

The use of Fish Oil was limited with controversial results [32, 33].

We are still in the need of large randomized controlled trials with a long duration (5 years seems optimal) to draw definite conclusions on the treatment of IGAN.

#### **10. Pathogenesis and patho-physiology of primary IgA nephropathy**

Very significant progress has been made during the last decades. The key protein in this disease is the immunoglobulin A and more precisely the subgroup 1; in fact IgA1 is deposited in the mesangium but not IgA2 [34]. The major difference between IgA1 and IgA2 is the presence of an hinge region in IgA1 composed of 23 aminoacids with usually five sugar side chains linked to threonin or serin. There is now a consensus [35-39] about the fact that the main difference between IgA1 in Controls and in IGAN patients is the hypogalactosylation of these glycosylated side chains. The normal complete sugar chain is O-linked to threonine or serin and composed of one molecule of N-acetyl galactosamine (GalNac) and one molecule of Galactose; in addition a molecule of sialic acid can be bound in terminal to Galactose or in lateral to GalNac. In IGAN, more side chains are truncated with loss of terminal Galactose with its terminal sialic acid and is referred to the Galactose-deficient IgA1 (deGal-IgA1). This deGal-IgA1 represents the specific autoantigen in this disease and is present in the serum, within the circulating immune complexes and in the mesangial deposits [40]. This loss in terminal galactose is associated with a downregulation of the gene controlling the linkage of galactose to GalNac, C1GALT1 [41] and the description of specific polymorphism [42] raising the possibility of genetic predisposition [43].

The loss of terminal galactose unusually exposed the GalNac molecules, which become antigenic with elicitation of a specific antibody response [44, 45]: IgG and/or IgA anti O-GalNac, the specific auto-antibodies. It is now possible to measure the amount of circulating

Primary IgA Nephropathy: An Update in 2011 65

diagnosis, is 4 % for ARR=0; 8 % for ARR=1; 18 % for ARR=2; and 68 % for ARR=3 in our

The pathogenesis of the disease has also made significant progress: it is an **auto-immune disease** with a known **auto-antigen**, the Galactose-deficient IgA1, which can elicit a specific **auto-antibody** response, IgG and IgA anti-O-GalNac. There is formation of specific **immune complexes** which are **circulating** and then **deposited** in the mesangium with creation of the disease. These recent findings will have significant future applications in the diagnosis and

[1] Berger J, Hinglais N. Intercapillary deposits of IgA-IgG. J Urol Nephrol 1968;74:694-5. [2] Berger J. IgA glomerular deposits in renal disease. Transplant Proc 1969; 1: 939-44.

[5] Berthoux FC, Mohey H, Afiani A. Natural history of primary IgA nephropathy. Semin

[6] Alamartine E, Sabatier JC, Guerin C, et al. Prognostic factors in mesangial IgA

[7] Koyama A, Igarashi M, Kobayashi M. Natural history and risk factors for

[8] Radford MG, Donadio JV, Bergstralh EJ, Grande JP. Predicting renal outcome in IgA

[9] D'Amico G. Natural history of idiopathic IgA nephropathy: role of clinical and

[10] Bartosik LP, Lajoie G, Sugar L, Cattran DC. Predicting progression in IgA nephropathy.

[11] Li PK, Ho KK, Szeto CC, et al. Prognostic indicators of IgA nephropathy in the Chineseclinical and pathological perspectives. Nephrol Dial Transplant 2002; 17: 64-9. [12] Berthoux F, Mohey H, Laurent B, Mariat C, Afiani A, Thibaudin L. Predicting the risk for Dialysis or Death in IgA nephropathy. J Am Soc Nephrol 2011; 22: 752-61. [13] Alamartine E, Sabatier JC, Berthoux FC. Comparison of pathological lesions on repeated

[14] Bonnet F, Deprele C, Sassolas A, et al. Excessive body weight as a new independent risk

[15] Donadio JV, Bergstralh EJ, Grande JP, Rademcher DM. Proteinuria patterns and their

[16] Syrjänen J, Mustonen J, Pasternack A. Hypertriglyceridaemia and hyperuricaemia are

histological prognostic factors. Am J Kidney Dis 2000; 36: 227-37.

glomerulonephritis: an extensive study with univariate and multivariate analyses.

immunoglobulin A nephropathy in Japan. Research Group on Progressive Renal

renal biopsies in 73 patients with primary IgA glomerulonephritis : value of quantitative scoring and approach to final prognosis. Clin Nephrol 1990; 34: 45- 51.

factor for clinical and pathological progression in primary IgA nephritis. Am J

association with subsequent end-stage renal disease in IgA nephropathy. Nephrol

risk factors for progression of IgA nephropathy. Nephrol Dial Transplant 2000;

[3] Donadio JV, Grande JP. IgA nephropathy. N Engl J Med 2002; 347: 738-48. [4] Barratt J, Feehally J. IgA nephropathy. J Am Soc Nephrol 2005; 16: 2088-97.

prospective cohort adequately treated/managed on long term.

in the treatment.

**13. References** 

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autoantigen and autoantibodies in patients sera and to discriminate between Controls and IGAN patients [46, 47].

The mesangial deposition of deGal-IgA1 is dependent on physical characteristics of this molecule (more sticky) and the presence of transferrin receptor (CD71) which is able to bind IgA1 variants [48]. After binding, the deGal-IgA1 and the deposited immune complexes are able to activate the different mediators of inflammation both cellular [49] and in fluid phase. There is also the CD89 system [50] that we personally consider as an amplification loop. This is a specific receptor for IgA1 variants, FcαRI, present on circulating monocytes but not in the mesangium; this receptor is able to bind immune complexes with further amplification and longer persistence in the circulation, and may play an additional pathogenic role.

### **11. Renal transplantation in patients with biopsy-proven IGAN on native kidneys**

In cases which progressed to dialysis, renal transplantation should be a strong option and overall the results are similar to the other recipients matched for age and gender. However, there are two specific situations which have strong pathogenic implication.

First, a silent IgA nephropathy can be present on grafted kidney from apparently normal donors leading to the discovery of mesangial IgA deposits on graft biopsies performed early after transplantation; it was demonstrated in few cases that these deposits can regress and disappear demonstrating a contrario that the disease has a systemic (blood) transmission.

Second, the original disease may reappear (**recurrence**) on the normal grafted kidney after few years and despite immunosuppression [51-59]. The cumulative incidence of clinicpathological recurrence is high reaching 35 % or more at 10 years post-transplant [56] and may lead to graft losses in up to 17 % at 10 y [57]. The factors associated to recurrence are not well understood: living donors, better HLA matching, short duration of the original disease, etc. There is yet no specific treatment for the recurrent disease; however in a retrospective study [56] we demonstrated that induction treatment with ATG seems able to reduce the incidence of recurrence in comparison to no induction or to induction with Basiliximab. A prospective randomized controlled trial comparing rabbit ATG to Basiliximab has already started, the PIRAT study: Prevention in IgA nephropathy recipients of full Recurrence After renal Transplantation according to induction immunosuppressive therapy: ATG versus Basiliximab. It seems now mandatory to carefully measure the serum levels of the auto-antigen and the auto-antibodies at time of grafting to check for any predicting value of these parameters for recurrence.

### **12. Conclusions**

IgA nephropathy is a frequent disease whose individual prognosis can be totally different: no significant progression for 50 years versus progression to dialysis in few months or years. The clinical challenge is to accurately predict the long term individual prognosis at time of diagnosis (at time of the renal biopsy which is still mandatory for this purpose). We have made significant progress with our new concept: the **Absolute Renal Risk of Dialysis/Death** (ARR); it takes in account the presence or not of three independent, simplified and dichotomous risk factors: - **arterial hypertension**; - **proteinuria ≥1g/d**; and severe histopathological lesions appreciated by the **Global Optical Score ≥ 8** (range from 0 to 20). The cumulative incidence rate of primary event (dialysis or death) at 10 years postdiagnosis, is 4 % for ARR=0; 8 % for ARR=1; 18 % for ARR=2; and 68 % for ARR=3 in our prospective cohort adequately treated/managed on long term.

The pathogenesis of the disease has also made significant progress: it is an **auto-immune disease** with a known **auto-antigen**, the Galactose-deficient IgA1, which can elicit a specific **auto-antibody** response, IgG and IgA anti-O-GalNac. There is formation of specific **immune complexes** which are **circulating** and then **deposited** in the mesangium with creation of the disease. These recent findings will have significant future applications in the diagnosis and in the treatment.

#### **13. References**

64 An Update on Glomerulopathies – Clinical and Treatment Aspects

autoantigen and autoantibodies in patients sera and to discriminate between Controls and

The mesangial deposition of deGal-IgA1 is dependent on physical characteristics of this molecule (more sticky) and the presence of transferrin receptor (CD71) which is able to bind IgA1 variants [48]. After binding, the deGal-IgA1 and the deposited immune complexes are able to activate the different mediators of inflammation both cellular [49] and in fluid phase. There is also the CD89 system [50] that we personally consider as an amplification loop. This is a specific receptor for IgA1 variants, FcαRI, present on circulating monocytes but not in the mesangium; this receptor is able to bind immune complexes with further amplification and longer persistence in the circulation, and may play an additional pathogenic role.

**11. Renal transplantation in patients with biopsy-proven IGAN on native** 

there are two specific situations which have strong pathogenic implication.

predicting value of these parameters for recurrence.

**12. Conclusions** 

In cases which progressed to dialysis, renal transplantation should be a strong option and overall the results are similar to the other recipients matched for age and gender. However,

First, a silent IgA nephropathy can be present on grafted kidney from apparently normal donors leading to the discovery of mesangial IgA deposits on graft biopsies performed early after transplantation; it was demonstrated in few cases that these deposits can regress and disappear demonstrating a contrario that the disease has a systemic (blood) transmission. Second, the original disease may reappear (**recurrence**) on the normal grafted kidney after few years and despite immunosuppression [51-59]. The cumulative incidence of clinicpathological recurrence is high reaching 35 % or more at 10 years post-transplant [56] and may lead to graft losses in up to 17 % at 10 y [57]. The factors associated to recurrence are not well understood: living donors, better HLA matching, short duration of the original disease, etc. There is yet no specific treatment for the recurrent disease; however in a retrospective study [56] we demonstrated that induction treatment with ATG seems able to reduce the incidence of recurrence in comparison to no induction or to induction with Basiliximab. A prospective randomized controlled trial comparing rabbit ATG to Basiliximab has already started, the PIRAT study: Prevention in IgA nephropathy recipients of full Recurrence After renal Transplantation according to induction immunosuppressive therapy: ATG versus Basiliximab. It seems now mandatory to carefully measure the serum levels of the auto-antigen and the auto-antibodies at time of grafting to check for any

IgA nephropathy is a frequent disease whose individual prognosis can be totally different: no significant progression for 50 years versus progression to dialysis in few months or years. The clinical challenge is to accurately predict the long term individual prognosis at time of diagnosis (at time of the renal biopsy which is still mandatory for this purpose). We have made significant progress with our new concept: the **Absolute Renal Risk of Dialysis/Death** (ARR); it takes in account the presence or not of three independent, simplified and dichotomous risk factors: - **arterial hypertension**; - **proteinuria ≥1g/d**; and severe histopathological lesions appreciated by the **Global Optical Score ≥ 8** (range from 0 to 20). The cumulative incidence rate of primary event (dialysis or death) at 10 years post-

IGAN patients [46, 47].

**kidneys** 


Primary IgA Nephropathy: An Update in 2011 67

[33] Donadio JV, Grande JP. The role of fish oil / omega-3 fatty acids in the treatment of IgA

[34] Conley ME, Cooper MD, Michael AF. Selective deposition of immunoglobulin A1 in

[35] Hiki Y, Tanaka A, Kokubo T, Iwase H, Nishikido J, Hotta K, Kobayashi Y. Analyses of

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[37] Allen AC, Bailey EM, Barratt J, Buck KS, Feehally J. Analysis of IgA1 O-glycans in IgA

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[40] Allen AC, Bailey EM, Brenchley PE, Buck KS, Barratt J, Feehally J. Mesangial IgA1 in

[41] Inoue T, Sugiyama H, Kikumoto Y, Fukuoka N, Maeshima Y, Hattori H, Fukushima K,

[42] Li GS, Zhang H, Lv JC, Shen Y, Wang HY. Variants of C1GALT1 gene are associated with the genetic susceptibility to IgA nephropathy. Kidney Int 2007; 71:448-53 [43] Gharavi AG, Moldoveanu Z, Wyatt RJ, Barker CV, Woodford SY, Lifton RP, Mestecky J,

[44] Tomana M, Novak J, Julian BA, Matousovic K, Konecny K, Mestecky J. Circulating

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Y, Maeda K. Mass spectrometry proves under-O-glycosylation of glomerular IgA1

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IgA nephropathy exhibits aberrant O-glycosylation: observations in three patients.

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Anreddy SR, Hall S, Hastings MC, Lau KK, Cook WJ, Novak J. Patients with IgA nephropathy have increased serum galactose-deficient IgA1 levels. Kidney Int 2007;

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[27] Kanno Y, Okada H, Saruta T, Suzuki H. Blood pressure reduction associated with

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[30] Pozzi C, Andrulli S, Del Vecchio L, et al. Corticosteroid effectiveness in IgA

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**5** 

*1Greece 2Italy* 

**IgA Nephropathy: Insights into** 

*1Aristotle University, Thessaloniki, 2Renal Unit, University of Bari, Bari,* 

*In memoriam of Prof. Efstathios Alexopoulos* 

**Genetic Basis and Treatment Options** 

Dimitrios Kirmizis1, Aikaterini Papagianni1 and Francesco Paolo Schena2

IgA nephropathy (IgAN), is the most common primary glomerulonephritis worldwide. On light microscopy the picture can vary from slight mesangial hypertrophy to extra capillary proliferation of glomeruli, with sclerosis and interstitial fibrosis. On immunofluorescence staining of kidney sections the disease is characterized by mesangial deposits of IgA, predominantly polymeric IgA (pIgA) of the IgA1 subclass, and often co-deposition of complement factor C3, properdin and IgG. It is important however to realize that, although IgA mesangial deposits are necessary for the diagnosis of IgAN, the latter is not obligatory in every individual with mesangial IgA deposits. Thus, IgA deposits may also be seen in subjects with no evidence of renal disease [Suzuki et al, 2003; Waldherr et al, 1989] at an incidence that ranges from 3 to 16 percent. Furthermore, there are also a number of reports documenting IgA deposition in other forms of glomerulonephritis, particularly thin basement membrane disease, lupus nephritis, minimal change disease, and diabetic

IgAN occurs at any age, but most commonly the age of onset is in the second or third decade of life. Males are more often affected than females, with a male:female ratio of 2:1. Most patients with IgAN present microscopic hematuria with or without mild proteinuria. About 40% of patients have episodes of macroscopic hematuria. This is sometimes preceded by infections, most commonly upper respiratory tract infections, a phenomenon known as "synpharyngitic" hematuria. Other infections like gastrointestinal or urinary tract infections have also been reported to precede macroscopic hematuria. Proteinuria is common and can

IgAN has been considered a benign disease for a long time, but nowadays it is clear that 30- 40% of patients may develop renal failure with significant socioeconomical consequenses. In Western Europe and the United States of America 7-10% of the patients on renal replacement therapy suffer from IgAN. The severity of histological lesions, especially diffuse proliferative glomerulonephritis, marked capsular adhesions, fibrocellular crescents, glomerular hyalinosis and severe sclerosis, as well as tubulointerstitial damage correlate

nephropathy, a finding which is most probably casual rather than causal.

vary from mild proteinuria to nephrotic syndrome.

**1. Introduction** 


## **IgA Nephropathy: Insights into Genetic Basis and Treatment Options**

Dimitrios Kirmizis1, Aikaterini Papagianni1 and Francesco Paolo Schena2

*1Aristotle University, Thessaloniki, 2Renal Unit, University of Bari, Bari, 1Greece 2Italy* 

*In memoriam of Prof. Efstathios Alexopoulos* 

### **1. Introduction**

68 An Update on Glomerulopathies – Clinical and Treatment Aspects

[47] Novak J, Julian BA, Tomana M, Mestecky J. IgA glycosylation and IgA immune complexes in the pathogenesis of IgA nephropathy. Semin Nephrol. 2008; 28:78-87 [48] Moura IC, Arcos-Fajardo M, Sadaka C, Leroy V, Benhamou M, Novak J, Vrtovsnik F,

[49] Coppo R, Fonsato V, Balegno S, Ricotti E, Loiacono E, Camilla R, Peruzzi L, Amore A,

[50] Launay P, Grossetête B, Arcos-Fajardo M, Gaudin E, Torres SP, Beaudoin L, Patey-

[51] Berger J, Yaneva H, Nabarra B, Barbanel C. Recurrence of mesangial deposition of IgA

[52] Hariharan S, Peddi VR, Savin VJ, et al. Recurrent and de novo renal diseases after renal

[53] Chandrakantan A, Ratanapanichkich P, Said M, Barker CV, Julian BA. Recurrent IgA

[54] Wang AY, Lai FM, Yu AW, et al. Recurrent IgA nephropathy in renal transplant

[55] Ponticelli C, Traversi L, Feliciani A, et al. Kidney transplantation in patients with IgA

[56] Berthoux F, El Deeb S, Mariat C, Diconne E, Laurent B, Thibaudin L. Antithymocyte

recipients with primary IgA nephropathy. Transplantation 2008; 85: 1505. [57] Briganti EM, Russ GK, Mc Neil JJ, et al. Risk of renal allograft loss from recurrent

[58] Ponticelli C, Glassock RJ. Posttransplant recurrence of primary glomerulonephritis. Clin

[59] Han SS, Huh W, Park SK, Ahn C, Han JS, Kim S, Kim YS. Impact of recurrent disease

mycophenolate mofetil. Nephrol Dial Transplant 2005; 20: 1214.

mesangial glomerulonephritis. Kidney Int 2001; 60 (5): 1948.

glomerulonephritis. New Engl J Med 2002; 347: 103

with IgA nephropathy. Transpl Int 2010; 23:169-75.

Am Soc Nephrol 2004; 15:622-34

transgenic mice. J Exp Med 2000 ; 5;191:1999-2009.

after renal transplantation. Kidney Int 1975; 7: 232.

allografts. Am J Kidney Dis 2001;38: 588.

J Am Soc Nephrol 2010; 5: 2363-72.

Kidney Int 2010; 77:417-27.

1998; 31: 928.

Haddad E, Chintalacharuvu KR, Monteiro RC. Glycosylation and size of IgA1 are essential for interaction with mesangial transferrin receptor in IgA nephropathy. J

Bussolati B, Camussi G. Aberrantly glycosylated IgA1 induces mesangial cells to produce platelet-activating factor that mediates nephrin loss in cultured podocytes.

Mariaud de Serre N, Lehuen A, Monteiro RC. Fc alpha receptor (CD89) mediates the development of immunoglobulin A (IgA) nephropathy (Berger's disease). Evidence for pathogenic soluble receptor-Iga complexes in patients and CD89

transplantation: a report from the renal allograft disease registry. Am J Kidney Dis

nephropathy after renal transplantation despite immunosuppressive regimens with

globulin (ATG) induction therapy and disease recurrence in renal transplant

and chronic allograft nephropathy on the long-term allograft outcome in patients

IgA nephropathy (IgAN), is the most common primary glomerulonephritis worldwide. On light microscopy the picture can vary from slight mesangial hypertrophy to extra capillary proliferation of glomeruli, with sclerosis and interstitial fibrosis. On immunofluorescence staining of kidney sections the disease is characterized by mesangial deposits of IgA, predominantly polymeric IgA (pIgA) of the IgA1 subclass, and often co-deposition of complement factor C3, properdin and IgG. It is important however to realize that, although IgA mesangial deposits are necessary for the diagnosis of IgAN, the latter is not obligatory in every individual with mesangial IgA deposits. Thus, IgA deposits may also be seen in subjects with no evidence of renal disease [Suzuki et al, 2003; Waldherr et al, 1989] at an incidence that ranges from 3 to 16 percent. Furthermore, there are also a number of reports documenting IgA deposition in other forms of glomerulonephritis, particularly thin basement membrane disease, lupus nephritis, minimal change disease, and diabetic nephropathy, a finding which is most probably casual rather than causal.

IgAN occurs at any age, but most commonly the age of onset is in the second or third decade of life. Males are more often affected than females, with a male:female ratio of 2:1. Most patients with IgAN present microscopic hematuria with or without mild proteinuria. About 40% of patients have episodes of macroscopic hematuria. This is sometimes preceded by infections, most commonly upper respiratory tract infections, a phenomenon known as "synpharyngitic" hematuria. Other infections like gastrointestinal or urinary tract infections have also been reported to precede macroscopic hematuria. Proteinuria is common and can vary from mild proteinuria to nephrotic syndrome.

IgAN has been considered a benign disease for a long time, but nowadays it is clear that 30- 40% of patients may develop renal failure with significant socioeconomical consequenses. In Western Europe and the United States of America 7-10% of the patients on renal replacement therapy suffer from IgAN. The severity of histological lesions, especially diffuse proliferative glomerulonephritis, marked capsular adhesions, fibrocellular crescents, glomerular hyalinosis and severe sclerosis, as well as tubulointerstitial damage correlate

IgA Nephropathy: Insights into Genetic Basis and Treatment Options 71

altered genes with modest genetic effects but high prevalence. Recently, a genome-wide association study was carried out in cohorts of Chinese and European IgAN patients (A.G.Gharawi et al, 2011). Five loci (3 in the major histocompatibility complex at chromosome 6p21, a common deletion of CGHR1 and CFHR3 at chromosome 1q32 and one locus at chromosome 22q12) were identified. They explain 4-7% of the disease variance.

Genome-wide linkage analysis is used successfully to identify major disease/susceptibility genes but has limited power to detect genes of modest effect. Linkage studies involve recruitment of families with multiple affected individuals. In a typical whole-genome linkage scan, up to 400 microsatellites, or equivalently approximately 10,000 SNP markers, equally spaced across the genome, are typed in families to interrogate marker cosegregation with a disease phenotype. The advantage of genome-wide linkage studies is that they do not require a priori assumption about disease pathogenesis. These studies are very sensitive to phenotype misspecification, however their power is limited to detecting rare genetic

Linkage studies of IgAN are faced with multiple challenges. Familial forms of IgAN are frequently underrecognized because the associated urinary abnormalities in affected family members are often mild or intermittent. Moreover, once familial disease is documented, systematic screening by renal biopsy cannot be justified among asymptomatic at-risk relatives, necessitating reliance on less accurate phenotypes, such as microscopic hematuria, to diagnose affection. Additionally, IgAN has been observed to co-occur in families with thin basement membrane disease (TBMD), an autosomal dominant disease caused by heterozygous mutations in the collagen type IV genes (COL4A3/COL4A4) [Frasca et al, 2004]. Short of kidney biopsy or direct sequencing of the very large collagen genes, TBMD cannot be reliably excluded among relatives of IgAN patients. Finally, because urinary abnormalities may manifest intermittently, one also cannot unequivocally classify at-risk relatives as unaffected, necessitating affected-only linkage analysis. The inability to classify affected and unaffected individuals accurately is commonly encountered in linkage studies of complex traits, leading to decreased study power. Increasing sample size by including additional families is also not necessarily helpful in these situations because the diagnosis of IgAN likely encompasses several disease subsets, such that expansion to larger sample size can paradoxically reduce analytic power due to increased heterogeneity [Durner et al, 1992;

To date, four genome-wide linkage studies of familial IgAN have been reported [Gharavi et al, 2000; Bisceglia et al, 2006; Paterson et al, 2007, Feehally et al, 2010]. Families in these studies have all been ascertained via at least two cases with biopsy-documented IgAN, with additional family members diagnosed as affected based on clinical evidence (renal failure or multiple documentation of hematuria/proteinuria). In the first study, 30 families with two or more affected members were examined [Gharavi et al, 2000]; multipoint linkage analysis under the assumption of genetic heterogeneity yielded a peak LOD score of 5.6 on chromosome 6q22-23 (locus named IGAN1), with 60% of families linked. The remainder of families linked to chromosome 3p24-23 with a suggestive LOD of 2.8. This study demonstrated that IgAN is genetically heterogeneous but argued for the existence of a single locus with a major effect in some families. In the second genome-wide linkage study 22 Italian IgAN families were enrolled [Bisceglia et al, 2006] (see section 2.3). The third linkage

**2.1 Genome-wide linkage analysis studies** 

Cavalli-Sforza & King, 1986].

variants with a relatively large effect on the risk of disease.

with poor renal outcome [Schena, 1998]. Next to the gravity of histological lesions, unfavourable outcome is associated with persistent hematuria and proteinuria of more than 1g/day, decreased glomerular filtration rate (GFR) at the time of the diagnosis, and hypertension. Although several laboratory tests have been reported to correlate with clinical outcome, so far no reliable biomarker has been identified to predict outcome in IgAN. Recurrence of IgAN after renal transplantation is common. This finding, along with the observation that IgA depositions disappear from a kidney of an IgAN patient, after transplantation of this kidney to a non IgAN patient, are suggestive of a rather systemic disease.

### **2. Genetics of IgAN**

The strongest evidence for the existence of genetic factors in the development and/or progression of IgAN comes from descriptions of familial IgAN, largely in white populations [Julian et al, 1985], and recent studies suggest that familial and sporadic IgAN may share a common pathogenic mechanism and similar outcomes [Izzi et al, 2006]. Τhe genetic predisposition may be independent of environmental factors and may reflect an inherited susceptibility to develop mesangial glomerulonephritis.

IgAN does not exhibit classic single-gene Mendelian inheritance pattern [Frimat & Kessler, 2002]. The complex genetic pattern of IgAN is reflected by the multiple pathways involved in its immunopathogenesis, namely multiple discrete immunologic abnormalities related to the abnormal overproduction and release of mucosal pIgA1 in the systemic compartment and possibly other protein functional abnormalities related to a propensity for mesangial deposition of pIgA1. It is therefore probable that the disease-associated genetic variations at identified *IGAN* loci, instead of occuring in the form of "classic" nonsense/missense/splice site mutations and deletions/insertions that affect protein structure and function, may be rather of the type specific single-nucleotide polymorphism (SNP) alleles in non-coding regions or synonymous SNPs in coding regions. The latter function as *cis*-acting elements that alter the transcriptional activity of a disease gene and/or messenger RNA stability and, therefore, the expression level of the encoded protein. It is interesting that recent studies indicate that 30% to 50% of human genes with coding SNPs can present allelic variation in gene expression [Hoogendoorn et al, 2003; Lo et al, 2003]. From this point of view, the most comprehensive theory is that several genetic loci contribute significantly to the disease susceptibility that underlie the primary immunologic defects observed in IgAN. Each locus may occur at a different prevalence rate in different racial/ethnic groups. Variations at these major genetic loci may not be sufficient for the development and progression of IgAN and the contribution from a potentially large number of modifying genes with modest genetic effects but high prevalence is probably needed as well. The various allelic combinations of these loci may underlie the different disease phenotypes (disease development and progression, nephritic vs. nephrotic clinical presentation, histopathologic subclass, severity of disease, responsiveness of proteinuria to angiotensin-converting enzyme [ACE] inhibitors and/or angiotensin II receptor blockers [ARBs], etc.) observed in IgAN. For diseases with complex genetic pattern, it has been shown that the optimal analysis approach is the combination of linkage, association and sequence approaches. Until now, two basic approaches have been used in genetic studies of IgAN: a) genome-wide linkage analysis study, a methodology that has been used successfully to identify major disease/susceptibility loci, b) candidate-gene association study, mainly used to identify altered genes with modest genetic effects but high prevalence. Recently, a genome-wide association study was carried out in cohorts of Chinese and European IgAN patients (A.G.Gharawi et al, 2011). Five loci (3 in the major histocompatibility complex at chromosome 6p21, a common deletion of CGHR1 and CFHR3 at chromosome 1q32 and one locus at chromosome 22q12) were identified. They explain 4-7% of the disease variance.

#### **2.1 Genome-wide linkage analysis studies**

70 An Update on Glomerulopathies – Clinical and Treatment Aspects

with poor renal outcome [Schena, 1998]. Next to the gravity of histological lesions, unfavourable outcome is associated with persistent hematuria and proteinuria of more than 1g/day, decreased glomerular filtration rate (GFR) at the time of the diagnosis, and hypertension. Although several laboratory tests have been reported to correlate with clinical outcome, so far no reliable biomarker has been identified to predict outcome in IgAN. Recurrence of IgAN after renal transplantation is common. This finding, along with the observation that IgA depositions disappear from a kidney of an IgAN patient, after transplantation of this kidney to a non IgAN patient, are suggestive of a rather systemic

The strongest evidence for the existence of genetic factors in the development and/or progression of IgAN comes from descriptions of familial IgAN, largely in white populations [Julian et al, 1985], and recent studies suggest that familial and sporadic IgAN may share a common pathogenic mechanism and similar outcomes [Izzi et al, 2006]. Τhe genetic predisposition may be independent of environmental factors and may reflect an inherited

IgAN does not exhibit classic single-gene Mendelian inheritance pattern [Frimat & Kessler, 2002]. The complex genetic pattern of IgAN is reflected by the multiple pathways involved in its immunopathogenesis, namely multiple discrete immunologic abnormalities related to the abnormal overproduction and release of mucosal pIgA1 in the systemic compartment and possibly other protein functional abnormalities related to a propensity for mesangial deposition of pIgA1. It is therefore probable that the disease-associated genetic variations at identified *IGAN* loci, instead of occuring in the form of "classic" nonsense/missense/splice site mutations and deletions/insertions that affect protein structure and function, may be rather of the type specific single-nucleotide polymorphism (SNP) alleles in non-coding regions or synonymous SNPs in coding regions. The latter function as *cis*-acting elements that alter the transcriptional activity of a disease gene and/or messenger RNA stability and, therefore, the expression level of the encoded protein. It is interesting that recent studies indicate that 30% to 50% of human genes with coding SNPs can present allelic variation in gene expression [Hoogendoorn et al, 2003; Lo et al, 2003]. From this point of view, the most comprehensive theory is that several genetic loci contribute significantly to the disease susceptibility that underlie the primary immunologic defects observed in IgAN. Each locus may occur at a different prevalence rate in different racial/ethnic groups. Variations at these major genetic loci may not be sufficient for the development and progression of IgAN and the contribution from a potentially large number of modifying genes with modest genetic effects but high prevalence is probably needed as well. The various allelic combinations of these loci may underlie the different disease phenotypes (disease development and progression, nephritic vs. nephrotic clinical presentation, histopathologic subclass, severity of disease, responsiveness of proteinuria to angiotensin-converting enzyme [ACE] inhibitors and/or angiotensin II receptor blockers [ARBs], etc.) observed in IgAN. For diseases with complex genetic pattern, it has been shown that the optimal analysis approach is the combination of linkage, association and sequence approaches. Until now, two basic approaches have been used in genetic studies of IgAN: a) genome-wide linkage analysis study, a methodology that has been used successfully to identify major disease/susceptibility loci, b) candidate-gene association study, mainly used to identify

disease.

**2. Genetics of IgAN** 

susceptibility to develop mesangial glomerulonephritis.

Genome-wide linkage analysis is used successfully to identify major disease/susceptibility genes but has limited power to detect genes of modest effect. Linkage studies involve recruitment of families with multiple affected individuals. In a typical whole-genome linkage scan, up to 400 microsatellites, or equivalently approximately 10,000 SNP markers, equally spaced across the genome, are typed in families to interrogate marker cosegregation with a disease phenotype. The advantage of genome-wide linkage studies is that they do not require a priori assumption about disease pathogenesis. These studies are very sensitive to phenotype misspecification, however their power is limited to detecting rare genetic variants with a relatively large effect on the risk of disease.

Linkage studies of IgAN are faced with multiple challenges. Familial forms of IgAN are frequently underrecognized because the associated urinary abnormalities in affected family members are often mild or intermittent. Moreover, once familial disease is documented, systematic screening by renal biopsy cannot be justified among asymptomatic at-risk relatives, necessitating reliance on less accurate phenotypes, such as microscopic hematuria, to diagnose affection. Additionally, IgAN has been observed to co-occur in families with thin basement membrane disease (TBMD), an autosomal dominant disease caused by heterozygous mutations in the collagen type IV genes (COL4A3/COL4A4) [Frasca et al, 2004]. Short of kidney biopsy or direct sequencing of the very large collagen genes, TBMD cannot be reliably excluded among relatives of IgAN patients. Finally, because urinary abnormalities may manifest intermittently, one also cannot unequivocally classify at-risk relatives as unaffected, necessitating affected-only linkage analysis. The inability to classify affected and unaffected individuals accurately is commonly encountered in linkage studies of complex traits, leading to decreased study power. Increasing sample size by including additional families is also not necessarily helpful in these situations because the diagnosis of IgAN likely encompasses several disease subsets, such that expansion to larger sample size can paradoxically reduce analytic power due to increased heterogeneity [Durner et al, 1992; Cavalli-Sforza & King, 1986].

To date, four genome-wide linkage studies of familial IgAN have been reported [Gharavi et al, 2000; Bisceglia et al, 2006; Paterson et al, 2007, Feehally et al, 2010]. Families in these studies have all been ascertained via at least two cases with biopsy-documented IgAN, with additional family members diagnosed as affected based on clinical evidence (renal failure or multiple documentation of hematuria/proteinuria). In the first study, 30 families with two or more affected members were examined [Gharavi et al, 2000]; multipoint linkage analysis under the assumption of genetic heterogeneity yielded a peak LOD score of 5.6 on chromosome 6q22-23 (locus named IGAN1), with 60% of families linked. The remainder of families linked to chromosome 3p24-23 with a suggestive LOD of 2.8. This study demonstrated that IgAN is genetically heterogeneous but argued for the existence of a single locus with a major effect in some families. In the second genome-wide linkage study 22 Italian IgAN families were enrolled [Bisceglia et al, 2006] (see section 2.3). The third linkage

IgA Nephropathy: Insights into Genetic Basis and Treatment Options 73

indexed on PubMed (e.g., components of the renin-angiotensin-aldosterone pathway, mediators of inflammation and/or vascular tone, components of the mesangial matrix, and various receptors for polymeric IgA1 expressed in mesangial cells) [Kiryluk et al, 2010]. Of these, 39 (31%) studies examined genetic polymorphisms in association with susceptibility to IgAN, 40 (32%) examined an association with disease severity, progression, or complications, and 44 (35%) examined both susceptibility and risk of progression. Many

Approximately one third of all studies involved polymorphisms in the renin-angiotensinaldosterone system (RAAS). A widely studied example of the dilemma of repeated nonreplication of results is represented by genetic case-control association studies of the angiotensin I–converting enzyme (*ACE*) gene insertion/deletion (I/D) polymorphism in the development and/or progression of IgAN, as well as a whole host of common human diseases and conditions, including cardiovascular disease, complications of diabetes such as retinopathy and nephropathy, glomerular, tubulointerstitial, and renal cystic renal diseases, and even renal allograft survival [Hsu SI et al, 2000; Hsu SI, 2001; Schena et al, 2001]. The interest in studying the *ACE* I/D polymorphism is based on evidence for "biologic plausibility." Rigat and colleagues reported in 1990 that the *ACE* I/D polymorphism in intron 16 of the human *ACE* gene accounts for half of the variation in serum ACE levels in a white study cohort [Rigat et al, 1990], and this is due to the presence of a transcriptional

There have been numerous population-based studies that either support or refute an association between the D allele and progression of renal disease in these conditions [Hsu SI et al, 2000; Hsu SI, 2001]. Recent meta-analyses have concluded that the D allele is not associated with renal disease progression in patients with IgAN or diabetic nephropathy [Schena et al, 2001; Kunz et al, 1998]. Despite more than a dozen generally small genetic case-control studies of the *ACE* I/D polymorphism in both white and Asian IgAN cohorts have been done, no definite conclusions can be drawn from them regarding the association between the D allele or DD genotype and development and/or progression of IgAN. Population-based genetic association studies of other genes encoding proteins in the RAAS such as angiotensinogen (AGT) and the angiotensin II type 1 receptor (ATR1), as well as renin (*REN*) and aldosterone synthase (CYP11b2), have also generated conflicting results, as have similar studies of the "expanded" RAAS that includes 11b-hydroxysteroid dehydrogenase type 2 (11bHSD2) and the mineralocorticoid receptor (MLR) [Poch E et al, 2001]. In general, the approach has been to genotype a single common polymorphism in each gene with the use of polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP). It is remarkable that to date, the role of the RAAS, whose components ACE and ATR1 are the targets of ACE inhibitors and angiotensin-II receptor blockers (ARB), respectively, has not been convincingly demonstrated by any genetic

In general, most of these studies were of poor quality and severely underpowered and, therefore, negative findings were almost universally inconclusive. Overall, the average size of case–control cohorts per study was 182 cases and 171 controls. Many studies used ad hoc controls derived from unscreened blood donors who were poorly matched to the cases in terms of ancestry and geography. The potential impact of confounding by population stratification was ignored by the majority of studies, despite the fact that the tools for quantification of this problem have been developed. An additional matter of concern is the

candidate-gene association studies are lacking in functional genetics.

repressor element in the I allele [Hunley et al, 1996].

association study.

scan was based on a unique large pedigree with 14 affected relatives (two individuals with biopsy defined diagnosis and 12 with hematuria/proteinuria on urine dipstick) [Paterson et al, 2007]. Linkage to chromosome 2q36 was detected with a maximal multipoint LOD score of 3.47. Most reported linkage intervals did not contain obvious candidate genes, but the 2q36 locus encompasses the COL4A3 and COL4A4 genes, which are mutated in TBMD. Together with the high penetrance of hematuria, this finding suggests that affected individuals in the 2q36-linked family may belong to an IgAN subtype that overlaps with TBMD. Finally, the fourth genome-wide analysis, carried out in a cohort of IgAN patients selected from the UK Glomerulonephritis DNA Bank, the region of the MHC (major histocompatibility complex). The strongest association signal included a combination of DQ loci and HLA-B. This study suggests that HLA region contains some alleles that predispose to the disease in the UK population. In conclusion, four genome-wide linkage studies, carried out in four different IgAN patient populations, demonstrate different chromosomal traits linked to the disease.

None of the genes underlying these linkage loci has been identified until now. The underlying reasons are numerous, including the phenotyping difficulties discussed above; the presence of locus heterogeneity, which limits the ability to precisely map the disease interval and find additional linked families to refine loci; the contribution from non-coding susceptibility alleles (e.g. point mutations or structural genomic variants within intronic or promoter regions), which usually escape detection if mutational screening is confined to exonic regions. It is expected that the availability of inexpensive Next-Gen sequencing will enable comprehensive interrogation of linkage intervals, facilitating identification of disease-risk alleles.

In addition, future studies of this kind that aim at dissection of increasingly genetically homogeneous cohorts must consider the importance of defining distinct clinical subtypes of IgAN that may exist within the single pathologic ascertainment criterion currently used to diagnose IgAN: light microscopic evidence of mesangial deposits of IgA by immunofluorescence. As with all family-based genetic studies, there is a high degree of dependency on access to sufficient numbers of clinically well-phenotyped and genetically informative cohorts. To address the paucity of cohorts with biopsy-proven IgAN available for the conduct of linkage-based, association-based, and sequence-based approaches, the European IgAN consortium has published the details of its IgAN Biobank resource [Schena et al, 2005].

#### **2.2 Candidate-gene association studies**

Candidate-gene association studies examine polymorphisms in only specific genes that are selected based on a priori assumption about their involvement in the disease pathogenesis, and they are highly sensitive to population stratification, multiple testing, and reporting bias. As a result, most candidate-gene association studies in the literature have not been replicated [Ioannidis et al, 2001; Hsu SI et al, 2000; Hsu SI, 2001; Frimat & Kessler, 2002], an issue which questions the validity and the methodology used in these studies [Hsu & Feehally, 2008]. Not surprisingly, candidate-gene studies for IgAN have also been largely unrevealing. Many candidate genes have been proposed, but for most of them no solid a priori evidence for their involvement in IgAN existed, whereas most were studied in the context of IgAN progression rather than causality. Over the last 15 years, there were more than 120 candidate-gene association studies for IgAN published in the English literature and

scan was based on a unique large pedigree with 14 affected relatives (two individuals with biopsy defined diagnosis and 12 with hematuria/proteinuria on urine dipstick) [Paterson et al, 2007]. Linkage to chromosome 2q36 was detected with a maximal multipoint LOD score of 3.47. Most reported linkage intervals did not contain obvious candidate genes, but the 2q36 locus encompasses the COL4A3 and COL4A4 genes, which are mutated in TBMD. Together with the high penetrance of hematuria, this finding suggests that affected individuals in the 2q36-linked family may belong to an IgAN subtype that overlaps with TBMD. Finally, the fourth genome-wide analysis, carried out in a cohort of IgAN patients selected from the UK Glomerulonephritis DNA Bank, the region of the MHC (major histocompatibility complex). The strongest association signal included a combination of DQ loci and HLA-B. This study suggests that HLA region contains some alleles that predispose to the disease in the UK population. In conclusion, four genome-wide linkage studies, carried out in four different IgAN patient populations, demonstrate different chromosomal

None of the genes underlying these linkage loci has been identified until now. The underlying reasons are numerous, including the phenotyping difficulties discussed above; the presence of locus heterogeneity, which limits the ability to precisely map the disease interval and find additional linked families to refine loci; the contribution from non-coding susceptibility alleles (e.g. point mutations or structural genomic variants within intronic or promoter regions), which usually escape detection if mutational screening is confined to exonic regions. It is expected that the availability of inexpensive Next-Gen sequencing will enable comprehensive interrogation of linkage intervals, facilitating identification of

In addition, future studies of this kind that aim at dissection of increasingly genetically homogeneous cohorts must consider the importance of defining distinct clinical subtypes of IgAN that may exist within the single pathologic ascertainment criterion currently used to diagnose IgAN: light microscopic evidence of mesangial deposits of IgA by immunofluorescence. As with all family-based genetic studies, there is a high degree of dependency on access to sufficient numbers of clinically well-phenotyped and genetically informative cohorts. To address the paucity of cohorts with biopsy-proven IgAN available for the conduct of linkage-based, association-based, and sequence-based approaches, the European IgAN consortium has published the details of its IgAN Biobank resource [Schena

Candidate-gene association studies examine polymorphisms in only specific genes that are selected based on a priori assumption about their involvement in the disease pathogenesis, and they are highly sensitive to population stratification, multiple testing, and reporting bias. As a result, most candidate-gene association studies in the literature have not been replicated [Ioannidis et al, 2001; Hsu SI et al, 2000; Hsu SI, 2001; Frimat & Kessler, 2002], an issue which questions the validity and the methodology used in these studies [Hsu & Feehally, 2008]. Not surprisingly, candidate-gene studies for IgAN have also been largely unrevealing. Many candidate genes have been proposed, but for most of them no solid a priori evidence for their involvement in IgAN existed, whereas most were studied in the context of IgAN progression rather than causality. Over the last 15 years, there were more than 120 candidate-gene association studies for IgAN published in the English literature and

traits linked to the disease.

disease-risk alleles.

et al, 2005].

**2.2 Candidate-gene association studies** 

indexed on PubMed (e.g., components of the renin-angiotensin-aldosterone pathway, mediators of inflammation and/or vascular tone, components of the mesangial matrix, and various receptors for polymeric IgA1 expressed in mesangial cells) [Kiryluk et al, 2010]. Of these, 39 (31%) studies examined genetic polymorphisms in association with susceptibility to IgAN, 40 (32%) examined an association with disease severity, progression, or complications, and 44 (35%) examined both susceptibility and risk of progression. Many candidate-gene association studies are lacking in functional genetics.

Approximately one third of all studies involved polymorphisms in the renin-angiotensinaldosterone system (RAAS). A widely studied example of the dilemma of repeated nonreplication of results is represented by genetic case-control association studies of the angiotensin I–converting enzyme (*ACE*) gene insertion/deletion (I/D) polymorphism in the development and/or progression of IgAN, as well as a whole host of common human diseases and conditions, including cardiovascular disease, complications of diabetes such as retinopathy and nephropathy, glomerular, tubulointerstitial, and renal cystic renal diseases, and even renal allograft survival [Hsu SI et al, 2000; Hsu SI, 2001; Schena et al, 2001]. The interest in studying the *ACE* I/D polymorphism is based on evidence for "biologic plausibility." Rigat and colleagues reported in 1990 that the *ACE* I/D polymorphism in intron 16 of the human *ACE* gene accounts for half of the variation in serum ACE levels in a white study cohort [Rigat et al, 1990], and this is due to the presence of a transcriptional repressor element in the I allele [Hunley et al, 1996].

There have been numerous population-based studies that either support or refute an association between the D allele and progression of renal disease in these conditions [Hsu SI et al, 2000; Hsu SI, 2001]. Recent meta-analyses have concluded that the D allele is not associated with renal disease progression in patients with IgAN or diabetic nephropathy [Schena et al, 2001; Kunz et al, 1998]. Despite more than a dozen generally small genetic case-control studies of the *ACE* I/D polymorphism in both white and Asian IgAN cohorts have been done, no definite conclusions can be drawn from them regarding the association between the D allele or DD genotype and development and/or progression of IgAN. Population-based genetic association studies of other genes encoding proteins in the RAAS such as angiotensinogen (AGT) and the angiotensin II type 1 receptor (ATR1), as well as renin (*REN*) and aldosterone synthase (CYP11b2), have also generated conflicting results, as have similar studies of the "expanded" RAAS that includes 11b-hydroxysteroid dehydrogenase type 2 (11bHSD2) and the mineralocorticoid receptor (MLR) [Poch E et al, 2001]. In general, the approach has been to genotype a single common polymorphism in each gene with the use of polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP). It is remarkable that to date, the role of the RAAS, whose components ACE and ATR1 are the targets of ACE inhibitors and angiotensin-II receptor blockers (ARB), respectively, has not been convincingly demonstrated by any genetic association study.

In general, most of these studies were of poor quality and severely underpowered and, therefore, negative findings were almost universally inconclusive. Overall, the average size of case–control cohorts per study was 182 cases and 171 controls. Many studies used ad hoc controls derived from unscreened blood donors who were poorly matched to the cases in terms of ancestry and geography. The potential impact of confounding by population stratification was ignored by the majority of studies, despite the fact that the tools for quantification of this problem have been developed. An additional matter of concern is the

IgA Nephropathy: Insights into Genetic Basis and Treatment Options 75

were significantly more frequently transmitted from heterozygous parents to patients than expected in the extended TDT analysis (increased co-transmission in 232 Chinese families, *P* < 0.001). In addition, haplotype relative risk (HRR) analyses showed that these same SNP alleles were more often transmitted to patients (HRR = 1.568, *P* < 0.014 for the 2093C allele; HRR = 2.114, *P* < 0.001 for the 2180T allele) [Li YJ et al, 2004]. The same group using a similar approach recently reported that the *Megsin* 23167G SNP variant is associated with both susceptibility and progression of IgAN in 435 Chinese patients and their family members using TDT and HRR analyses [Takei et al, 2006]. The GG genotype was found to be associated with severe histologic lesions and disease progression. Megsin is a member of the serpin (serine proteinase inhibitor) superfamily that is upregulated in the context of mesangial proliferation and extracellular matrix expansion in IgAN, and therefore represents a strong candidate gene for susceptibility to IgAN. Lately, the gene encoding Cosmc, a C1Gal-T1 chaperone protein which also mediates IgA O-galactosylation was studied as a candidate gene involved in the pathogenesis of IgAN but no evidence for a role

The IgAN Biobank, coordinated by F.P. Schena, contains at minimum 72 multiplex extended pedigrees, 159 trios, and 1,068 cases and 1,040 matched controls. All subjects enrolled are white and belong to various geographic areas from Germany, Italy, and Greece [Schena et al, 2005]. Aiming at a genome wide linkage study, which has been considered the most promising approach to identify IgAN susceptibility genes, a group of investigators constituted the European IgAN Consortium which was initially funded by the European Union. DNA samples of IgAN patients and relatives belonging to 74 multiple extended pedigrees were collected. Moreover, 166 trios (affected sons or daughters and their healthy parents), 1,085 patients with biopsy-proven IgAN and 1,125 healthy subjects were included in the Biobank. In combination with linkage analysis, family based candidate gene association studies were also applied in an efford to discover responsible genes and overcome obstacles inherent the genetic analysis of complex traits

*Linkage Analysis Studies* – The European IgAN Consortium performed the first genome-wide scan involving 22 Italian multiplex IgAN families [Bisceglia L et al, 2006]. A total of 186 individuals (59 affected and 127 unaffected) were genotyped and included in a two-stage linkage analysis. The regions 4q26–31 and 17q12–22 exhibited the strongest evidence of linkage by non-parametric analysis (best p values of 0.0025 and 0.0045, respectively). These localizations were also supported by multipoint parametric analysis where a peak LOD score of 1.83 (α=0.50) and of 2.56 (α=0.65), respectively, were obtained using the affected only dominant model, and by allowing for the presence of genetic heterogeneity. These regions became the second (IGAN2) and third (IGAN3) genetic locus candidates to contain causative and/or susceptibility genes for familial IgAN. Other regions did not reach the threshold of a suggestive or significant LOD score; however, the enrolment of additional IgAN families means that these chromosomal regions may be explored in the near future. The above results provide further evidence for genetic heterogeneity among IgAN families. Evidence of linkage to multiple chromosomal regions is consistent with both an oligo/polygenic and a multiple susceptibility gene model for familial IgAN with

for Cosmc mutations was reported [Malycha et al, 2009].

**2.3 IgAN consortium** 

such as IgAN.

lack of adequate correction (permutation testing) for multiple, non-independent tests which would be anticipated as long as several of these studies tested several hypotheses (multiple polymorphisms, multiple phenotypes, or multiple genetic models). Other major problems included inadequate or variable SNP coverage of candidate genomic areas, with several studies examining only a single polymorphism. Thus far, only one group attempted to survey the entire genome, yet the results have not been replicated, are inconclusive and difficult to interpret, as long as an underpowered cohort was studied with inadequate coverage of ~80,000 SNPs [Obara et al, 2003; Ohtsubo et al, 2005]. Moreover, 77% of all published candidate-gene studies reported positive findings, an observation that is likely explained by a combination of high rate of false positives and a strong publication bias, whereas the statistical effect of the study of the same patient cohorts for multiple polymorphisms has never been accounted for in the literature [Kiryluk et al, 2010]. Most findings were not reproduced in other populations. None of the above problems is unique to the field of IgAN and for these reasons, new general guidelines aimed at improving the design and execution of genetic association studies have recently been formulated [von Elm et al, 2007; Little et al, 2009].

In the post-genomic era, there has been a renewed interest in conducting genetic association studies, especially SNP-based, whole-genome association studies, to identify genetic variations associated with the development and/or progression of a number of common human diseases. This renewed interest reflects the important finding that linkage disequilibrium (LD), the phenomenon that particular alleles at nearby sites can co-occur on the same haplotype more often than expected by chance [Goldstein, 2001; Wahl et al, 2003] is highly structured into discrete blocks separated by hotspots for recombination. The haplotype block model for LD has important implications for the way in which genetic association studies should now be conducted, and may explain at least in part the problem of repeated non-replication of results that has plagued such studies in the past. Based on the haplotype block model of LD, the ACE I/D polymorphism is a single marker variant in the ACE gene, whereas in unknown yet whether the D allele defines a simple population of subjects at risk for disease or not. The lumping of subgroups defined by haplotypes that share the D allele may explain at least in part the basis for discrepant reports of genetic association with disease.

Nowadays the common SNP haplotype block model is considered essential for the credibility of a study [Couser, 2003] and genetic association studies, especially family based, that employ one or more methodologically valid approaches and satisfy the minimum rigorous conditions for a reliable genetic association study are viewed as studies with solid documentation. These include mainly studies employing biologic plausibility, haplotype relative risk analysis to identify statistically significant "at-risk haplotype[s]" associated with small *P* values, use of family-based methodologies, such as the transmission equilibrium test (TDT/sib-TDT) or the family-based association test (FBAT) to directly study trios/sib-trios and extended families or to verify the absence of significant population stratification bias (admixture) inherent in population-based case-control association studies, and the study of moderately large i.e., adequately powered] cohorts. To date, very few studies examining candidate genes have employed the family-based TDT study methodology and/or analysis of "at-risk" haplotypes, reflecting the emergence of the first studies to attempt to satisfy minimum criteria for a valid association study.

A family- and haplotype-based association study employing the TDT methodology has shown that 2093C and 2180T SNP variants in the 3'-untranslated region of the *Megsin* gene were significantly more frequently transmitted from heterozygous parents to patients than expected in the extended TDT analysis (increased co-transmission in 232 Chinese families, *P* < 0.001). In addition, haplotype relative risk (HRR) analyses showed that these same SNP alleles were more often transmitted to patients (HRR = 1.568, *P* < 0.014 for the 2093C allele; HRR = 2.114, *P* < 0.001 for the 2180T allele) [Li YJ et al, 2004]. The same group using a similar approach recently reported that the *Megsin* 23167G SNP variant is associated with both susceptibility and progression of IgAN in 435 Chinese patients and their family members using TDT and HRR analyses [Takei et al, 2006]. The GG genotype was found to be associated with severe histologic lesions and disease progression. Megsin is a member of the serpin (serine proteinase inhibitor) superfamily that is upregulated in the context of mesangial proliferation and extracellular matrix expansion in IgAN, and therefore represents a strong candidate gene for susceptibility to IgAN. Lately, the gene encoding Cosmc, a C1Gal-T1 chaperone protein which also mediates IgA O-galactosylation was studied as a candidate gene involved in the pathogenesis of IgAN but no evidence for a role for Cosmc mutations was reported [Malycha et al, 2009].

#### **2.3 IgAN consortium**

74 An Update on Glomerulopathies – Clinical and Treatment Aspects

lack of adequate correction (permutation testing) for multiple, non-independent tests which would be anticipated as long as several of these studies tested several hypotheses (multiple polymorphisms, multiple phenotypes, or multiple genetic models). Other major problems included inadequate or variable SNP coverage of candidate genomic areas, with several studies examining only a single polymorphism. Thus far, only one group attempted to survey the entire genome, yet the results have not been replicated, are inconclusive and difficult to interpret, as long as an underpowered cohort was studied with inadequate coverage of ~80,000 SNPs [Obara et al, 2003; Ohtsubo et al, 2005]. Moreover, 77% of all published candidate-gene studies reported positive findings, an observation that is likely explained by a combination of high rate of false positives and a strong publication bias, whereas the statistical effect of the study of the same patient cohorts for multiple polymorphisms has never been accounted for in the literature [Kiryluk et al, 2010]. Most findings were not reproduced in other populations. None of the above problems is unique to the field of IgAN and for these reasons, new general guidelines aimed at improving the design and execution of genetic association studies have recently been formulated [von Elm

In the post-genomic era, there has been a renewed interest in conducting genetic association studies, especially SNP-based, whole-genome association studies, to identify genetic variations associated with the development and/or progression of a number of common human diseases. This renewed interest reflects the important finding that linkage disequilibrium (LD), the phenomenon that particular alleles at nearby sites can co-occur on the same haplotype more often than expected by chance [Goldstein, 2001; Wahl et al, 2003] is highly structured into discrete blocks separated by hotspots for recombination. The haplotype block model for LD has important implications for the way in which genetic association studies should now be conducted, and may explain at least in part the problem of repeated non-replication of results that has plagued such studies in the past. Based on the haplotype block model of LD, the ACE I/D polymorphism is a single marker variant in the ACE gene, whereas in unknown yet whether the D allele defines a simple population of subjects at risk for disease or not. The lumping of subgroups defined by haplotypes that share the D allele may explain at least in part the basis for discrepant reports of genetic

Nowadays the common SNP haplotype block model is considered essential for the credibility of a study [Couser, 2003] and genetic association studies, especially family based, that employ one or more methodologically valid approaches and satisfy the minimum rigorous conditions for a reliable genetic association study are viewed as studies with solid documentation. These include mainly studies employing biologic plausibility, haplotype relative risk analysis to identify statistically significant "at-risk haplotype[s]" associated with small *P* values, use of family-based methodologies, such as the transmission equilibrium test (TDT/sib-TDT) or the family-based association test (FBAT) to directly study trios/sib-trios and extended families or to verify the absence of significant population stratification bias (admixture) inherent in population-based case-control association studies, and the study of moderately large i.e., adequately powered] cohorts. To date, very few studies examining candidate genes have employed the family-based TDT study methodology and/or analysis of "at-risk" haplotypes, reflecting the emergence of the first

A family- and haplotype-based association study employing the TDT methodology has shown that 2093C and 2180T SNP variants in the 3'-untranslated region of the *Megsin* gene

studies to attempt to satisfy minimum criteria for a valid association study.

et al, 2007; Little et al, 2009].

association with disease.

The IgAN Biobank, coordinated by F.P. Schena, contains at minimum 72 multiplex extended pedigrees, 159 trios, and 1,068 cases and 1,040 matched controls. All subjects enrolled are white and belong to various geographic areas from Germany, Italy, and Greece [Schena et al, 2005]. Aiming at a genome wide linkage study, which has been considered the most promising approach to identify IgAN susceptibility genes, a group of investigators constituted the European IgAN Consortium which was initially funded by the European Union. DNA samples of IgAN patients and relatives belonging to 74 multiple extended pedigrees were collected. Moreover, 166 trios (affected sons or daughters and their healthy parents), 1,085 patients with biopsy-proven IgAN and 1,125 healthy subjects were included in the Biobank. In combination with linkage analysis, family based candidate gene association studies were also applied in an efford to discover responsible genes and overcome obstacles inherent the genetic analysis of complex traits such as IgAN.

*Linkage Analysis Studies* – The European IgAN Consortium performed the first genome-wide scan involving 22 Italian multiplex IgAN families [Bisceglia L et al, 2006]. A total of 186 individuals (59 affected and 127 unaffected) were genotyped and included in a two-stage linkage analysis. The regions 4q26–31 and 17q12–22 exhibited the strongest evidence of linkage by non-parametric analysis (best p values of 0.0025 and 0.0045, respectively). These localizations were also supported by multipoint parametric analysis where a peak LOD score of 1.83 (α=0.50) and of 2.56 (α=0.65), respectively, were obtained using the affected only dominant model, and by allowing for the presence of genetic heterogeneity. These regions became the second (IGAN2) and third (IGAN3) genetic locus candidates to contain causative and/or susceptibility genes for familial IgAN. Other regions did not reach the threshold of a suggestive or significant LOD score; however, the enrolment of additional IgAN families means that these chromosomal regions may be explored in the near future. The above results provide further evidence for genetic heterogeneity among IgAN families. Evidence of linkage to multiple chromosomal regions is consistent with both an oligo/polygenic and a multiple susceptibility gene model for familial IgAN with

IgA Nephropathy: Insights into Genetic Basis and Treatment Options 77

involved gene, respectively [Preston et al, 2004]. The expression levels of these genes were then examined on expanded RNA samples from other renal biopsies, leukocyte samples and cultured primary cells. Data demonstrated the involvement of the genes GABP and STAT3 in cluster I, and gp330 (megalin), MBP45K, MEF2, Oct1 and GABX in cluster II. The use of laser-capture microdissection applied to renal biopsy samples in combination with differential gene expression analysis is expected to provide novel knowledge in the search

*Candidate genes association studies* – The IgAN Consortium takes care of the collection of biological samples from large homogeneous cohorts of IgAN patients, their parents and their first degree relatives, and family-based association studies are preferred to analyze the role of some candidate genes. A family-based association study, including 53 patients, 45 complete trios, 4 incomplete trios and 36 discordant siblings, evaluated the impact of some Th1/Th2/Th3/TR-type lymphocyte and monocyte/macrophage cytokines on IgAN susceptibility [Schena FP et al, 2006]. Cytokine gene polymorphisms with a potential regulatory role on their production were investigated using the family-based association test (FBAT): IFN intron-1 CA-repeat at position 1349–1373; IL-13 –1055C/T; TGF 915G/C; IL-10 5'-proximal and distal microsatellites; TNF –308G/A, –238G/A. The FBAT multi-allelic analysis showed an association between IFN polymorphism and susceptibility to IgAN (p=0.03). The bi-allelic analysis showed that the 13-CA repeat allele was preferentially transmitted to the affected individuals (p=0.006; Bonferroni p=0.04). The direct sequencing of IFN amplicons showed a strict association between the 13-CA repeat allele and the A variant of the +874T/A SNP (rs2430561) directly adjacent to the 5' end of the microsatellite. The in vitro production of IFN evaluated in PBMC from 10 genotyped patients demonstrated a correlation between the +874A allele and a lower production of IFNγ (p=0.028). Notably, the +874A variant was associated with transcriptional downregulation of INFγ gene promoter activity, consistent with the known role of NF-κB in the

The occurrence of the +874A variant is responsible for the low production of IFNγ and predisposes to a preferential Th2-mediated immune response. The predominance of this variant in individuals with IgAN may be responsible for the onset of the disease. This unbalanced Th2 cytokine production in response to upper respiratory tract infections may be a significant pathogenic factor in human IgAN. The prevalence of Th2 cytokines may also explain the abnormality in IgA1 glycosylation occurring in IgAN patients and the concomitant formation of circulating IgA1-IgG immune complexes. Hyperfunction of Th2 cells and cytokine polarity are linked to a more nephritogenic pattern of IgA1 glycosylation in the animal model, and the decreased glycosylation of IgA1 elicited by Th2 cytokines is blunted in vitro by the addition of IFNγ [Ebihara et al, 2008]. The core 1 β1,3-galactosyltransferase (C1Gal-T1) is suspected to be involved in the abnormal glycosylation process of IgA1 in IgAN. With the genetic characterization of the enzymes responsible for O-glycosylation of IgA1, it has been possible to study changes in the Oglycosylation of IgA1 at a genetic level. Most recently two groups [Pirulli et al, 2009; Li GS et al, 2007] have independently found that SNPs in the C1Gal-T1 gene are associated with a genetic susceptibility to IgAN in Chinese and Italian populations, albeit it is not clear how these polymorphisms relate to changes in the functional activity of C1Gal-T1. The C1Gal-T1 gene complete sequence analysis was performed in 284 IgAN patients and 234 healthy controls. A statistically significant association of the genotype 1365G/G with

for IgAN candidate genes.

transcriptional regulation of the INFγ gene.

small/moderate effects in determining the pathological phenotype. The analysis of the known genes located in these two novel loci (positional information procedure), carried out consulting the National Center for Biotechnology Information, identified some potential candidate genes such as the transient receptor potential channel 3 (TRPC3) gene, the interleukin-2 (IL-2) gene, and the IL-21 gene located in 4q26–31, which could be largely involved in the unbalanced Th1/Th2 immune response reported in IgAN patients. In addition, the histone deacetylase 5 (HD5) gene and the granulin (GRN) gene located on the 17q12–22 region, which could be involved in the immune-response deregulation, will also considered. Family-based association studies, evaluating the distribution of these candidate gene polymorphisms, are in progress.

*Microarray Studies* – Different high-throughput gene analysis techniques can be used for obtaining transcriptome profiling of renal diseases. Microarray analysis represents the best and the latest approach to gain information on global gene expression. Genome-wide linkage analyses have identified at least three locus candidates containing IgAN susceptibility genes, although no specific gene(s) have been discovered. Microarrays are now in use to fingerprint the pathological process.

A published study postulated that changes in gene expression patterns in circulating leukocytes of IgAN patients may correlate with renal disease activity [Preston et al, 2004]. The investigators identified 14 upregulated genes. The BTG2, NCUBE1, FLJ2948, SRPK1, LYZ, GIG2 and IL-8 genes correlated with serum creatinine levels and the PMAIP1, SRPK1, SSI-3, LYZ and PTGS2 genes correlated with higher values of creatinine clearance, thus implying that the latter group of genes may provide a protective effect, while the overexpression of other genes such as B3GNT5, AXUD1 and GIG-2 indicated a worse prognosis. This gene signature reflected kidney function and did not correlate with hematuria or proteinuria. The authors concluded that studies carried out on large populations of IgAN patients will be necessary to confirm that the leukocyte gene expression profile can be used as a marker for diagnosis and for predicting outcome. The European IgAN Consortium has recently organized a protocol for studying gene expression in peripheral blood mononuclear cells (PBMC) and their subpopulations from IgAN patients with different clinical and histological patterns. Cox et al [2010] conducted a whole-genome expression study to identify genes and pathways differently modulated in peripheral blood leukocytes of IgAN patients. Gene expression of leukocytes demonstrated the hyperactivity of two important pathways as the canonical WNT- catenin and the PI3k/Akt pathways. The abnormal WNT signalling was also confirmed in IgAN patient's monocytes and to a less extent in B lymphocytes. Low gene expression of inversion and phosphatase and tensin homolog (PTEN) are responsible for the hyperactivation of these two pathways that enhance cell proliferation through lymphoid enhancer factor-1 (LEF-1) of which the gene is located within our previous described region 4q26-31 linked to IgAN [Bisceglia et al, 2006]. Finally, the hyperactivation of the PJ3k/Akt pathway is in linkage with the upregulation of the immunoproteasome in peripheral blood mononuclear cells of IgAN patients, reported by Coppo et al, [2009].

Expression profiling using serial analysis of gene expression (SAGE) and microarray techniques allows global description of expressed genes present in renal tissue. This is a high throughput genomics technology which enables the simultaneous determination of a large number of genes from tissue samples. Preston et al identified 13 upregulated genes in IgAN renal biopsy samples. The cluster analysis identified 3 clusters with 7, 12 and 1

small/moderate effects in determining the pathological phenotype. The analysis of the known genes located in these two novel loci (positional information procedure), carried out consulting the National Center for Biotechnology Information, identified some potential candidate genes such as the transient receptor potential channel 3 (TRPC3) gene, the interleukin-2 (IL-2) gene, and the IL-21 gene located in 4q26–31, which could be largely involved in the unbalanced Th1/Th2 immune response reported in IgAN patients. In addition, the histone deacetylase 5 (HD5) gene and the granulin (GRN) gene located on the 17q12–22 region, which could be involved in the immune-response deregulation, will also considered. Family-based association studies, evaluating the distribution of these candidate

*Microarray Studies* – Different high-throughput gene analysis techniques can be used for obtaining transcriptome profiling of renal diseases. Microarray analysis represents the best and the latest approach to gain information on global gene expression. Genome-wide linkage analyses have identified at least three locus candidates containing IgAN susceptibility genes, although no specific gene(s) have been discovered. Microarrays are

A published study postulated that changes in gene expression patterns in circulating leukocytes of IgAN patients may correlate with renal disease activity [Preston et al, 2004]. The investigators identified 14 upregulated genes. The BTG2, NCUBE1, FLJ2948, SRPK1, LYZ, GIG2 and IL-8 genes correlated with serum creatinine levels and the PMAIP1, SRPK1, SSI-3, LYZ and PTGS2 genes correlated with higher values of creatinine clearance, thus implying that the latter group of genes may provide a protective effect, while the overexpression of other genes such as B3GNT5, AXUD1 and GIG-2 indicated a worse prognosis. This gene signature reflected kidney function and did not correlate with hematuria or proteinuria. The authors concluded that studies carried out on large populations of IgAN patients will be necessary to confirm that the leukocyte gene expression profile can be used as a marker for diagnosis and for predicting outcome. The European IgAN Consortium has recently organized a protocol for studying gene expression in peripheral blood mononuclear cells (PBMC) and their subpopulations from IgAN patients with different clinical and histological patterns. Cox et al [2010] conducted a whole-genome expression study to identify genes and pathways differently modulated in peripheral blood leukocytes of IgAN patients. Gene expression of leukocytes demonstrated the hyperactivity of two important pathways as the canonical WNT- catenin and the PI3k/Akt pathways. The abnormal WNT signalling was also confirmed in IgAN patient's monocytes and to a less extent in B lymphocytes. Low gene expression of inversion and phosphatase and tensin homolog (PTEN) are responsible for the hyperactivation of these two pathways that enhance cell proliferation through lymphoid enhancer factor-1 (LEF-1) of which the gene is located within our previous described region 4q26-31 linked to IgAN [Bisceglia et al, 2006]. Finally, the hyperactivation of the PJ3k/Akt pathway is in linkage with the upregulation of the immunoproteasome in peripheral blood mononuclear cells of IgAN patients, reported by

Expression profiling using serial analysis of gene expression (SAGE) and microarray techniques allows global description of expressed genes present in renal tissue. This is a high throughput genomics technology which enables the simultaneous determination of a large number of genes from tissue samples. Preston et al identified 13 upregulated genes in IgAN renal biopsy samples. The cluster analysis identified 3 clusters with 7, 12 and 1

gene polymorphisms, are in progress.

Coppo et al, [2009].

now in use to fingerprint the pathological process.

involved gene, respectively [Preston et al, 2004]. The expression levels of these genes were then examined on expanded RNA samples from other renal biopsies, leukocyte samples and cultured primary cells. Data demonstrated the involvement of the genes GABP and STAT3 in cluster I, and gp330 (megalin), MBP45K, MEF2, Oct1 and GABX in cluster II. The use of laser-capture microdissection applied to renal biopsy samples in combination with differential gene expression analysis is expected to provide novel knowledge in the search for IgAN candidate genes.

*Candidate genes association studies* – The IgAN Consortium takes care of the collection of biological samples from large homogeneous cohorts of IgAN patients, their parents and their first degree relatives, and family-based association studies are preferred to analyze the role of some candidate genes. A family-based association study, including 53 patients, 45 complete trios, 4 incomplete trios and 36 discordant siblings, evaluated the impact of some Th1/Th2/Th3/TR-type lymphocyte and monocyte/macrophage cytokines on IgAN susceptibility [Schena FP et al, 2006]. Cytokine gene polymorphisms with a potential regulatory role on their production were investigated using the family-based association test (FBAT): IFN intron-1 CA-repeat at position 1349–1373; IL-13 –1055C/T; TGF 915G/C; IL-10 5'-proximal and distal microsatellites; TNF –308G/A, –238G/A. The FBAT multi-allelic analysis showed an association between IFN polymorphism and susceptibility to IgAN (p=0.03). The bi-allelic analysis showed that the 13-CA repeat allele was preferentially transmitted to the affected individuals (p=0.006; Bonferroni p=0.04). The direct sequencing of IFN amplicons showed a strict association between the 13-CA repeat allele and the A variant of the +874T/A SNP (rs2430561) directly adjacent to the 5' end of the microsatellite. The in vitro production of IFN evaluated in PBMC from 10 genotyped patients demonstrated a correlation between the +874A allele and a lower production of IFNγ (p=0.028). Notably, the +874A variant was associated with transcriptional downregulation of INFγ gene promoter activity, consistent with the known role of NF-κB in the transcriptional regulation of the INFγ gene.

The occurrence of the +874A variant is responsible for the low production of IFNγ and predisposes to a preferential Th2-mediated immune response. The predominance of this variant in individuals with IgAN may be responsible for the onset of the disease. This unbalanced Th2 cytokine production in response to upper respiratory tract infections may be a significant pathogenic factor in human IgAN. The prevalence of Th2 cytokines may also explain the abnormality in IgA1 glycosylation occurring in IgAN patients and the concomitant formation of circulating IgA1-IgG immune complexes. Hyperfunction of Th2 cells and cytokine polarity are linked to a more nephritogenic pattern of IgA1 glycosylation in the animal model, and the decreased glycosylation of IgA1 elicited by Th2 cytokines is blunted in vitro by the addition of IFNγ [Ebihara et al, 2008]. The core 1 β1,3-galactosyltransferase (C1Gal-T1) is suspected to be involved in the abnormal glycosylation process of IgA1 in IgAN. With the genetic characterization of the enzymes responsible for O-glycosylation of IgA1, it has been possible to study changes in the Oglycosylation of IgA1 at a genetic level. Most recently two groups [Pirulli et al, 2009; Li GS et al, 2007] have independently found that SNPs in the C1Gal-T1 gene are associated with a genetic susceptibility to IgAN in Chinese and Italian populations, albeit it is not clear how these polymorphisms relate to changes in the functional activity of C1Gal-T1. The C1Gal-T1 gene complete sequence analysis was performed in 284 IgAN patients and 234 healthy controls. A statistically significant association of the genotype 1365G/G with

IgA Nephropathy: Insights into Genetic Basis and Treatment Options 79

 Patients with more severe or rapidly progressive disease (eg, proteinuria > 1g or proteinuria persisting despite ACE inhibitor/ARB therapy, rising serum creatinine, and/or renal biopsy with more severe histologic findings, but no significant chronic changes) may benefit from immunosuppressive therapy in addition to non-

 In the future, the Oxford histologic classification system, once validated, is anticipated to become a useful prognostic tool that could lead in the future our therapeutic choices [Working Group of the International IgAN Network and the Renal Pathology Society,

Up to date, there are no specific markers to identify continued immunologic activity. Instead, the clinical parameters typically used as the main therapeutic criterion are the urine sediment, protein excretion and the serum creatinine concentration. Persistent hematuria is generally a marker of persistent immunologic activity, but not necessarily of progressive disease. This finding may be a sign of a "smoldering" segmental necrotizing lesion, suggestive of "capillaritis." Hematuria alone does not require any form of therapy. Proteinuria, rather than hematuria alone, is a marker of more severe disease [Donadio et al, 2002]. Increasing proteinuria may be due to one of two factors: ongoing active disease; and secondary glomerular injury due to non-immunologic progression. It is often not possible to distinguish between these two possibilities, except for a rapid increase in protein excretion which is only seen with active disease. Protein excretion most often falls with ACE inhibitor/ARB therapy and the degree of proteinuria is, as described below, one of the end points of such therapy. Protein excretion also may fall spontaneously, particularly during recovery from an acute episode and perhaps in children, and following effective immunosuppressive therapy. Finally, serum creatinine, unless it is rapidly rising, permits an estimation of the GFR. As noted above, most patients with chronic IgAN have stable or slowly progressive disease. The rate of GFR decline is often as low as 1 to 3 mL/min per year, a change that will not detectably raise the serum creatinine above normal levels for many years [Rekola et al, 1991]. Thus, a stable and even normal serum creatinine does not

The establishment of accurate biomarkers is necessary for the optimal categorization and treatment of patients with IgAN. Over the last few years specific urine biomolecules have been proposed as probable biomarkers to be used in the prognosis and therapeutic strategy in patients with IgAN. Two recent studies identified urine epidermal growth factor and monocyte chemoattractant protein-1 as strong independent predictors of renal outcome in patients with IgAN [Torres et al, 2008; Stangou et al, 2009]. These and other biomolecules

Three main non-immunosuppressive therapies are in use in IgAN [Barratt & Feehally, 2006;

ACE inhibitors or ARB, to control blood pressure and to slow down progression of the

Statin therapy, for lipid-lowering in selected patients, to lower cardiovascular risk and

are being validated as probable biomarkers of IgAN in studies underway.

Fish oil (omega-3 fatty acids) may also be beneficial in certain cases.

immunosuppressive interventions to slow disease progression.

2010].

**3.1 Monitoring disease activity** 

necessarily indicate stable disease.

**3.2 Non-immunosuppressive therapies** 

possibly reduce disease progression.

Appel & Waldman, 2006]:

renal disease.

susceptibility to IgAN (χ2=17.58, p<0.0001, odds ratio 2.57 [95% CI: 1.64–4.04]), but not with the progression of the disease, was found [Pirulli et al, 2009]. The latter case-control association study demonstrates that the low expression of C1Gal-T1 seems to confer susceptibility to IgAN.

In conclusion, to date the Consortium has identified two loci (located on chromosomes 4q26–31 and 17q12–22), in addition to a previous study which described the first IgAN locus on chromosome 6q22–23. The functional mapping of genes involved in the disease proceeds from the identification of susceptibility loci identified by linkage analysis (step 1) to the isolation of candidate genes within gene disease-susceptibility loci, after obtaining information by microarray analysis carried out on peripheral leukocytes and renal tissue samples (step 2). Next steps will be the design of RNA interference agents against selected genes (step 3) and the application of systematically tested effect of RNA agents on functional cellular assay (step 4). The above combined high-throughput technologies will give information on the pathogenic mechanisms of IgAN. In addition, these data may indicate potential targets for screening, prevention and early diagnosis of the disease and more appropriate and effective treatment.

### **3. Treatment of IgAN**

Treatment strategy for IgAN remains a controversial issue, even more as published randomized controlled trials (RCTs) on this topic are few and most studies are underpowered to provide definitive information. Furthermore, the disease heterogeneity, its clinical course along with the slow rate of GFR decline (1 to 3 mL/min per year) seen in many patients hinders the ability to perform adequate studies. An additional obstacle is the fact that a significant percentage of the patients have only minimal clinical presentation, such as isolated microhematuria, no or minimal proteinuria and normal GFR, and are often not biopsied or even identified. Still no treatment is known to modify mesangial deposition of IgA, which obviously reflects our incomplete knowledge of immunopathogenesis of IgAN [Barratt et al, 2007], and available treatment options are directed mostly at downstream immune and inflammatory events that may lead on to renal scarring. Therefore, as more pathogenetic details, the genetic substrate and heterogeneity of IgAN become increasingly understood, novel treatment strategies with solid therapeutic targets are anticipated, as long as the traditional therapies used until today seem symptomatic rather than etiologic. The discovery and establishment of novel biomarkers associated with the disease activity and outcome will provide the prognostic and therapeutic tool for more accurate and clear therapeutic targeting.

However, there seems to be a consensus regarding patient selection for the different therapeutic approaches. Patient selection for therapy is based in part upon the perceived risk of progressive kidney disease:


### **3.1 Monitoring disease activity**

78 An Update on Glomerulopathies – Clinical and Treatment Aspects

susceptibility to IgAN (χ2=17.58, p<0.0001, odds ratio 2.57 [95% CI: 1.64–4.04]), but not with the progression of the disease, was found [Pirulli et al, 2009]. The latter case-control association study demonstrates that the low expression of C1Gal-T1 seems to confer

In conclusion, to date the Consortium has identified two loci (located on chromosomes 4q26–31 and 17q12–22), in addition to a previous study which described the first IgAN locus on chromosome 6q22–23. The functional mapping of genes involved in the disease proceeds from the identification of susceptibility loci identified by linkage analysis (step 1) to the isolation of candidate genes within gene disease-susceptibility loci, after obtaining information by microarray analysis carried out on peripheral leukocytes and renal tissue samples (step 2). Next steps will be the design of RNA interference agents against selected genes (step 3) and the application of systematically tested effect of RNA agents on functional cellular assay (step 4). The above combined high-throughput technologies will give information on the pathogenic mechanisms of IgAN. In addition, these data may indicate potential targets for screening, prevention and early diagnosis of the disease and more

Treatment strategy for IgAN remains a controversial issue, even more as published randomized controlled trials (RCTs) on this topic are few and most studies are underpowered to provide definitive information. Furthermore, the disease heterogeneity, its clinical course along with the slow rate of GFR decline (1 to 3 mL/min per year) seen in many patients hinders the ability to perform adequate studies. An additional obstacle is the fact that a significant percentage of the patients have only minimal clinical presentation, such as isolated microhematuria, no or minimal proteinuria and normal GFR, and are often not biopsied or even identified. Still no treatment is known to modify mesangial deposition of IgA, which obviously reflects our incomplete knowledge of immunopathogenesis of IgAN [Barratt et al, 2007], and available treatment options are directed mostly at downstream immune and inflammatory events that may lead on to renal scarring. Therefore, as more pathogenetic details, the genetic substrate and heterogeneity of IgAN become increasingly understood, novel treatment strategies with solid therapeutic targets are anticipated, as long as the traditional therapies used until today seem symptomatic rather than etiologic. The discovery and establishment of novel biomarkers associated with the disease activity and outcome will provide the prognostic and therapeutic tool for more

However, there seems to be a consensus regarding patient selection for the different therapeutic approaches. Patient selection for therapy is based in part upon the perceived

 Patients with isolated hematuria, no or minimal proteinuria (<500mg/day), a normal GFR and no signs of progressive disease, such as increasing proteinuria, blood pressure,

 Patients with persistent proteinuria (>500 mg/day), normal or only slightly reduced GFR (>50mL/min) that is not declining rapidly, and only mild to moderate histologic findings on renal biopsy are managed with general interventions to slow progression with ACE-inhibitors or angiotensin receptor blockers (ARB) or with combination

susceptibility to IgAN.

appropriate and effective treatment.

accurate and clear therapeutic targeting.

and/or serum creatinine, are typically not treated.

therapy of corticosteroid (6 months) and ACE-inhibitors forever.

risk of progressive kidney disease:

**3. Treatment of IgAN** 

Up to date, there are no specific markers to identify continued immunologic activity. Instead, the clinical parameters typically used as the main therapeutic criterion are the urine sediment, protein excretion and the serum creatinine concentration. Persistent hematuria is generally a marker of persistent immunologic activity, but not necessarily of progressive disease. This finding may be a sign of a "smoldering" segmental necrotizing lesion, suggestive of "capillaritis." Hematuria alone does not require any form of therapy. Proteinuria, rather than hematuria alone, is a marker of more severe disease [Donadio et al, 2002]. Increasing proteinuria may be due to one of two factors: ongoing active disease; and secondary glomerular injury due to non-immunologic progression. It is often not possible to distinguish between these two possibilities, except for a rapid increase in protein excretion which is only seen with active disease. Protein excretion most often falls with ACE inhibitor/ARB therapy and the degree of proteinuria is, as described below, one of the end points of such therapy. Protein excretion also may fall spontaneously, particularly during recovery from an acute episode and perhaps in children, and following effective immunosuppressive therapy. Finally, serum creatinine, unless it is rapidly rising, permits an estimation of the GFR. As noted above, most patients with chronic IgAN have stable or slowly progressive disease. The rate of GFR decline is often as low as 1 to 3 mL/min per year, a change that will not detectably raise the serum creatinine above normal levels for many years [Rekola et al, 1991]. Thus, a stable and even normal serum creatinine does not necessarily indicate stable disease.

The establishment of accurate biomarkers is necessary for the optimal categorization and treatment of patients with IgAN. Over the last few years specific urine biomolecules have been proposed as probable biomarkers to be used in the prognosis and therapeutic strategy in patients with IgAN. Two recent studies identified urine epidermal growth factor and monocyte chemoattractant protein-1 as strong independent predictors of renal outcome in patients with IgAN [Torres et al, 2008; Stangou et al, 2009]. These and other biomolecules are being validated as probable biomarkers of IgAN in studies underway.

#### **3.2 Non-immunosuppressive therapies**

Three main non-immunosuppressive therapies are in use in IgAN [Barratt & Feehally, 2006; Appel & Waldman, 2006]:


IgA Nephropathy: Insights into Genetic Basis and Treatment Options 81

disease or diabetes, where an increase in adverse side effects (including a possible increase in mortality) in patients who received combination therapy with an ACE inhibitor and ARB was shown, compared to those who received monotherapy

Chronic kidney disease is associated with a marked increase in cardiovascular risk, and is now considered a coronary artery disease risk equivalent. Furthermore, lipid-lowering with statins has been associated with a slower rate of loss of GFR in patients in some patients with mild to moderate CKD, and there are indications for such a beneficial effect of statins in patients with IgAN as well [Kano et al, 2003]. Therefore, it seems rational that all patients with decreased kidney function and/or hypercholesterolemia should receive lipid-lowering therapy with a statin, with treatment goals similar to that in patients with underlying

The rationale for using fish oil (omega-3 fatty acids) in IgAN is based on the premise that they may limit the production or action of cytokines and eicosanoids evoked by the initial or by repeated immunologic renal injury, and the resulting production of mediators involved in renal damage [Donadio, 1991]. Randomized controlled trials evaluating fish oil in patients with IgAN have reported conflicting results [Donadio et al, 1994; Alexopoulos et al, 2004; Donadio et al, 1999, 2001; Bennett et al, 1989; Pettersson et al, 1994; Hogg et al, 2006; Ferraro et al, 2009]. In the largest and most well documented and conducted randomized trial in 106 patients with baseline creatinine clearance 80 mL/min and protein excretion of 2.5 to 3 g/day, Donadio et al found better patient and renal outcomes at 4 years, extended also at >6 years, in patients having received 12g of fish oil for 2 years, compared to patients having received a similar amount of olive oil [Donadio et al, 1994, 1999]. Similarly, in a controlled study of 14 IgAN patients and 14 controls, a low dose of fish oil (0.85 g eicosapentaenoic acid and 0.57 g phytohemaglutinin) was found effective in slowing renal progression in high-risk patients with IgAN and particularly those with advanced renal disease [Alexopoulos et al, 2004]. On the other hand, in the randomized controlled trial by the Southwest Pediatric Nephrology Study Group in 96 patients with IgAN, mean GFR >100 mL/min per 1.73 m2 and proteinuria 1.4 to 2.2 g/day, no significant benefit in the renal outcome was found in patients assigned to omega-3 fatty acids (4 g/day) for two years compared to the patients assigned to either alternate day prednisone or placebo [Hogg et al, 2006]. On the basis of the existing data, fish oil can be tried in addition to ACE inhibitors or ARBs in patients with protein excretion >500 to 1000 mg/day, a gradual reduction in GFR,

The optimal role of immunosuppressive therapy in IgAN is uncertain [Barratt & Feehally, 2006; Appel & Waldman, 2006]. A variety of regimens have been used, mostly consisting of corticosteroids alone or with other immunosuppressive drugs. The available studies are not conclusive since most are relatively small and have limited follow-up, and the results are sometimes conflicting [D'Amico, 1992; Alamartine et al, 1991; Alexopoulos, 2004; Strippoli et al, 2003; Laville & Alamartine, 2004; Ballardie, 2004; Julian, 2000; Dillon, 2001]. There is

[ONTARGET Investigators, 2008; Mann et al, 2008].

and mild to moderate histologic lesions [Alexopoulos E, 2004].

**3.3 Immunosuppressive therapy** 

**3.2.2 Lipid-lowering therapy** 

coronary heart disease.

**3.2.3 Fish oil** 

#### **3.2.1 Angiotensin inhibition**

The progression of IgAN may be slowed by anti-hypertensive and anti-proteinuric therapy that can minimize secondary glomerular injury [Kanno et al, 2000]. The treatment goals with angiotensin inhibition are the same as those in other forms of proteinuric chronic kidney disease as described in the K/DOQI guidelines [K/DOQI, 2004]. ACE inhibitors and ARBs act by reducing the intraglomerular pressure and by directly improving the size-selective properties of the glomerular capillary wall, both of which contribute to reducing protein excretion [Remuzzi et al, 1991; Maschio et al, 1994].

Both observational studies [Cattran et al, 1994; Kanno et al, 2005] and small randomized trials [Maschio et al, 1994; Praga et al, 2003; Li PK et al, 2006] have provided suggestive evidence that ACE inhibitors or ARBs are more effective than other antihypertensive drugs in slowing the progressive decline in GFR in IgAN as they are in other forms of chronic proteinuric kidney disease. Praga et al in their randomized trial in 44 IgAN patients with proteinuria (≥0.5 g/day, mean 1.9 g/day) and a serum creatinine concentration ≤1.5 mg/dL at baseline, found a significant decrease in proteinuria in the enalapril group (1.9 g/day at baseline to 0.9 g/day at the last visit) and a significantly higher renal survival, defined as less than a 50 percent increase in the serum creatinine concentration, at 6 years of follow up [Praga et al, 2003]. More recently, Li et al in their double-blind randomized placebocontrolled HKVIN trial in 109 Chinese patients with protein excretion ≥1 g/day (mean ~2.0 g/day), found a better renal survival, defined as doubling of serum creatinine or ESRD, a significant improvement in proteinuria (33 % reduction in proteinuria) and a slower rate of decline in GFR (4.6 versus 6.9 mL/min per year) in the valsartan group compared to placebo [Li PK et al, 2006]. Similarly, the IgACE trial in 65 young patients with moderate proteinuria (between 1 and 3.5 grams/day per 1.73 m2) and creatinine clearance >50 mL/min per 1.73 m2 revealed a better renal survival (fewer patients with >30% decline in renal function) and significant improvement in proteinuria at 38 months of follow-up in the benazepril group compared to the placebo group [Coppo, 2007].

Normotensive patients who excrete less than 500 mg of protein per day are not typically treated with angiotensin inhibition. However, because most patients progress slowly over time, monitoring of the serum creatinine and protein excretion at yearly intervals is recommended. Angiotensin inhibition should be started if there is evidence of progressive disease and protein excretion above 500 mg/day.

The addition of an ARB to an ACE inhibitor in patients with IgAN seems to exert a further antiproteinuric effect [Russo et al, 1999, 2001], albeit there are no randomized trials showing that this regimen improves renal outcomes. This finding is consistent with metaanalyses of trials in different glomerular diseases, the largest of which found a significant 18 to 25% greater reduction in proteinuria with combined ACE inhibitors and ARBs compared to monotherapy [Kunz et al, 2008; Catapano et al, 2008]. The rationale for this combination therapy is the assumption that ARBs would counteract theAT1-mediated effect of residual angiotensin II formation by non-ACE enzymes like chymase, whereas ACE inhibitors would additionally increase the level of kinins. Furthermore, ACE inhibitors as well as ARBs would synergistically elevate the levels of angiotensin, which also might promote vasodilation. Finally, combining both drug classes might simply provide a higher degree of blockade of the classic renin-angiotensin system pathways [Alexopoulos, 2004]. However, any anticipated benefits from this combination should be weighted against possible adverse effects in individual patients; this is important especially given the findings from the ONTARGET trial in 25,620 patients with vascular

disease or diabetes, where an increase in adverse side effects (including a possible increase in mortality) in patients who received combination therapy with an ACE inhibitor and ARB was shown, compared to those who received monotherapy [ONTARGET Investigators, 2008; Mann et al, 2008].

#### **3.2.2 Lipid-lowering therapy**

Chronic kidney disease is associated with a marked increase in cardiovascular risk, and is now considered a coronary artery disease risk equivalent. Furthermore, lipid-lowering with statins has been associated with a slower rate of loss of GFR in patients in some patients with mild to moderate CKD, and there are indications for such a beneficial effect of statins in patients with IgAN as well [Kano et al, 2003]. Therefore, it seems rational that all patients with decreased kidney function and/or hypercholesterolemia should receive lipid-lowering therapy with a statin, with treatment goals similar to that in patients with underlying coronary heart disease.

### **3.2.3 Fish oil**

80 An Update on Glomerulopathies – Clinical and Treatment Aspects

The progression of IgAN may be slowed by anti-hypertensive and anti-proteinuric therapy that can minimize secondary glomerular injury [Kanno et al, 2000]. The treatment goals with angiotensin inhibition are the same as those in other forms of proteinuric chronic kidney disease as described in the K/DOQI guidelines [K/DOQI, 2004]. ACE inhibitors and ARBs act by reducing the intraglomerular pressure and by directly improving the size-selective properties of the glomerular capillary wall, both of which contribute to reducing protein

Both observational studies [Cattran et al, 1994; Kanno et al, 2005] and small randomized trials [Maschio et al, 1994; Praga et al, 2003; Li PK et al, 2006] have provided suggestive evidence that ACE inhibitors or ARBs are more effective than other antihypertensive drugs in slowing the progressive decline in GFR in IgAN as they are in other forms of chronic proteinuric kidney disease. Praga et al in their randomized trial in 44 IgAN patients with proteinuria (≥0.5 g/day, mean 1.9 g/day) and a serum creatinine concentration ≤1.5 mg/dL at baseline, found a significant decrease in proteinuria in the enalapril group (1.9 g/day at baseline to 0.9 g/day at the last visit) and a significantly higher renal survival, defined as less than a 50 percent increase in the serum creatinine concentration, at 6 years of follow up [Praga et al, 2003]. More recently, Li et al in their double-blind randomized placebocontrolled HKVIN trial in 109 Chinese patients with protein excretion ≥1 g/day (mean ~2.0 g/day), found a better renal survival, defined as doubling of serum creatinine or ESRD, a significant improvement in proteinuria (33 % reduction in proteinuria) and a slower rate of decline in GFR (4.6 versus 6.9 mL/min per year) in the valsartan group compared to placebo [Li PK et al, 2006]. Similarly, the IgACE trial in 65 young patients with moderate proteinuria (between 1 and 3.5 grams/day per 1.73 m2) and creatinine clearance >50 mL/min per 1.73 m2 revealed a better renal survival (fewer patients with >30% decline in renal function) and significant improvement in proteinuria at 38 months of follow-up in the benazepril

Normotensive patients who excrete less than 500 mg of protein per day are not typically treated with angiotensin inhibition. However, because most patients progress slowly over time, monitoring of the serum creatinine and protein excretion at yearly intervals is recommended. Angiotensin inhibition should be started if there is evidence of progressive

The addition of an ARB to an ACE inhibitor in patients with IgAN seems to exert a further antiproteinuric effect [Russo et al, 1999, 2001], albeit there are no randomized trials showing that this regimen improves renal outcomes. This finding is consistent with metaanalyses of trials in different glomerular diseases, the largest of which found a significant 18 to 25% greater reduction in proteinuria with combined ACE inhibitors and ARBs compared to monotherapy [Kunz et al, 2008; Catapano et al, 2008]. The rationale for this combination therapy is the assumption that ARBs would counteract theAT1-mediated effect of residual angiotensin II formation by non-ACE enzymes like chymase, whereas ACE inhibitors would additionally increase the level of kinins. Furthermore, ACE inhibitors as well as ARBs would synergistically elevate the levels of angiotensin, which also might promote vasodilation. Finally, combining both drug classes might simply provide a higher degree of blockade of the classic renin-angiotensin system pathways [Alexopoulos, 2004]. However, any anticipated benefits from this combination should be weighted against possible adverse effects in individual patients; this is important especially given the findings from the ONTARGET trial in 25,620 patients with vascular

**3.2.1 Angiotensin inhibition** 

excretion [Remuzzi et al, 1991; Maschio et al, 1994].

group compared to the placebo group [Coppo, 2007].

disease and protein excretion above 500 mg/day.

The rationale for using fish oil (omega-3 fatty acids) in IgAN is based on the premise that they may limit the production or action of cytokines and eicosanoids evoked by the initial or by repeated immunologic renal injury, and the resulting production of mediators involved in renal damage [Donadio, 1991]. Randomized controlled trials evaluating fish oil in patients with IgAN have reported conflicting results [Donadio et al, 1994; Alexopoulos et al, 2004; Donadio et al, 1999, 2001; Bennett et al, 1989; Pettersson et al, 1994; Hogg et al, 2006; Ferraro et al, 2009]. In the largest and most well documented and conducted randomized trial in 106 patients with baseline creatinine clearance 80 mL/min and protein excretion of 2.5 to 3 g/day, Donadio et al found better patient and renal outcomes at 4 years, extended also at >6 years, in patients having received 12g of fish oil for 2 years, compared to patients having received a similar amount of olive oil [Donadio et al, 1994, 1999]. Similarly, in a controlled study of 14 IgAN patients and 14 controls, a low dose of fish oil (0.85 g eicosapentaenoic acid and 0.57 g phytohemaglutinin) was found effective in slowing renal progression in high-risk patients with IgAN and particularly those with advanced renal disease [Alexopoulos et al, 2004]. On the other hand, in the randomized controlled trial by the Southwest Pediatric Nephrology Study Group in 96 patients with IgAN, mean GFR >100 mL/min per 1.73 m2 and proteinuria 1.4 to 2.2 g/day, no significant benefit in the renal outcome was found in patients assigned to omega-3 fatty acids (4 g/day) for two years compared to the patients assigned to either alternate day prednisone or placebo [Hogg et al, 2006]. On the basis of the existing data, fish oil can be tried in addition to ACE inhibitors or ARBs in patients with protein excretion >500 to 1000 mg/day, a gradual reduction in GFR, and mild to moderate histologic lesions [Alexopoulos E, 2004].

#### **3.3 Immunosuppressive therapy**

The optimal role of immunosuppressive therapy in IgAN is uncertain [Barratt & Feehally, 2006; Appel & Waldman, 2006]. A variety of regimens have been used, mostly consisting of corticosteroids alone or with other immunosuppressive drugs. The available studies are not conclusive since most are relatively small and have limited follow-up, and the results are sometimes conflicting [D'Amico, 1992; Alamartine et al, 1991; Alexopoulos, 2004; Strippoli et al, 2003; Laville & Alamartine, 2004; Ballardie, 2004; Julian, 2000; Dillon, 2001]. There is

IgA Nephropathy: Insights into Genetic Basis and Treatment Options 83

et al, 1989]. Nephrotic syndrome can also occur with severe chronic IgAN and relatively advanced disease on renal biopsy. These patients do not seem to benefit from corticosteroid

Combined immunosuppressive therapy should only be attempted in patients with more severe active disease as defined by a more rapidly progressive clinical course and/or histologic evidence of severe active inflammation (eg, crescent formation). Early therapy is important because improvement is rare when the baseline serum creatinine concentration is greater than 3.0 mg/dL in the absence of crescentic glomerulonephritis [Alexopoulos E,

*Corticosteroids plus azathioprine* — Whether the addition of azathioprine provide any benefit to that of corticosteroids is still debatable. In a multicenter randomized trial by Pozzi et al in 207 patients with plasma creatinine ≤ 2.0 mg/dL and protein excretion >1.0 g/day, at a median follow-up of 4.9 years, there was no difference neither in renal survival time, defined as the time to 50% increase in plasma creatinine from baseline, nor in the decrease in proteinuria between patients who received corticosteroids alone or along with azathioprine (1.5 mg/kg /day for six months) [Pozzi et al, 2010]. However, in the above study, only a rather small percentage of patients were receiving either ACE inhibitors or ARBs, and even fewer patients were receiving statins. Most recently, Stangou et al published a randomized, yet underpowered study in 22 patients with IgAN and eGRF≥30mL/min, urine protein ≥1g/day, blood pressure <130/80mmHg, who failed to respond to previous treatment with renin-angiotensin system inhibitors and poly-unsaturated fatty acids administered for at least 6 months. During the 5th year after the diagnosis was made, the patients were randomized to receive either methylprednisolone alone, or methyl-prednisolone in combination with azathioprine, for 12months, while treatment with renin-angiotensin system inhibitors and poly-unsaturated fatty acids continued unchanged in both groups. Both, steroid treatment alone, or steroids in combination with azathioprine were found to be equally effective in reducing the severity of proteinuria and stabilizing renal function

*Corticosteroids plus cyclophosphamide*— There are evidence suggesting that patients with severe or progressive disease (eg, rising creatinine, nephrotic range proteinuria, and/or marked proliferation without crescents) who do not have significant chronic damage on kidney biopsy may benefit from combined immunosuppressive therapy with prednisone and cyclophosphamide. This regimen was evaluated in a study of Ballardie et al in 38 patients with IgAN and initially impaired renal function (baseline serum creatinine between 1.5 and 2.8 mg/dL, mean baseline protein excretion 4.0 to 4.5 g/day, no crescents on biopsy) that was declining at a relatively moderate rate (by at least 15% per year). Compared with the control group, the patients treated with combination therapy (prednisolone 40mg/day tapered to 10mg/day by two years plus low-dose cyclophosphamide for 3 months followed by low-dose azathioprine for at least 2 years) had a significant reduction in protein excretion during the first six months of therapy that persisted during follow-up (eg, reached 1.8 g/day in treatment group versus unchanged at 4.4 g/day in controls at one year). Renal survival was significantly higher in the treatment group at two (82 versus 68 percent) and five years (72 versus 6 percent). [Ballardie et al,

therapy alone [Mustonen et al, 1983; Lai KN et al, 1986].

**3.3.2 Combined immunosuppressive therapy** 

2004].

[Stangou et al, 2011].

2002].

rather consensus that mild, stable, or very slowly progressive IgAN should not be treated with corticosteroids or other immunosuppressive therapies, given the limited evidence of benefit and their known toxicity from chronic use [Floege & Eitner, 2005; Locatelli et al, 1999]. Corticosteroid or other immunosuppressive therapy should only be attempted in patients with clinical and histologic evidence of active inflammation (eg, hematuria and/or proliferative or necrotizing glomerular changes). Patients with chronic kidney disease with significant tubulointerstitial fibrosis and glomerulosclerosis are not likely to benefit from such therapy and are likely to be harmed from the side effects.

#### **3.3.1 Corticosteroids**

Current evidence regarding the potential benefit of corticosteroid therapy in IgAN are rather limited, as long as most of the studies performed are uncontrolled retrospective observations; moreover, the few available randomized controlled trials are rather small and in most of them the standard recommendations for proteinuria and blood pressure goals were not followed, limiting thus the applicability of the findings to current practice. Whatsoever, these studies point towards a beneficial effect of corticosteroid therapy (administered for 6 up to >24 months) in proteinuria and perhaps in renal survival [Floege & Eitner, 2005; Nolin & Courteau, 1999; Kobayashi Y et al, 1989, 1996; Pozzi et al, 1999, 2004; Tamoura et al, 2001; Katafuchi et al, 2003; Hotta et al, 2001; Moriyama et al, 2004; Lv et al, 2009, Manno et al, 2009], probably preferentially in individuals with preserved kidney function (eg, creatinine clearance above 50 mL/min) [Kobayashi et al, 1989, 1996; Pozzi et al, 1999, 2004]. Two randomised clinical trials demonstrated the benefit of the combination therapy with corticosteroids and ACE inhibitors on long-term follow-up in proteinuric IgAN patients. Lv et al (2009) evaluated the efficacy of the combination therapy versus ACEinhibitors alone in a small number of IgAN patients with mild or moderate histologic lesions and with a follow-up period that was too short to evaluate the renal survival. Data demonstrated that the combination therapy reduced better the urinary protein excretion than the administration of ACE-inhibitors alone.

Even more, Manno et al enrolled 97 IgAN patients with moderate histologic lesion (see IgAN classification of F.P. Schena in Manno et al., 2007) daily proteinuria more than 1.0g and estimated GFR more than 50 ml/min/1.73m2. Patients were randomly allocated to receive a 6-month course of oral prednisone plus ramipril or ramipril alone for the total duration of the follow-up (96 months). The combination of corticosteroids and ramipril provided less probability of renal disease progression because induced the decline of GFR and daily proteinuria. In an interesting recent meta-analysis of randomized and quasirandomized controlled trials of corticosteroid treatment in IgAN against treatment without steroids, Zhou et al found that steroid therapy, especially long-term, is associated with a significant benefit in proteinuria and renal survival [Zhou et al, 2011].

In addition, the IgAN patients seemingly to benefit from prednisone therapy are those with nephrotic syndrome, little or no hematuria, preserved kidney function, minimal glomerular changes on light microscopy, and diffuse fusion of glomerular epithelial cell foot processes on electron microscopy. These histologic findings are characteristic of minimal change disease, and these patients behave accordingly, frequently developing a remission with corticosteroids and occasionally requiring cytotoxics for frequently relapsing proteinuria [Mustonen et al, 1983; Lai KN et al, 1986; Cheng et al, 1989]. Mesangial IgA deposits in these patients often disappear or are greatly reduced following steroid-induced remission [Cheng et al, 1989]. Nephrotic syndrome can also occur with severe chronic IgAN and relatively advanced disease on renal biopsy. These patients do not seem to benefit from corticosteroid therapy alone [Mustonen et al, 1983; Lai KN et al, 1986].

#### **3.3.2 Combined immunosuppressive therapy**

82 An Update on Glomerulopathies – Clinical and Treatment Aspects

rather consensus that mild, stable, or very slowly progressive IgAN should not be treated with corticosteroids or other immunosuppressive therapies, given the limited evidence of benefit and their known toxicity from chronic use [Floege & Eitner, 2005; Locatelli et al, 1999]. Corticosteroid or other immunosuppressive therapy should only be attempted in patients with clinical and histologic evidence of active inflammation (eg, hematuria and/or proliferative or necrotizing glomerular changes). Patients with chronic kidney disease with significant tubulointerstitial fibrosis and glomerulosclerosis are not likely to benefit from

Current evidence regarding the potential benefit of corticosteroid therapy in IgAN are rather limited, as long as most of the studies performed are uncontrolled retrospective observations; moreover, the few available randomized controlled trials are rather small and in most of them the standard recommendations for proteinuria and blood pressure goals were not followed, limiting thus the applicability of the findings to current practice. Whatsoever, these studies point towards a beneficial effect of corticosteroid therapy (administered for 6 up to >24 months) in proteinuria and perhaps in renal survival [Floege & Eitner, 2005; Nolin & Courteau, 1999; Kobayashi Y et al, 1989, 1996; Pozzi et al, 1999, 2004; Tamoura et al, 2001; Katafuchi et al, 2003; Hotta et al, 2001; Moriyama et al, 2004; Lv et al, 2009, Manno et al, 2009], probably preferentially in individuals with preserved kidney function (eg, creatinine clearance above 50 mL/min) [Kobayashi et al, 1989, 1996; Pozzi et al, 1999, 2004]. Two randomised clinical trials demonstrated the benefit of the combination therapy with corticosteroids and ACE inhibitors on long-term follow-up in proteinuric IgAN patients. Lv et al (2009) evaluated the efficacy of the combination therapy versus ACEinhibitors alone in a small number of IgAN patients with mild or moderate histologic lesions and with a follow-up period that was too short to evaluate the renal survival. Data demonstrated that the combination therapy reduced better the urinary protein excretion

Even more, Manno et al enrolled 97 IgAN patients with moderate histologic lesion (see IgAN classification of F.P. Schena in Manno et al., 2007) daily proteinuria more than 1.0g and estimated GFR more than 50 ml/min/1.73m2. Patients were randomly allocated to receive a 6-month course of oral prednisone plus ramipril or ramipril alone for the total duration of the follow-up (96 months). The combination of corticosteroids and ramipril provided less probability of renal disease progression because induced the decline of GFR and daily proteinuria. In an interesting recent meta-analysis of randomized and quasirandomized controlled trials of corticosteroid treatment in IgAN against treatment without steroids, Zhou et al found that steroid therapy, especially long-term, is associated with a

In addition, the IgAN patients seemingly to benefit from prednisone therapy are those with nephrotic syndrome, little or no hematuria, preserved kidney function, minimal glomerular changes on light microscopy, and diffuse fusion of glomerular epithelial cell foot processes on electron microscopy. These histologic findings are characteristic of minimal change disease, and these patients behave accordingly, frequently developing a remission with corticosteroids and occasionally requiring cytotoxics for frequently relapsing proteinuria [Mustonen et al, 1983; Lai KN et al, 1986; Cheng et al, 1989]. Mesangial IgA deposits in these patients often disappear or are greatly reduced following steroid-induced remission [Cheng

such therapy and are likely to be harmed from the side effects.

than the administration of ACE-inhibitors alone.

significant benefit in proteinuria and renal survival [Zhou et al, 2011].

**3.3.1 Corticosteroids** 

Combined immunosuppressive therapy should only be attempted in patients with more severe active disease as defined by a more rapidly progressive clinical course and/or histologic evidence of severe active inflammation (eg, crescent formation). Early therapy is important because improvement is rare when the baseline serum creatinine concentration is greater than 3.0 mg/dL in the absence of crescentic glomerulonephritis [Alexopoulos E, 2004].

*Corticosteroids plus azathioprine* — Whether the addition of azathioprine provide any benefit to that of corticosteroids is still debatable. In a multicenter randomized trial by Pozzi et al in 207 patients with plasma creatinine ≤ 2.0 mg/dL and protein excretion >1.0 g/day, at a median follow-up of 4.9 years, there was no difference neither in renal survival time, defined as the time to 50% increase in plasma creatinine from baseline, nor in the decrease in proteinuria between patients who received corticosteroids alone or along with azathioprine (1.5 mg/kg /day for six months) [Pozzi et al, 2010]. However, in the above study, only a rather small percentage of patients were receiving either ACE inhibitors or ARBs, and even fewer patients were receiving statins. Most recently, Stangou et al published a randomized, yet underpowered study in 22 patients with IgAN and eGRF≥30mL/min, urine protein ≥1g/day, blood pressure <130/80mmHg, who failed to respond to previous treatment with renin-angiotensin system inhibitors and poly-unsaturated fatty acids administered for at least 6 months. During the 5th year after the diagnosis was made, the patients were randomized to receive either methylprednisolone alone, or methyl-prednisolone in combination with azathioprine, for 12months, while treatment with renin-angiotensin system inhibitors and poly-unsaturated fatty acids continued unchanged in both groups. Both, steroid treatment alone, or steroids in combination with azathioprine were found to be equally effective in reducing the severity of proteinuria and stabilizing renal function [Stangou et al, 2011].

*Corticosteroids plus cyclophosphamide*— There are evidence suggesting that patients with severe or progressive disease (eg, rising creatinine, nephrotic range proteinuria, and/or marked proliferation without crescents) who do not have significant chronic damage on kidney biopsy may benefit from combined immunosuppressive therapy with prednisone and cyclophosphamide. This regimen was evaluated in a study of Ballardie et al in 38 patients with IgAN and initially impaired renal function (baseline serum creatinine between 1.5 and 2.8 mg/dL, mean baseline protein excretion 4.0 to 4.5 g/day, no crescents on biopsy) that was declining at a relatively moderate rate (by at least 15% per year). Compared with the control group, the patients treated with combination therapy (prednisolone 40mg/day tapered to 10mg/day by two years plus low-dose cyclophosphamide for 3 months followed by low-dose azathioprine for at least 2 years) had a significant reduction in protein excretion during the first six months of therapy that persisted during follow-up (eg, reached 1.8 g/day in treatment group versus unchanged at 4.4 g/day in controls at one year). Renal survival was significantly higher in the treatment group at two (82 versus 68 percent) and five years (72 versus 6 percent). [Ballardie et al, 2002].

IgA Nephropathy: Insights into Genetic Basis and Treatment Options 85

tonsillectomy may be effective in inducing remission of proteinuria and hematuria in patients with IgAN (ie, proteinuria >500 mg/day). However, there are no randomized trials of tonsillectomy in IgAN, the design of the above studies precludes any definitive conclusions regarding the overall efficacy of tonsillectomy in IgAN, while other studies

*Low antigen diet* — The rationale for using a low antigen diet in IgAN, ie diet free of gluten, dairy products, eggs, and most meats, is that dietary macromolecules may be responsible for activating the mucosal IgA system. When given to 21 consecutive patients with IgAN, protein excretion fell markedly in all 12 patients whose baseline rate was more than 1g/day. In addition, repeat renal biopsy showed significant reductions in mesangial IgA and complement deposition and mesangial cellularity [Ferri et al, 1993]. However, the benefits in the above study have not been confirmed and a report using a gluten-free diet alone for several years was unable to document improvement in either proteinuria or renal function despite a reduction in the level of circulating IgA-containing immune complexes [Coppo et

*Intravenous immune globulin* — At least part of the rationale for intravenous immune globulin (IVIG) therapy in IgAN comes from the observation that a partial IgG deficiency, which could be corrected with IVIG, may predispose to infections that trigger flare-ups of the renal disease [Rostoker et al, 1989, 1994]. Despite the promising findings from two small studies with the administration of high-dose IVIG in severe IgAN, characterized by heavy proteinuria and a relatively rapid decline in GFR (reduction in protein excretion, prevention of GFR decline, decreased inflammatory activity and IgA deposition on repeat renal biopsy) [Rostoker et al, 1994; Rasche et al, 2006], these findings need to be confirmed by larger

Genetic susceptibility for IgAN exhibits a complex genetic pattern. To date various groups and the European IgAN Consortium have identified several loci. The extensive genetic studies under way with the use of delicate, high-throughput technologies will give information on the pathogenic mechanisms of IgAN. In addition, these data may indicate potential targets for screening, prevention and early diagnosis of the disease and more

 Patients with isolated hematuria, no or minimal proteinuria (<0.5g/day), and a normal GFR need no treatment. Such patients should be periodically monitored at 6 to 12

 Patients with persistent proteinuria (>0.5 or >1 g/day), should be treated with angiotensin inhibition (ACE inhibitor or ARB), with a target of a minimum reduction in protein excretion of at least 50 to 60% from baseline values and a goal protein excretion

All patients who meet criteria for angiotensin inhibition may also be considered as

 Patients with persistent nephrotic syndrome and/or chronic kidney disease who have dyslipidemia should be treated with a statin, primarily for cardiovascular protection.

Summarizing the most updated data, following are concise treatment guidelines:

month intervals to assess disease progression that might warrant therapy.

Corticosteroid therapy for at least 6 months is indicated in the following cases:

reported negative results [Rasche et al, 1999].

al, 1990].

studies.

**4. Conclusion** 

appropriate and effective treatment.

of <0.5 or <1 g/day.

candidates to receive fish oil.

Uncontrolled reports in patients with crescentic, rapidly progressive glomerulonephritis suggest possible benefit from regimens similar to those used in idiopathic crescentic glomerulonephritis: intravenous pulse methylprednisolone followed by oral prednisone, intravenous or oral cyclophosphamide, and/or plasmapheresis [Welch et al, 1988; Lai KN et al, 1987a; Rocatello et al, 1995; McIntyre et al, 2001; Tumlin et al, 2003]. Corticosteroids may act in this setting by diminishing acute inflammatory injury rather than by correcting the abnormality in IgA production [Galla, 1995]. In a study by Rocatello et al, although a substantial clinical improvement was found with the administration of aggressive combination therapy (including pulse methylprednisolone, oral cyclophosphamide, and plasmapheresis) for 2 months in six patients with crescentic glomerulonephritis due to IgAN [Rocatello et al, 1995], yet cellular crescents failed to remit in repeat renal biopsy, whereas the disease continued to progress in half of the patients after therapy was discontinued. Limited data for a more prolonged course of aggressive immunosuppressive therapy (pulse methylprednisolone 15mg/kg/day for 3 days, followed by oral prednisolone 1 mg/kg/ day for 60 days gradually tapered, along with monthly iv cyclophosphamide (0.5 g/m2) for 6 months) point towards a significant improvement in the serum creatinine and in protein excretion along with a significant reversion of cellular crescents and endocapillary proliferation [Tumlin et al, 2003].

These limited data suggest that patients with active crescentic glomerulonephritis who do not have significant chronic damage on kidney biopsy may benefit from therapy that initially includes intravenous cyclophosphamide. This is consistent with the benefit noted with a similar regimen in other forms of crescentic glomerulonephritis.

#### **3.3.3 Other immunosuppressive agents**

*Cyclosporine* — Cyclosporine has been investigated in small studies, and resulted in reduced proteinuria. In a recent study, Shin et al reported a significant benefit of cyclosporine therapy in proteinuria reduction and renal pathology regression in 14 children with IgAN and near normal creatinine clearance [Shin et al, 2010]. However, there are important issues of concern regarding its use in IgAN treatment, with most important the associated nephrotoxicity, which can lead to harmful effects on renal function [Lai KN et al, 1987b; Cattran, 1991], as well as the rapid disease relapse after drug discontinuation.

*Mycophenolate mofetil* — Small, prospective placebo-controlled randomized trials of mycophenolate mofetil (MMF) in which the patients were also treated with ACE inhibitors, have produced conflicting results, ranging from no benefit [Maes et al, 2004; Frisch et al, 2005] to a reduction in proteinuria and decrease in rate of decline of GFR [Tang et al, 2010]. A short course (< 6 months) of MMF in patients with persistent proteinuria (>1.5 g/day) and well-maintained renal function (serum creatinine <1.5 mg/dL) in addition to ACE inhibitor/ARB therapy may be considered in patients with well-preserved renal histology on biopsy. Current evidence does not support the use of MMF in patients with advanced disease (serum creatinine >2.5 to 3 mg/dL) [Cattran & Appel, 2011].

#### **3.4 Other possible interventions**

*Tonsillectomy* — Several retrospective studies have suggested that tonsillectomy, usually in combination with some immunosuppressive therapy, is associated with improved renal survival among patients with relatively mild renal injury [Hotta et al, 2001; Xie et al, 2003; Komatsu et al, 2008]. These non-randomized studies provide some evidence that tonsillectomy may be effective in inducing remission of proteinuria and hematuria in patients with IgAN (ie, proteinuria >500 mg/day). However, there are no randomized trials of tonsillectomy in IgAN, the design of the above studies precludes any definitive conclusions regarding the overall efficacy of tonsillectomy in IgAN, while other studies reported negative results [Rasche et al, 1999].

*Low antigen diet* — The rationale for using a low antigen diet in IgAN, ie diet free of gluten, dairy products, eggs, and most meats, is that dietary macromolecules may be responsible for activating the mucosal IgA system. When given to 21 consecutive patients with IgAN, protein excretion fell markedly in all 12 patients whose baseline rate was more than 1g/day. In addition, repeat renal biopsy showed significant reductions in mesangial IgA and complement deposition and mesangial cellularity [Ferri et al, 1993]. However, the benefits in the above study have not been confirmed and a report using a gluten-free diet alone for several years was unable to document improvement in either proteinuria or renal function despite a reduction in the level of circulating IgA-containing immune complexes [Coppo et al, 1990].

*Intravenous immune globulin* — At least part of the rationale for intravenous immune globulin (IVIG) therapy in IgAN comes from the observation that a partial IgG deficiency, which could be corrected with IVIG, may predispose to infections that trigger flare-ups of the renal disease [Rostoker et al, 1989, 1994]. Despite the promising findings from two small studies with the administration of high-dose IVIG in severe IgAN, characterized by heavy proteinuria and a relatively rapid decline in GFR (reduction in protein excretion, prevention of GFR decline, decreased inflammatory activity and IgA deposition on repeat renal biopsy) [Rostoker et al, 1994; Rasche et al, 2006], these findings need to be confirmed by larger studies.

### **4. Conclusion**

84 An Update on Glomerulopathies – Clinical and Treatment Aspects

Uncontrolled reports in patients with crescentic, rapidly progressive glomerulonephritis suggest possible benefit from regimens similar to those used in idiopathic crescentic glomerulonephritis: intravenous pulse methylprednisolone followed by oral prednisone, intravenous or oral cyclophosphamide, and/or plasmapheresis [Welch et al, 1988; Lai KN et al, 1987a; Rocatello et al, 1995; McIntyre et al, 2001; Tumlin et al, 2003]. Corticosteroids may act in this setting by diminishing acute inflammatory injury rather than by correcting the abnormality in IgA production [Galla, 1995]. In a study by Rocatello et al, although a substantial clinical improvement was found with the administration of aggressive combination therapy (including pulse methylprednisolone, oral cyclophosphamide, and plasmapheresis) for 2 months in six patients with crescentic glomerulonephritis due to IgAN [Rocatello et al, 1995], yet cellular crescents failed to remit in repeat renal biopsy, whereas the disease continued to progress in half of the patients after therapy was discontinued. Limited data for a more prolonged course of aggressive immunosuppressive therapy (pulse methylprednisolone 15mg/kg/day for 3 days, followed by oral prednisolone 1 mg/kg/ day for 60 days gradually tapered, along with monthly iv cyclophosphamide (0.5 g/m2) for 6 months) point towards a significant improvement in the serum creatinine and in protein excretion along with a significant reversion of cellular crescents and endocapillary

These limited data suggest that patients with active crescentic glomerulonephritis who do not have significant chronic damage on kidney biopsy may benefit from therapy that initially includes intravenous cyclophosphamide. This is consistent with the benefit noted

*Cyclosporine* — Cyclosporine has been investigated in small studies, and resulted in reduced proteinuria. In a recent study, Shin et al reported a significant benefit of cyclosporine therapy in proteinuria reduction and renal pathology regression in 14 children with IgAN and near normal creatinine clearance [Shin et al, 2010]. However, there are important issues of concern regarding its use in IgAN treatment, with most important the associated nephrotoxicity, which can lead to harmful effects on renal function [Lai KN et al, 1987b;

*Mycophenolate mofetil* — Small, prospective placebo-controlled randomized trials of mycophenolate mofetil (MMF) in which the patients were also treated with ACE inhibitors, have produced conflicting results, ranging from no benefit [Maes et al, 2004; Frisch et al, 2005] to a reduction in proteinuria and decrease in rate of decline of GFR [Tang et al, 2010]. A short course (< 6 months) of MMF in patients with persistent proteinuria (>1.5 g/day) and well-maintained renal function (serum creatinine <1.5 mg/dL) in addition to ACE inhibitor/ARB therapy may be considered in patients with well-preserved renal histology on biopsy. Current evidence does not support the use of MMF in patients with advanced

*Tonsillectomy* — Several retrospective studies have suggested that tonsillectomy, usually in combination with some immunosuppressive therapy, is associated with improved renal survival among patients with relatively mild renal injury [Hotta et al, 2001; Xie et al, 2003; Komatsu et al, 2008]. These non-randomized studies provide some evidence that

with a similar regimen in other forms of crescentic glomerulonephritis.

Cattran, 1991], as well as the rapid disease relapse after drug discontinuation.

disease (serum creatinine >2.5 to 3 mg/dL) [Cattran & Appel, 2011].

proliferation [Tumlin et al, 2003].

**3.3.3 Other immunosuppressive agents** 

**3.4 Other possible interventions** 

Genetic susceptibility for IgAN exhibits a complex genetic pattern. To date various groups and the European IgAN Consortium have identified several loci. The extensive genetic studies under way with the use of delicate, high-throughput technologies will give information on the pathogenic mechanisms of IgAN. In addition, these data may indicate potential targets for screening, prevention and early diagnosis of the disease and more appropriate and effective treatment.

Summarizing the most updated data, following are concise treatment guidelines:


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Reich HN, Troyanov S, Scholey JW, et al (2007). Remission of proteinuria improves prognosis in IgA nephropathy. J Am Soc Nephrol, 18, 12, 3177-3183. Rekola S, Bergstrand A, Bucht H (1991). Deterioration of GFR in IgA nephropathy as

Remuzzi A, Perticucci E, Ruggenenti P, et al (1991). Angiotensin converting enzyme

Rigat B,Hubert C, Alhenc-Gelas F, et al (1990). An insertion/deletion polymorphism in the

Roccatello D, Ferro M, Coppo R, et al (1995). Report on intensive treatment of extracapillary

Rostoker G, Pech MA, Del Prato S, et al (1989). Serum IgG subclasses and IgM imbalances in

Rostoker G, Desvaux-Belghiti D, Pilatte Y, et al (1994). High-dose immunoglobulin therapy

Russo D, Pisani A, Balletta MM, et al (1999). Additive antiproteinuric effect of converting

Russo D, Minutolo R, Pisani A, et al (2001). Coadministration of losartan and enalapril

Schena F.P. IgA nephropathies, In: *Oxford Textbook of Clinical Nephrology,* A.M. Davison, J. S.

Oxford University Press, 2nd ed., 1998, New York, U.S.A.

measured by 51Cr-EDTA clearance. *Kidney Int*, 40, 6, 1050-1054.

in IgA nephropathy. *Clin Nephrol*, 51, 3, 147-152.

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enzyme levels. *J Clin Invest*, 86, 4,1343–1346.

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angiotensin I-converting enzyme gene accounting for half the variance of serum

glomerulonephritis with focus on crescentic IgA nephropathy. *Nephrol Dial* 

adult IgA mesangial glomerulonephritis and idiopathic Henoch-Schoenlein

for severe IgA nephropathy and Henoch-Schönlein purpura. *Ann Intern Med*, 120, 6,

enzyme inhibitor and losartan in normotensive patients with IgA nephropathy. *Am* 

exerts additive antiproteinuric effect in IgA nephropathy. *Am J Kidney Dis*, 38, 1, 18-

Cameron, J.-R. Gruenfeld, D.N.S. Kerr, E. Ritz, C. G. Winearls (Ed.), 537-570,


**6** 

*Italy* 

Maria Pia Rastaldi *Renal Research Laboratory,* 

**Rapidly Progressive Glomerulonephritis** 

*Policlinico & Fondazione D'Amico per la Ricerca sulle Malattie Renali Milano,* 

Rapidly Progressive Glomerulonephritis are a group of renal diseases which are still posing serious threat to human health and survival. They are all characterised by acute and rapid deterioration of renal function. Renal biopsy reveals extracapillary glomerulonephritis, most frequently circumferential and diffuse, and immunofluorescence findings continue to represent the most important clue toward precise diagnosis. In the last years, with the development of new technologies and more targeted animal models, several discoveries have been made, that can help in better understanding the pathogenesis and perspectively defining new molecular targets for novel therapies, which are still required to improve the

Introduced for the first time by Ellis in 1942 (Ellis, 1942), the term Rapidly Progressive Glomerulonephritis (RPGN) clinically describes a heterogeneous group of glomerulonephritis characterised by worsening of kidney function that, if not adequately and timely treated, rapidly progresses to end stage renal disease. From the pathological point of view, these diseases are classified as extracapillary or crescentic glomerulonephritis, generally showing extracapillary proliferation in more than 50% of glomeruli. Besides renal biopsy, which is mandatory to make the diagnosis and guide therapeutic decisions, clinical symptoms, biochemical exams, and the observation of the urinary sediment are relevant to

Observation of the urinary sediment in the acute phase of disease allows to detect in the vast majority of cases marked erythrocytic cylindruria, mild to moderate leukocyturia, presence of tubular epithelial cells and tubular epithelial cell casts. Fatty casts and leukocyte casts can be detected in about one third of cases (Fogazzi, 2009). Progressive disappearance of these features follows successful therapeutic intervention, and their reappearance frequently precedes disease relapses, making the urinary sediment an important exam not only at

Despite the amelioration of prognosis obtained with introduction of high doses of steroids, immunosuppressive agents, and plasma exchange, these diseases are still life-threatening

**1. Introduction** 

prognosis of these patients.

the diagnostic process.

diagnosis but also during the patient's follow-up.

and a high percentage of subjects have a poor renal outcome.

**2. Definition** 

*Fondazione IRCCS Ca' Granda Ospedale Maggiore* 


## **Rapidly Progressive Glomerulonephritis**

### Maria Pia Rastaldi

*Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico & Fondazione D'Amico per la Ricerca sulle Malattie Renali Milano, Italy* 

### **1. Introduction**

94 An Update on Glomerulopathies – Clinical and Treatment Aspects

Working Group of the International IgA Nephropathy Network and the Renal Pathology

Xie Y, Nishi S, Ueno M, et al (2003). The efficacy of tonsillectomy on long-term renal survival in patients with IgA nephropathy. *Kidney Int*, 63, 5, 1861-1867. Zhou YH, Tang LG, Guo SL, et al (2011). Steroids in the treatment of IgA nephropathy to the

77, 10, 921-927.

4, e18788.

Society, Coppo R, Troyanov S, et al (2010). The Oxford IgA nephropathy clinicopathological classification is valid for children as well as adults. *Kidney Int*,

improvement of renal survival: a systematic review and meta-analysis. *PLoS One*, 6,

Rapidly Progressive Glomerulonephritis are a group of renal diseases which are still posing serious threat to human health and survival. They are all characterised by acute and rapid deterioration of renal function. Renal biopsy reveals extracapillary glomerulonephritis, most frequently circumferential and diffuse, and immunofluorescence findings continue to represent the most important clue toward precise diagnosis. In the last years, with the development of new technologies and more targeted animal models, several discoveries have been made, that can help in better understanding the pathogenesis and perspectively defining new molecular targets for novel therapies, which are still required to improve the prognosis of these patients.

### **2. Definition**

Introduced for the first time by Ellis in 1942 (Ellis, 1942), the term Rapidly Progressive Glomerulonephritis (RPGN) clinically describes a heterogeneous group of glomerulonephritis characterised by worsening of kidney function that, if not adequately and timely treated, rapidly progresses to end stage renal disease. From the pathological point of view, these diseases are classified as extracapillary or crescentic glomerulonephritis, generally showing extracapillary proliferation in more than 50% of glomeruli. Besides renal biopsy, which is mandatory to make the diagnosis and guide therapeutic decisions, clinical symptoms, biochemical exams, and the observation of the urinary sediment are relevant to the diagnostic process.

Observation of the urinary sediment in the acute phase of disease allows to detect in the vast majority of cases marked erythrocytic cylindruria, mild to moderate leukocyturia, presence of tubular epithelial cells and tubular epithelial cell casts. Fatty casts and leukocyte casts can be detected in about one third of cases (Fogazzi, 2009). Progressive disappearance of these features follows successful therapeutic intervention, and their reappearance frequently precedes disease relapses, making the urinary sediment an important exam not only at diagnosis but also during the patient's follow-up.

Despite the amelioration of prognosis obtained with introduction of high doses of steroids, immunosuppressive agents, and plasma exchange, these diseases are still life-threatening and a high percentage of subjects have a poor renal outcome.

Rapidly Progressive Glomerulonephritis 97

RPGN can also complicate any primary form of GN, most frequently MPGN and IgA nephropathy. A part from the immunofluorescence findings, diagnosis is also guided by clinical and biochemical exams, such as the evaluation of complement components for the diagnosis of MPGN, and the presence of purpura in cases of Henoch-Schoenlein syndrome. It remains to be said, as a word of caution, that it is not infrequent to find a completely negative immunofluorescence or immunofluorescence findings particularly difficult to interpret in cases with very severe extracapillary proliferation or necrotic lesions, because of

Though the common element characterising this group of diseases is the presence of extracapillary proliferation, morphological findings can be very diverse, according to the stage of the disease and also to the underlying disease, likely reflecting the different

By light microscopy, variable degrees of necrotising extracapillary lesions can be observed,

Fig. 1. Anti-GBM nephritis. A large area of necrosis of the glomerular tuft is surrounded by

Extracapillary lesions are composed by monocytes, epithelioid macrophages and epithelial cells. Glomeruli not involved by these lesions and the parts of the glomerulus not affected by necrosis can present normal features, but more commonly they show mild to moderate mesangial proliferation, some degree of mesangial matrix expansion, and increased leukocyte infiltration. Intraglomerular inflammatory cells are mostly monocyte-

In about 50% of cases, multinucleated giant cells can be detected either in the crescent or in the periglomerular inflammatory infiltrate, forming the so-called granuloma-like lesions.

macrophages with variable numbers of T-lymphocytes (Ferrario, 1985; Bolton, 1987).

a circumferential crescent. Inflammatory cells surround the glomerulus.

the consequent compression or destruction of the glomerular tuft.

**4.1 Anti-GBM nephritis and Goodpasture's Syndrome** 

which range from focal and segmental to global and diffuse (Fig 1).

**4. Morphological findings** 

pathogenesis of glomerular lesions.

### **3. Classification**

Along the years, different schemes for classifying RPGN have been proposed. Among them, the classification which is still largely accepted and mostly utilised was proposed by Couser (Couser, 1988), and defines disease groups on the basis of immunofluorescent findings. Clinical features and haematological exams are as well very important in reaching a precise diagnosis (Table 1).


Table 1. Classification of RPGN according to immunofluorescence findings

Linear deposition of IgG along the glomerular basement membrane associated to circulating anti-GBM antibodies allow the diagnosis of anti-GBM disease. If pulmonary haemorrhage is present, the diagnosis becomes of Goodpasture's Syndrome.

When immunofluorescence on renal biopsy material demonstrates absence of immune deposits or scanty immune deposition in the glomerulus, in association to the presence of circulating ANCA antibodies, a diagnosis of pauci-immune ANCA-associated renal vasculitis is made. In these cases, necrotising crescentic GN can be associated to clinical symptoms of systemic vasculitis. A prevalent involvement of the upper respiratory tract is highly suggestive for a diagnosis of Wegener's Granulomatosis, whereas the presence of only general systemic symptoms, such as fever, is highly suggestive of Renal Limited Vasculitis. Rarely presenting as RPGN, Churg-Strauss syndrome is diagnosed when asthma and increased circulating eosinophils are present.

Importantly, there is a percentage (10-30%, according to the literature) (Chen, 2009) of renal vasculitis which are negative for ANCA antibodies. A part from subjects with circulating AECA (anti-endothelial cell antibodies), that according to a recent study may be present in about 50% of cases (Cong, 2008), diagnosis is mainly based on clinical and biopsy findings and exclusion of other causes.

Among cases of RPGN with granular immune deposition in glomeruli, the most frequent are Post-Streptococcal/post-infectious nephritis and extracapillary GN observed in cases of Lupus Nephritis. In these diseases, besides clinical symptoms, diagnosis is made thanks to the presence of autoantibodies (ASLO and anti-DNAseB in PSGN, and ANA in SLE).

Along the years, different schemes for classifying RPGN have been proposed. Among them, the classification which is still largely accepted and mostly utilised was proposed by Couser (Couser, 1988), and defines disease groups on the basis of immunofluorescent findings. Clinical features and haematological exams are as well very important in reaching a precise

Light microscopy Necrotising extracapillary or pure extracapillary glomerulonephritis

Clinical features Lung haemorrhage Systemic symptoms Systemic symptoms

Linear deposition of IgG along the glomerular basement membrane associated to circulating anti-GBM antibodies allow the diagnosis of anti-GBM disease. If pulmonary haemorrhage is

When immunofluorescence on renal biopsy material demonstrates absence of immune deposits or scanty immune deposition in the glomerulus, in association to the presence of circulating ANCA antibodies, a diagnosis of pauci-immune ANCA-associated renal vasculitis is made. In these cases, necrotising crescentic GN can be associated to clinical symptoms of systemic vasculitis. A prevalent involvement of the upper respiratory tract is highly suggestive for a diagnosis of Wegener's Granulomatosis, whereas the presence of only general systemic symptoms, such as fever, is highly suggestive of Renal Limited Vasculitis. Rarely presenting as RPGN, Churg-Strauss syndrome is diagnosed when asthma

Importantly, there is a percentage (10-30%, according to the literature) (Chen, 2009) of renal vasculitis which are negative for ANCA antibodies. A part from subjects with circulating AECA (anti-endothelial cell antibodies), that according to a recent study may be present in about 50% of cases (Cong, 2008), diagnosis is mainly based on clinical and biopsy findings

Among cases of RPGN with granular immune deposition in glomeruli, the most frequent are Post-Streptococcal/post-infectious nephritis and extracapillary GN observed in cases of Lupus Nephritis. In these diseases, besides clinical symptoms, diagnosis is made thanks to

the presence of autoantibodies (ASLO and anti-DNAseB in PSGN, and ANA in SLE).

Wegener's granulomatosis Microscopic polyangiitis Renal limited vasculitis Churg-Strauss syndrome

deposits Granular deposits

Autoantibodies Complement components

Post-streptococcal GN Post-infectious GN SLE nephritis IgA GN/HS syndrome Primary MPGN Other primary GN

fluorescence Linear IgG staining None or minimal

GBM antibodies ANCA

Table 1. Classification of RPGN according to immunofluorescence findings

Circulating anti-

Anti-GBM disease Goodpasture's syndrome

present, the diagnosis becomes of Goodpasture's Syndrome.

and increased circulating eosinophils are present.

and exclusion of other causes.

**3. Classification** 

diagnosis (Table 1).

Immuno-

Hemato-chemical exams

Diagnosis

RPGN can also complicate any primary form of GN, most frequently MPGN and IgA nephropathy. A part from the immunofluorescence findings, diagnosis is also guided by clinical and biochemical exams, such as the evaluation of complement components for the diagnosis of MPGN, and the presence of purpura in cases of Henoch-Schoenlein syndrome. It remains to be said, as a word of caution, that it is not infrequent to find a completely negative immunofluorescence or immunofluorescence findings particularly difficult to interpret in cases with very severe extracapillary proliferation or necrotic lesions, because of the consequent compression or destruction of the glomerular tuft.

### **4. Morphological findings**

Though the common element characterising this group of diseases is the presence of extracapillary proliferation, morphological findings can be very diverse, according to the stage of the disease and also to the underlying disease, likely reflecting the different pathogenesis of glomerular lesions.

### **4.1 Anti-GBM nephritis and Goodpasture's Syndrome**

By light microscopy, variable degrees of necrotising extracapillary lesions can be observed, which range from focal and segmental to global and diffuse (Fig 1).

Fig. 1. Anti-GBM nephritis. A large area of necrosis of the glomerular tuft is surrounded by a circumferential crescent. Inflammatory cells surround the glomerulus.

Extracapillary lesions are composed by monocytes, epithelioid macrophages and epithelial cells. Glomeruli not involved by these lesions and the parts of the glomerulus not affected by necrosis can present normal features, but more commonly they show mild to moderate mesangial proliferation, some degree of mesangial matrix expansion, and increased leukocyte infiltration. Intraglomerular inflammatory cells are mostly monocytemacrophages with variable numbers of T-lymphocytes (Ferrario, 1985; Bolton, 1987). In about 50% of cases, multinucleated giant cells can be detected either in the crescent or in the periglomerular inflammatory infiltrate, forming the so-called granuloma-like lesions.

Rapidly Progressive Glomerulonephritis 99

Extracapillary and granuloma-like lesions are mainly made by inflammatory cells, mostly acutely activated monocyte-macrophages (Fig. 3, Fig 4) (Rastaldi, 1996; Rastaldi, 2000), whose entrance into the glomerulus seems to be facilitated by the de novo expression of the

Fig. 3. ANCA-associated vasculitis. Glomerular damage and periglomerular granuloma-like reaction are mainly composed by monocyte-macrophages, as witnessed by the positivity for

Fig. 4. ANCA-associated vasculitis. A vast glomerular granuloma-like reaction is strongly

positive for the marker of acutely activated monocyte-macrophages 27E10.

adhesion molecule VCAM-1 (Fig 5).

the marker CD68.

Generally well corresponding to the degree of glomerular damage, the tubulointerstitium shows variable extents of tubular atrophy, oedema, and interstitial inflammation. If the biopsy is timely performed, no interstitial fibrosis is observed.

Vascular lesions are not common, though necrotising arteritis and thrombotic microangiopathy have been reported occasionally in the literature (Dean, 1991; Stave, 1984).

Immunofluorescence is diagnostic, with the linear deposition of IgG along the glomerular basement membrane. This aspect can be best appreciated in glomeruli not particularly damaged, whereas it is more difficult to be seen when the glomerular capillary is largely destroyed by necrosis or compressed by extensive cellular crescents.

A combination of IgG and C3 can also be found, as well as a linear deposition of IgA or IgM (Gris, 1991; Peto, 2011) has been reported.

Linear IgG staining can be also detected along the Bowman's capsule, and along the tubular basement membranes. Additionally, the fibrinogen antiserum strongly stains the areas of necrosis in the tuft and within the crescents.

#### **4.2 ANCA-associated renal vasculitis**

Irrespective of diagnosis, identical renal microscopy features can be observed in Wegener's granulomatosis, microscopic polyangiitis, and renal limited vasculitis. Necrotising glomerulonephritis and extracapillary proliferation are the renal hallmark of these diseases, and can be found with variable degrees of association. Necrosis can be present alone in cases when renal biopsy is early performed, but more commonly is associated with segmental areas of extracapillary proliferation. Particularly compromised glomeruli show instead large areas of necrosis of the tuft and circumferential crescents, with frequent rupture of the Bowman's capsule and intense periglomerular leukocyte infiltration, so that the limit of the glomerular area is no more distinguishable (Fig 2), and the area has the aspect of a granulomatous reaction.

Fig. 2. ANCA-associated vasculitis. A large rupture of the Bowman's capsule can be observed.

Generally well corresponding to the degree of glomerular damage, the tubulointerstitium shows variable extents of tubular atrophy, oedema, and interstitial inflammation. If the

Vascular lesions are not common, though necrotising arteritis and thrombotic microangiopathy have been reported occasionally in the literature (Dean, 1991; Stave, 1984). Immunofluorescence is diagnostic, with the linear deposition of IgG along the glomerular basement membrane. This aspect can be best appreciated in glomeruli not particularly damaged, whereas it is more difficult to be seen when the glomerular capillary is largely

A combination of IgG and C3 can also be found, as well as a linear deposition of IgA or IgM

Linear IgG staining can be also detected along the Bowman's capsule, and along the tubular basement membranes. Additionally, the fibrinogen antiserum strongly stains the areas of

Irrespective of diagnosis, identical renal microscopy features can be observed in Wegener's granulomatosis, microscopic polyangiitis, and renal limited vasculitis. Necrotising glomerulonephritis and extracapillary proliferation are the renal hallmark of these diseases, and can be found with variable degrees of association. Necrosis can be present alone in cases when renal biopsy is early performed, but more commonly is associated with segmental areas of extracapillary proliferation. Particularly compromised glomeruli show instead large areas of necrosis of the tuft and circumferential crescents, with frequent rupture of the Bowman's capsule and intense periglomerular leukocyte infiltration, so that the limit of the glomerular area is no more distinguishable (Fig 2), and the area has the aspect of a

Fig. 2. ANCA-associated vasculitis. A large rupture of the Bowman's capsule can be

biopsy is timely performed, no interstitial fibrosis is observed.

destroyed by necrosis or compressed by extensive cellular crescents.

(Gris, 1991; Peto, 2011) has been reported.

necrosis in the tuft and within the crescents.

**4.2 ANCA-associated renal vasculitis** 

granulomatous reaction.

observed.

Extracapillary and granuloma-like lesions are mainly made by inflammatory cells, mostly acutely activated monocyte-macrophages (Fig. 3, Fig 4) (Rastaldi, 1996; Rastaldi, 2000), whose entrance into the glomerulus seems to be facilitated by the de novo expression of the adhesion molecule VCAM-1 (Fig 5).

Fig. 3. ANCA-associated vasculitis. Glomerular damage and periglomerular granuloma-like reaction are mainly composed by monocyte-macrophages, as witnessed by the positivity for the marker CD68.

Fig. 4. ANCA-associated vasculitis. A vast glomerular granuloma-like reaction is strongly positive for the marker of acutely activated monocyte-macrophages 27E10.

Rapidly Progressive Glomerulonephritis 101

Depending on the timing of renal biopsy, glomeruli can be affected by active lesions or by more sclerotic alterations. It is not infrequent to observe both types of lesions in the same

Fig. 7. ANCA-associated vasculitis. The glomerulus shows evident necrotic damage in the

Besides periglomerular infiltrates, focal perivascular inflammatory cells are frequently detected in the interstitium, and a diffuse interstitial leukocyte infiltration is also present, whose degree well corresponds to the extent of glomerular damage. Interstitial cells are

Prevalence of eosinophils, in association to the clinical symptoms of asthma, and increased numbers of circulating eosinophils, stand for a diagnosis of Churg-Strauss

By definition, in ANCA-associated renal vasculitis immune deposits are absent or few and scattered, hence the term pauci-immune glomerulonephritis. Instead, the fibrinogen antiserum strongly stains areas of necrosis of the tuft and fibrin deposits into the

Either post-streptococcal and other post-infectious glomerulonephritis can present with a

Several systemic infections, especially occult, such as infective endocarditis, infected atrioventricular shunts, visceral abscesses, and infected vascular prostheses, can be at the origin

By light microscopy necrotising lesions, but more frequently extracapillary damage without

Especially in case of streptococcal infections, the presence of numerous intraglomerular granulocytes (so-called glomerular exudative lesions) (Fig 8) is useful for diagnostic

upper part of the crescent, whereas the lower part is already fibrous.

rapidly progressive course, which is indicative of a poor prognosis.

of RPGN. Blood levels of complement can be reduced.

mainly monocyte-macrophages and T-lymphocytes.

**4.3 Post-infectious glomerulonephritis** 

necrosis of the tuft are observed.

syndrome.

crescents.

purposes.

renal biopsy and even in the same glomerulus (Fig 7).

The acute activation of cells composing the glomerular granuloma-like reaction differentiates this type of alteration from other kind of tissue granulomas, where acute macrophages have not been found (Bhardwaj, 1992).

Differently from those observed in other diseases, monocyte-macrophages present in renal vasculitis are proliferating cells, as we have shown by staining with antibodies against PCNA (Fig 6) and Ki67 (Rastaldi, 2000).

Fig. 5. ANCA-associated vasculitis. VCAM-1 de novo expression in damaged areas of the glomerulus.

Fig. 6. ANCA-associated vasculitis. PCNA labels numerous cells in and around the glomerulus.

The acute activation of cells composing the glomerular granuloma-like reaction differentiates this type of alteration from other kind of tissue granulomas, where acute

Differently from those observed in other diseases, monocyte-macrophages present in renal vasculitis are proliferating cells, as we have shown by staining with antibodies against

Fig. 5. ANCA-associated vasculitis. VCAM-1 de novo expression in damaged areas of the

Fig. 6. ANCA-associated vasculitis. PCNA labels numerous cells in and around the

macrophages have not been found (Bhardwaj, 1992).

PCNA (Fig 6) and Ki67 (Rastaldi, 2000).

glomerulus.

glomerulus.

Depending on the timing of renal biopsy, glomeruli can be affected by active lesions or by more sclerotic alterations. It is not infrequent to observe both types of lesions in the same renal biopsy and even in the same glomerulus (Fig 7).

Fig. 7. ANCA-associated vasculitis. The glomerulus shows evident necrotic damage in the upper part of the crescent, whereas the lower part is already fibrous.

Besides periglomerular infiltrates, focal perivascular inflammatory cells are frequently detected in the interstitium, and a diffuse interstitial leukocyte infiltration is also present, whose degree well corresponds to the extent of glomerular damage. Interstitial cells are mainly monocyte-macrophages and T-lymphocytes.

Prevalence of eosinophils, in association to the clinical symptoms of asthma, and increased numbers of circulating eosinophils, stand for a diagnosis of Churg-Strauss syndrome.

By definition, in ANCA-associated renal vasculitis immune deposits are absent or few and scattered, hence the term pauci-immune glomerulonephritis. Instead, the fibrinogen antiserum strongly stains areas of necrosis of the tuft and fibrin deposits into the crescents.

### **4.3 Post-infectious glomerulonephritis**

Either post-streptococcal and other post-infectious glomerulonephritis can present with a rapidly progressive course, which is indicative of a poor prognosis.

Several systemic infections, especially occult, such as infective endocarditis, infected atrioventricular shunts, visceral abscesses, and infected vascular prostheses, can be at the origin of RPGN. Blood levels of complement can be reduced.

By light microscopy necrotising lesions, but more frequently extracapillary damage without necrosis of the tuft are observed.

Especially in case of streptococcal infections, the presence of numerous intraglomerular granulocytes (so-called glomerular exudative lesions) (Fig 8) is useful for diagnostic purposes.

Rapidly Progressive Glomerulonephritis 103

Fig. 9. Primary IgA nephropathy. A circumferential crescent surrounds a glomerulus

Among the histological classes of SLE nephritis (Weening, 2004), RPGN is more frequently observed in classes III and IV. In these cases the occurrence of antineutrophil cytoplasmic antibodies is not uncommon and is thought to contribute to the development of necrotising

Extensive extracapillary proliferation has been rarely reported (Fig 10), whereas segmental necrotising extracapillary alterations are a rather common finding, but not always translate

Fig. 10. Rapidly progressive class IV SLE nephritis. A circumferential crescent surrounds a glomerulus affected by intense intracapillary proliferation, mesangial expansion, and

affected by mesangial proliferation and mesangial expansion.

**4.4.2 Systemic lupus erythematosus** 

in a RPGN clinical phenotype.

leukocyte infiltration.

and crescentic glomerular lesions (Sen, 2003).

In post-streptococcal GN, granular IgG and C3 deposits are the most common finding. IgG, C3, and IgM deposits can be observed in other post-infectious GN, at various locations, but primarily subendothelial and mesangial.

Fig. 8. Post-streptococcal GN. Numerous granulocytes can be observed in the glomerular tuft and in the crescent.

### **4.4 RPGN complicating primary and secondary glomerular diseases**

Though rarely, any primary or secondary glomerular diseases can be complicated by a rapidly progressive course and display necrotising crescentic glomerulonephritis at light microscopy. Very recently, a report has shown for the first time the appearance of RPGN complicating the course of AL amyloidosis (Crosthwaite, 2010). Cases of association of primary or secondary glomerulonephritis and anti-GBM disease or renal vasculitis have also been published, as well as cases of association of anti-GBM disease and ANCApositive renal vasculitis (Curioni, 2002; O'Connor, 2010). Diagnosis in these patients requires skilful and careful analysis of clinical features, renal biopsy findings, and hematochemical exams.

### **4.4.1 IgA nephropathy and Henoch-Schonlein purpura**

Less than 10% of patients with primary IgA nephropathy or Henoch-Schonlein syndrome have been reported with a truly rapidly progressive course (Ferrario, 1997).

Clinical features of cutaneous purpura or abdominal and joint pain, accompanied by the finding of a small vessel leukocytoclastic vasculitis, most frequently detected in skin biopsies, help in making a diagnosis of systemic disease.

Focal segmental or global and diffuse necrotising and extracapillary lesions of the glomerulus can be found, or extracapillary lesions can be present without necrosis of the glomerular tuft (Fig 9), which always presents variable degrees of mesangial proliferation and expansion of the mesangial matrix.

Immunofluorescence shows prevailing IgA mesangial deposits, possibly in combination with IgG and C3 deposition, especially in the systemic disease.

In post-streptococcal GN, granular IgG and C3 deposits are the most common finding. IgG, C3, and IgM deposits can be observed in other post-infectious GN, at various locations, but

Fig. 8. Post-streptococcal GN. Numerous granulocytes can be observed in the glomerular

Though rarely, any primary or secondary glomerular diseases can be complicated by a rapidly progressive course and display necrotising crescentic glomerulonephritis at light microscopy. Very recently, a report has shown for the first time the appearance of RPGN complicating the course of AL amyloidosis (Crosthwaite, 2010). Cases of association of primary or secondary glomerulonephritis and anti-GBM disease or renal vasculitis have also been published, as well as cases of association of anti-GBM disease and ANCApositive renal vasculitis (Curioni, 2002; O'Connor, 2010). Diagnosis in these patients requires skilful and careful analysis of clinical features, renal biopsy findings, and

Less than 10% of patients with primary IgA nephropathy or Henoch-Schonlein syndrome

Clinical features of cutaneous purpura or abdominal and joint pain, accompanied by the finding of a small vessel leukocytoclastic vasculitis, most frequently detected in skin

Focal segmental or global and diffuse necrotising and extracapillary lesions of the glomerulus can be found, or extracapillary lesions can be present without necrosis of the glomerular tuft (Fig 9), which always presents variable degrees of mesangial proliferation

Immunofluorescence shows prevailing IgA mesangial deposits, possibly in combination

**4.4 RPGN complicating primary and secondary glomerular diseases** 

have been reported with a truly rapidly progressive course (Ferrario, 1997).

**4.4.1 IgA nephropathy and Henoch-Schonlein purpura** 

biopsies, help in making a diagnosis of systemic disease.

with IgG and C3 deposition, especially in the systemic disease.

and expansion of the mesangial matrix.

primarily subendothelial and mesangial.

tuft and in the crescent.

hematochemical exams.

Fig. 9. Primary IgA nephropathy. A circumferential crescent surrounds a glomerulus affected by mesangial proliferation and mesangial expansion.

#### **4.4.2 Systemic lupus erythematosus**

Among the histological classes of SLE nephritis (Weening, 2004), RPGN is more frequently observed in classes III and IV. In these cases the occurrence of antineutrophil cytoplasmic antibodies is not uncommon and is thought to contribute to the development of necrotising and crescentic glomerular lesions (Sen, 2003).

Extensive extracapillary proliferation has been rarely reported (Fig 10), whereas segmental necrotising extracapillary alterations are a rather common finding, but not always translate in a RPGN clinical phenotype.

Fig. 10. Rapidly progressive class IV SLE nephritis. A circumferential crescent surrounds a glomerulus affected by intense intracapillary proliferation, mesangial expansion, and leukocyte infiltration.

Rapidly Progressive Glomerulonephritis 105

Instead, several experimental models implicate T-cell mediated immunity in the pathogenesis of anti-GBM disease, which is based on the following findings. In rats, anti-GBM disease can be induced by injecting alpha3(IV)NC1-specific CD4+Tcells (Wu, 2002). Anti-CD8 monoclonal antibodies reduce disease severity and antigen-specific CD8+Tcell clones have been found in diseased patients (Reynolds, 2002). Invariant natural killer cells (iNKT) could have a role as well, because the disease has a worse course in iNKT celldeficient mice (Mesnard, 2009). Finally, mice deficient in IL-23, which is important for the maintenance of Th17 cells, the CD4+Tcell subset producing IL17, are protected from anti-

The discovery of ANCAs (Falk, 1988) radically changed not only the diagnosis of small vessel vasculitis, but also introduced an important element for the study of the etiology and pathogenesis of this group of diseases. Major ANCA autoantigens are two proteins contained in azurophil granules of neutrophil granulocytes, MPO and PR3, which are mainly expressed during neutrophil development at the myeloblast and promyelocytic stage (Cowland, 1999). They are aberrantly expressed in mature neutrophils of ANCA

In vivo first evidence for a pathogenetic role of ANCA was demonstrated by injection of anti-MPO antibodies or anti-MPO lymphocytes, causing a pauci-immune focal necrotising extracapillary glomerulonephritis (Xiao, 2002). Subsequent research then showed that in this model neutrophil granulocytes are required, because mice depleted of neutrophils do not develop the disease, and disease worsening is obtained by priming neutrophils using a proinflammatory stimulus (Xiao, 2005). The model has been also useful in investigating the role of the alternative complement pathway, because the disease does not occur in C5 or Factor B

In an additional model, MPO-KO mice were first immunised with mouse MPO, determining production of anti-MPO antibodies. These mice having circulating anti-MPO antibodies were then irradiated and subsequently transplanted with MPO-wild type or MPO-KO bone marrow cells. A pauci-immune necrotising-crescentic glomerulonephritis developed only in mice engrafted with MPO-wild type cells, indicating the requirement for bone marrow

Along the years, composition of glomerular extracapillary proliferation has been, and still

Though the exact mechanism/s of crescent formation remain elusive, novel animal models have recently added important information, that will lead to further clarification of the molecular pathways involved and the potential identification of possible novel therapeutic

A word has first to be spent in stating that, morphologically speaking, extracapillary proliferation is a heterogeneous phenomenon. It has been shown by several investigators that presence or absence of necrosis of the glomerular capillary is relevant to the type of crescent. When necrosis is present, the crescent is more inflammatory, and mainly formed by monocyte-macrophages. In absence of tuft necrosis, the crescent has more epithelial and

patients, whereas are silenced in mature neutrophils of healthy subjects (Yang, 2004).

null mice, but it fully develops in C4-KO animals (Xiao, 2007).

derived cells in disease development (Schreiber, 2006).

remains, the object of intense investigation and discussion.

**5.3 Cells involved in crescent formation** 

targets.

less inflammatory features.

GBM disease (Ooi, 2009).

**5.2 ANCA-associated renal vasculitis** 

Immunofluorescence has the typical findings of lupus nephritis, according to the class of disease, with frequent "full house" deposition, and fibrinogen positivity in necrotic areas and crescents.

### **5. Pathogenesis and experimental models**

In recent years, thanks to the possibilities offered by molecular modelling, genetic studies, and the generation of novel animal models better reproducing human disease features, important advances have been made in understanding pathogenetic mechanisms underlying certain forms of RPGN, especially anti-GBM nephritis and ANCA-associated renal vasculitis.

Instead, it continues to be less clear why a rapidly progressive course can complicate virtually any type of primary and secondary glomerulonephritis.

### **5.1 Anti-GBM nephritis and Goodpasture's disease**

The seminal discovery in understanding the pathogenesis of the disease was the identification of the antigen that causes production of pathogenic autoantibodies (Saus, 1988).

Thereafter, injection of the recombinant antigen, i.e. the noncollagenous domain (NC1) of the alpha3chain of collagen type IV, was shown to induce a severe glomerulonephritis in Wistar-Kyoto rats (Sado, 1998), hence proving a direct relationship between the self-antigen sustaining autoantibody production and the disease.

More recently, a second class of autoantibodies has been described, which are specific for the alpha5NC1 domain, occur in 70% of affected patients, and seem to be associated with a worse renal prognosis (Pedchenko, 2010).

In the normal glomerular basement membrane, the NC1 domain is assembled in alpha345NC1 hexamers, whose quaternary organisation has been shown in a threedimensional model (Vanacore, 2008) as an ellipsoid-shaped structure composed by two NC1 trimers joined at the base by hydrophobic and hydrophilic interactions and reinforced by sulfilimine bonds. This crosslinked alpha345NC1 hexamer is inert to antibody binding. Anti-GBM antibodies in fact can bind only to dissociated monomer and dimer subunits that form after alteration of the hexamer and expose pathogenic neoepitopes. This explains why passive transfer of antibodies to the mouse, where hexamers in the GBM are completely crosslinked, does not result in glomerulonephritis (Luo, 2010).

The major epitopes within the alpha3 and alpha5 subunits have been identified as well, and named EA-alpha3, EA-alpha5, and EB-alpha3 (Netzer, 1999; Hellmark, 1999; Pedchenko, 2010).

Several questions, primarily regarding the causes of hexamer alteration that induce epitope exposure and antibody production, need to be answered. At present, the most accredited hypothesis is that environmental factors act in genetically predisposed subjects, leading to epitope alteration and antibody formation.

As for genetic predisposition, positive and negative associations with HLA molecules have been found, especially with the MHC class II HLA-DRB1\*1501 allele (Yang, 2009), which is strongly associated to anti-GBM disease.

A number of experimental data are in favour of a role played by FcR gene and the complement system, though their precise role in humans is still unclear.

Immunofluorescence has the typical findings of lupus nephritis, according to the class of disease, with frequent "full house" deposition, and fibrinogen positivity in necrotic areas

In recent years, thanks to the possibilities offered by molecular modelling, genetic studies, and the generation of novel animal models better reproducing human disease features, important advances have been made in understanding pathogenetic mechanisms underlying certain forms of RPGN, especially anti-GBM nephritis and ANCA-associated

Instead, it continues to be less clear why a rapidly progressive course can complicate

The seminal discovery in understanding the pathogenesis of the disease was the identification of the antigen that causes production of pathogenic autoantibodies (Saus,

Thereafter, injection of the recombinant antigen, i.e. the noncollagenous domain (NC1) of the alpha3chain of collagen type IV, was shown to induce a severe glomerulonephritis in Wistar-Kyoto rats (Sado, 1998), hence proving a direct relationship between the self-antigen

More recently, a second class of autoantibodies has been described, which are specific for the alpha5NC1 domain, occur in 70% of affected patients, and seem to be associated with a

In the normal glomerular basement membrane, the NC1 domain is assembled in alpha345NC1 hexamers, whose quaternary organisation has been shown in a threedimensional model (Vanacore, 2008) as an ellipsoid-shaped structure composed by two NC1 trimers joined at the base by hydrophobic and hydrophilic interactions and reinforced by sulfilimine bonds. This crosslinked alpha345NC1 hexamer is inert to antibody binding. Anti-GBM antibodies in fact can bind only to dissociated monomer and dimer subunits that form after alteration of the hexamer and expose pathogenic neoepitopes. This explains why passive transfer of antibodies to the mouse, where hexamers in the GBM are completely

The major epitopes within the alpha3 and alpha5 subunits have been identified as well, and named EA-alpha3, EA-alpha5, and EB-alpha3 (Netzer, 1999; Hellmark, 1999; Pedchenko,

Several questions, primarily regarding the causes of hexamer alteration that induce epitope exposure and antibody production, need to be answered. At present, the most accredited hypothesis is that environmental factors act in genetically predisposed subjects, leading to

As for genetic predisposition, positive and negative associations with HLA molecules have been found, especially with the MHC class II HLA-DRB1\*1501 allele (Yang, 2009), which is

A number of experimental data are in favour of a role played by FcR gene and the

complement system, though their precise role in humans is still unclear.

and crescents.

renal vasculitis.

1988).

2010).

**5. Pathogenesis and experimental models** 

virtually any type of primary and secondary glomerulonephritis.

**5.1 Anti-GBM nephritis and Goodpasture's disease** 

sustaining autoantibody production and the disease.

crosslinked, does not result in glomerulonephritis (Luo, 2010).

worse renal prognosis (Pedchenko, 2010).

epitope alteration and antibody formation.

strongly associated to anti-GBM disease.

Instead, several experimental models implicate T-cell mediated immunity in the pathogenesis of anti-GBM disease, which is based on the following findings. In rats, anti-GBM disease can be induced by injecting alpha3(IV)NC1-specific CD4+Tcells (Wu, 2002). Anti-CD8 monoclonal antibodies reduce disease severity and antigen-specific CD8+Tcell clones have been found in diseased patients (Reynolds, 2002). Invariant natural killer cells (iNKT) could have a role as well, because the disease has a worse course in iNKT celldeficient mice (Mesnard, 2009). Finally, mice deficient in IL-23, which is important for the maintenance of Th17 cells, the CD4+Tcell subset producing IL17, are protected from anti-GBM disease (Ooi, 2009).

#### **5.2 ANCA-associated renal vasculitis**

The discovery of ANCAs (Falk, 1988) radically changed not only the diagnosis of small vessel vasculitis, but also introduced an important element for the study of the etiology and pathogenesis of this group of diseases. Major ANCA autoantigens are two proteins contained in azurophil granules of neutrophil granulocytes, MPO and PR3, which are mainly expressed during neutrophil development at the myeloblast and promyelocytic stage (Cowland, 1999). They are aberrantly expressed in mature neutrophils of ANCA patients, whereas are silenced in mature neutrophils of healthy subjects (Yang, 2004).

In vivo first evidence for a pathogenetic role of ANCA was demonstrated by injection of anti-MPO antibodies or anti-MPO lymphocytes, causing a pauci-immune focal necrotising extracapillary glomerulonephritis (Xiao, 2002). Subsequent research then showed that in this model neutrophil granulocytes are required, because mice depleted of neutrophils do not develop the disease, and disease worsening is obtained by priming neutrophils using a proinflammatory stimulus (Xiao, 2005). The model has been also useful in investigating the role of the alternative complement pathway, because the disease does not occur in C5 or Factor B null mice, but it fully develops in C4-KO animals (Xiao, 2007).

In an additional model, MPO-KO mice were first immunised with mouse MPO, determining production of anti-MPO antibodies. These mice having circulating anti-MPO antibodies were then irradiated and subsequently transplanted with MPO-wild type or MPO-KO bone marrow cells. A pauci-immune necrotising-crescentic glomerulonephritis developed only in mice engrafted with MPO-wild type cells, indicating the requirement for bone marrow derived cells in disease development (Schreiber, 2006).

#### **5.3 Cells involved in crescent formation**

Along the years, composition of glomerular extracapillary proliferation has been, and still remains, the object of intense investigation and discussion.

Though the exact mechanism/s of crescent formation remain elusive, novel animal models have recently added important information, that will lead to further clarification of the molecular pathways involved and the potential identification of possible novel therapeutic targets.

A word has first to be spent in stating that, morphologically speaking, extracapillary proliferation is a heterogeneous phenomenon. It has been shown by several investigators that presence or absence of necrosis of the glomerular capillary is relevant to the type of crescent. When necrosis is present, the crescent is more inflammatory, and mainly formed by monocyte-macrophages. In absence of tuft necrosis, the crescent has more epithelial and less inflammatory features.

Rapidly Progressive Glomerulonephritis 107

All images in this chapter are taken from "Ferrario F, Rastaldi MP. Histopathological Atlas of Renal Diseases", which is publicly available at www.fondazionedamico.org. Fondazione

Bhardwaj, R.S., Zotz, C., Zwadlo-Klarwasser, G., Roth, J., Goebeler, M., Mahnke, K., Falk,

Bolton, W.K., Innes, D.J. Jr, Sturgill, B.C., & Kaiser, D.L. (1987). T-cells and macrophages in

Chen, M., Kallenberg, C.G., & Zhao, M.H. (2009). ANCA-negative pauci-immune crescentic

Cong, M., Chen, M., Zhang, J. J., Hu, Z. & Zhao, M. H. (2008). Anti-endothelial cell

Crosthwaite, A., Skene, A., & Mount, P. (2010). Rapidly progressive glomerulonephritis

Curioni, S., Ferrario, F., Rastaldi, M.P., Colleoni, N., Colasanti, G., & D'Amico, G. (2002).

Dean, S.E., Saba, S.R., & Ramírez, G. (1991). Systemic vasculitis in Goodpasture's syndrome.

Ding, M., Cui, S., Li, C., Jothy, S., Haase, V., Steer, B.M., Marsden, P.A., Pippin, J.,

Falk, R.J., & Jennette, J.C. (1988). Anti-neutrophil cytoplasmic autoantibodies with specificity

glomerulonephritis. *Nat Rev Nephrol*, Vol. 5, No. 6, pp. 313-318.

M., Meinardus-Hager, G., & Sorg, C. (1992). The calcium-binding proteins MRP8 and MRP14 form a membrane-associated heterodimer in a subset of monocytes/macrophages present in acute but absent in chronic inflammatory

rapidly progressive glomerulonephritis: clinicopathologic correlations. *Kidney Int*,

antibodies in antineutrophil cytoplasmic antibodies negative pauci-immune crescentic glomerulonephritis. *Nephrology (Carlton)* Vol. 13, No. 3, pp. 228–234. Couser, W.G. (1988). Rapidly progressive glomerulonephritis: classification, pathogenetic mechanisms, and therapy. *Am J Kidney Dis,* Vol. 11, No. 6, pp. 449-464. Cowland. J,B, & Borregaard, N. (1999). The individual regulation of granule protein mRNA

levels during neutrophil maturation explains the heterogeneity of neutrophil

complicating primary AL amyloidosis and multiple myeloma. *Nephrol Dial* 

Anti-GBM nephritis complicating diabetic nephropathy. *J Nephrol*, Vol. 15, No. 1,

Shankland, S., Rastaldi, M.P., Cohen, C.D., Kretzler, M., & Quaggin, S.E. (2006). Loss of the tumor suppressor Vhlh leads to upregulation of Cxcr4 and rapidly progressive glomerulonephritis in mice. *Nat Med*, Vol. 12, No. 9, pp. 1081-1087. Ellis, A. (1942). Natural history of Bright's disease. Clinical, histological and experimental

for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. *N Engl J Med*, Vol. 318, No. 25, pp. 1651-1657. Ferrario, F., Castiglione, A., Colasanti, G., Barbiano di Belgioioso, G., Bertoli, S., & D'Amico,

G. (1985). The detection of monocytes in human glomerulonephritis. *Kidney Int*,

D'Amico per la Ricerca sulle Malattie Renali is gratefully acknowledged.

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granules. *J Leukoc Biol*, Vol. 66, No. 6, pp. 989–995.

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**7. Acknowledgment** 

**8. References** 

If the presence of inflammatory cells and epithelioid macrophages has never been questioned either in animal models and in human disease, opposite data have been obtained when attempting to define the epithelial cell composition.

Until some years ago, both experimental and human studies aiming to study the cells contained in the crescents were mostly based on morphological findings and immunostaining. The conflicting results produced by these studies were due not only to the specific type of experimental model or of human disease under analysis, but especially to a dysregulated phenotype with loss of specific markers. In fact, both podocytes and parietal epithelial cells are likely to change their original resting phenotype once they start proliferating and filling the Bowman's space.

The advent of novel experimental models, though not generating unifying and conclusive data, is providing more convincing proofs of the participation of either podocytes or parietal epithelial cells, based on tagged expression of specific molecules.

Convincing evidence of podocyte contribution to crescent formation has been shown in a podocyte specific mouse model of Vhlh gene knockout (Ding, 2006). These mice showed rapidly progressive glomerulonephritis by 4 weeks of age and died by terminal renal failure after 3-4 weeks. Histology displayed a crescentic glomerulonephritis, and podocytes expressing tagged-ZO1 were found into the crescents. A part from showing podocyte participation in crescent formation, the model also identified a novel pathway potentially operating in extracapillary glomerulonephritis; deletion of Vhlh in fact resulted in stabilisation of hypoxia inducible factor-aplha (HIF1alpha) and consequent upregulation of target genes, among them the chemokine receptor CXCR4. Further, podocyte-specific expression of CXCR4 was sufficient to induce podocyte proliferation and crescent formation, and CXCR4 positivity was observed in glomeruli of human biopsies with necrotising extracapillary lesions, suggesting that the VHLH-HIF-CXCR4 pathway may have functional relevance also in humans.

The contribution of parietal epithelial cells to crescent formation has been recently shown in a mouse model where a construct containing 3 kb of the human podocalyxin (*hPODXL1*) 5′ flanking region and 0.3 kb of the rabbit *Podxl1* 5′ untranslated region were used to drive expression of rabbit podocalyxin, and transgene expression was detected exclusively within PECs but not in podocytes. In this model, injection of nephrotoxic serum caused extracapillary glomerulonephritis and cells within crescents could be clearly identified as of parietal origin (Smeets, 2009, a).

As a final consideration, recent work has demonstrated that the Bowman's capsule contains renal progenitors mainly located at the urinary pole of the glomerulus (Ronconi, 2009). If it is true that these cells are able to regenerate either tubular cells and podocytes, then their participation to crescent formation can be viewed as the pathological consequence of a tentative to repair glomerular damage in the course of inflammatory conditions (Smeets, 2009, b).

### **6. Conclusion**

RPGN still constitute a threat for human health and survival. Despite numerous improvements in understanding the pathogenesis of these diseases, numerous questions still remain unanswered and will need clarification before providing targeted, pathwaybased, novel therapeutics.

### **7. Acknowledgment**

All images in this chapter are taken from "Ferrario F, Rastaldi MP. Histopathological Atlas of Renal Diseases", which is publicly available at www.fondazionedamico.org. Fondazione D'Amico per la Ricerca sulle Malattie Renali is gratefully acknowledged.

#### **8. References**

106 An Update on Glomerulopathies – Clinical and Treatment Aspects

If the presence of inflammatory cells and epithelioid macrophages has never been questioned either in animal models and in human disease, opposite data have been obtained

Until some years ago, both experimental and human studies aiming to study the cells contained in the crescents were mostly based on morphological findings and immunostaining. The conflicting results produced by these studies were due not only to the specific type of experimental model or of human disease under analysis, but especially to a dysregulated phenotype with loss of specific markers. In fact, both podocytes and parietal epithelial cells are likely to change their original resting phenotype once they start

The advent of novel experimental models, though not generating unifying and conclusive data, is providing more convincing proofs of the participation of either podocytes or parietal

Convincing evidence of podocyte contribution to crescent formation has been shown in a podocyte specific mouse model of Vhlh gene knockout (Ding, 2006). These mice showed rapidly progressive glomerulonephritis by 4 weeks of age and died by terminal renal failure after 3-4 weeks. Histology displayed a crescentic glomerulonephritis, and podocytes expressing tagged-ZO1 were found into the crescents. A part from showing podocyte participation in crescent formation, the model also identified a novel pathway potentially operating in extracapillary glomerulonephritis; deletion of Vhlh in fact resulted in stabilisation of hypoxia inducible factor-aplha (HIF1alpha) and consequent upregulation of target genes, among them the chemokine receptor CXCR4. Further, podocyte-specific expression of CXCR4 was sufficient to induce podocyte proliferation and crescent formation, and CXCR4 positivity was observed in glomeruli of human biopsies with necrotising extracapillary lesions, suggesting that the VHLH-HIF-CXCR4 pathway may

The contribution of parietal epithelial cells to crescent formation has been recently shown in a mouse model where a construct containing 3 kb of the human podocalyxin (*hPODXL1*) 5′ flanking region and 0.3 kb of the rabbit *Podxl1* 5′ untranslated region were used to drive expression of rabbit podocalyxin, and transgene expression was detected exclusively within PECs but not in podocytes. In this model, injection of nephrotoxic serum caused extracapillary glomerulonephritis and cells within crescents could be clearly identified as of

As a final consideration, recent work has demonstrated that the Bowman's capsule contains renal progenitors mainly located at the urinary pole of the glomerulus (Ronconi, 2009). If it is true that these cells are able to regenerate either tubular cells and podocytes, then their participation to crescent formation can be viewed as the pathological consequence of a tentative to repair glomerular damage in the course of inflammatory conditions (Smeets,

RPGN still constitute a threat for human health and survival. Despite numerous improvements in understanding the pathogenesis of these diseases, numerous questions still remain unanswered and will need clarification before providing targeted, pathway-

when attempting to define the epithelial cell composition.

epithelial cells, based on tagged expression of specific molecules.

proliferating and filling the Bowman's space.

have functional relevance also in humans.

parietal origin (Smeets, 2009, a).

2009, b).

**6. Conclusion** 

based, novel therapeutics.


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**Part 2** 

**Infectious Glomerulopathies** 

**and Related Disorders** 

cause glomerulonephritis and vasculitis in mice. *J Clin Invest*, Vol. 110, No. 7, pp. 955-963.


## **Part 2**

**Infectious Glomerulopathies and Related Disorders** 

110 An Update on Glomerulopathies – Clinical and Treatment Aspects

Xiao, H., Heeringa, P., Liu, Z., Huugen, D., Hu, P., Maeda, N., Falk, R.J., & Jennette, J.C.

Xiao, H., Schreiber, A., Heeringa, P., Falk, R.J., & Jennette, J.C. (2007). Alternative

Yang, J.J., Pendergraft, W.F., Alcorta, D.A., Nachman, P.H., Hogan, S.L., Thomas, R.P.,

Yang, R., Cui, Z., Zhao, J., & Zhao, M.H. (2009). The role of HLA-DRB1 alleles on

myeloperoxidase antibodies. *Am J Pathol*, Vol. 167, No. 1, pp. 39-45.

cytoplasmic autoantibodies. *Am J Pathol*, Vol. 170, No. 1, pp. 52-64.

955-963.

2103–2114.

No. 2, pp. 245–250.

cause glomerulonephritis and vasculitis in mice. *J Clin Invest*, Vol. 110, No. 7, pp.

(2005). The role of neutrophils in the induction of glomerulonephritis by anti-

complement pathway in the pathogenesis of disease mediated by anti-neutrophil

Sullivan, P., Jennette, J.C., Falk, R.J., & Preston, G.A. (2004). Circumvention of normal constraints on granule protein gene expression in peripheral blood neutrophils and monocytes of patients with antineutrophil cytoplasmic autoantibody associated glomerulonephritis. *J Am Soc Nephrol*, Vol. 15, No. 8, pp.

susceptibility of Chinese patients with anti-GBM disease. *Clin Immunol*, Vol. 133,

**7** 

Gurmeet Singh

*Australia* 

**Post-Infectious Glomerulonephritis** 

*Northern Territory Medical Program, Flinders University, SA,* 

*Menzies School of Health Research, Cahrles Darwin University, Darwin, NT,* 

This chapter will provide a comprehensive review of post-infectious glomerulonephritis

The immunological response of the kidney to an insult results in glomerulonephritis. The insult can result from a large number of conditions, both infectious and non-infectious. Regardless of the initial insult, the outcome is similar in terms of pathology and clinical symptoms. The most common and most-studied cause is post streptococcal

focusing in particular on the changing epidemiology and long term outcome.

*Bacterial*: *Streptococcal (PSGN),* methicillin-resistant *Staphylococcus aureus* (MRSA), pneumococcal pneumonia, typhoid, secondary syphilis, meningococcemia, infective

*Viral:* Hepatitis B, infectious mononucleosis, mumps, measles, varicella, vaccinia,

*Primary glomerular diseases:* Membranoproliferative GN (MPGN), IgA nephropathy,

*Miscellaneous:* Gullian-barre syndrome, pertussis-tetanus vaccine, serum sickness

Autoimmune Neuropsychiatric Disorder Associated with Streptococcus) [1].

M*ultisystem systemic diseases:* Systemic lupus erythematosus, vasculitis, Henoch-Schönlein

Of all the bacterial pathogens, group A streptococcus (GAS) causes the widest range of illness in humans. These illnesses range from local infections of the skin and throat (impetigo and pharyngitis respectively) to invasive infections as well as the significant postinfectious immunological sequelae such as the well documented acute rheumatic fever and acute post-streptococcal glomerulonephritis and the lesser known PANDAS (Paediatric

Glomerulonephritis (PSGN). A list of causes is presented in Table 1.

purpura, Goodpasture syndrome, Wegener granulomatosis

Table 1. Causes of post-Infectious Glomerulonephritis

**2. Burden of disease and changing epidemiology** 

**1. Introduction** 

**INFECTIOUS** 

endocarditis, shunt nephritis, sepsis

*Parasitic:* Malaria, toxoplasmosis *Fungal:* cryptococccus imitis

**NON INFECTIOUS**

mesangial proliferative GN

echovirus, parvovirus, and coxsackievirus

## **Post-Infectious Glomerulonephritis**

### Gurmeet Singh

*Menzies School of Health Research, Cahrles Darwin University, Darwin, NT, Northern Territory Medical Program, Flinders University, SA, Australia* 

### **1. Introduction**

This chapter will provide a comprehensive review of post-infectious glomerulonephritis focusing in particular on the changing epidemiology and long term outcome.

The immunological response of the kidney to an insult results in glomerulonephritis. The insult can result from a large number of conditions, both infectious and non-infectious. Regardless of the initial insult, the outcome is similar in terms of pathology and clinical symptoms. The most common and most-studied cause is post streptococcal Glomerulonephritis (PSGN). A list of causes is presented in Table 1.

### **INFECTIOUS**

*Bacterial*: *Streptococcal (PSGN),* methicillin-resistant *Staphylococcus aureus* (MRSA), pneumococcal pneumonia, typhoid, secondary syphilis, meningococcemia, infective endocarditis, shunt nephritis, sepsis *Viral:* Hepatitis B, infectious mononucleosis, mumps, measles, varicella, vaccinia, echovirus, parvovirus, and coxsackievirus *Parasitic:* Malaria, toxoplasmosis *Fungal:* cryptococccus imitis

#### **NON INFECTIOUS**

*Primary glomerular diseases:* Membranoproliferative GN (MPGN), IgA nephropathy, mesangial proliferative GN M*ultisystem systemic diseases:* Systemic lupus erythematosus, vasculitis, Henoch-Schönlein purpura, Goodpasture syndrome, Wegener granulomatosis

*Miscellaneous:* Gullian-barre syndrome, pertussis-tetanus vaccine, serum sickness

Table 1. Causes of post-Infectious Glomerulonephritis

### **2. Burden of disease and changing epidemiology**

Of all the bacterial pathogens, group A streptococcus (GAS) causes the widest range of illness in humans. These illnesses range from local infections of the skin and throat (impetigo and pharyngitis respectively) to invasive infections as well as the significant postinfectious immunological sequelae such as the well documented acute rheumatic fever and acute post-streptococcal glomerulonephritis and the lesser known PANDAS (Paediatric Autoimmune Neuropsychiatric Disorder Associated with Streptococcus) [1].

Post-Infectious Glomerulonephritis 115

dwellings or areas with poor hygienic conditions, both urban and rural. These conditions are especially prevalent in Aboriginal peoples of Australia living in remote communities, in settings with a high burden of infectious disease and overcrowding [11, 12]. Sporadic cases of PSGN occur in the Northern Territory of Australia each year with outbreaks every 5-7 years [13]. PSGN in New Zealand occurs mostly in children of Pacific Island and Maori heritage (>85% of cases) [14]. Sporadic cases of PSGN also continue to be reported

The rates are higher in children than in adults, and PANDAS is described solely in the paediatric age group. PSGN primarily affects children, aged 2-12 years, with clinically detectable cases estimated to be 10% of children with pharyngitis and up to 25% of children with impetigo during epidemics [15, 16]. Children account for 50-90% of epidemic cases, with 5-10% occurring in people > 40 years and 10% in those below 2 years of age [5]. PSGN is uncommon below 3 years of age and rarely seen below 2 years [17]. This low incidence of PSGN is likely to be due to the decreased immunogenicity of children below 2 years of age, for although GAS pharyngitis is uncommon in children of this age, GAS skin infections are common. Decreased immunogenicity likely results in less robust immune complex

Males have more symptomatic disease, but this difference is no longer present when symptomatic and asymptomatic cases are considered together [19]. Spontaneous recovery occurs in almost all patients, including those who develop renal insufficiency during the

There are no large scale published studies of bacterial infections associated with GN other than streptococcal infection. These are limited to small cases series and individual cases reports. The most common of these are related to staphylococcal infections, both methicillin sensitive [20] and methicillin resistant [21, 22]. A case series of 10 cases, age range 21-65 years, of MRSA-associated glomerulonephritis reported polyclonal increases of IgA and IgG and massive T cell activation and suggested the role of the enterotoxin as a bacterial superantigen initiating the immunological response leading to the glomerulonephritis [22]. The histopathologic findings on immunofluorescence in the patients with MRSA infection with nephritis resemble those seen in IgA nephropathy [22]. Nephritis associated with endocarditis and ventricular shunts is associated with staphylococcal infection [22]. A number of infections can cause nephritis as listed in table 1. Most have been reported as case reports, such as a report of nephritis following malaria due to *falciparum vivax* infection in a 7 year old girl [23], and a report of nephritis following pneumococcal pneumonia in an adult

Hepatitis-B-associated glomerulonephritis (HBGN) is a distinct entity occurring frequently in hepatitis-B-prevalent areas of the world. The disease affects both adults and children who are chronic hepatitis-B-virus (HBV) carriers with or without a history of overt liver disease. The diagnosis is established by serologic evidence of HBV antigens/antibodies, presence of an immune complex glomerulonephritis, immunohistochemical localization of 1 or more HBV antigens and pertinent clinical history [25]. With the high incidence of hepatitis B in Asia, this entity assumes a greater public health importance. A study from China reported 205 cases from a single hospital from September 1995 to November 2008 [26]. In this series, the peak incidence of HBV-GN was between 20 -40 years of age, with a 3:1 predominance of males. The most common clinic manifestation was nephrotic syndrome and the most common pathology was membranous nephropathy. Decreased renal function was present in

10% of cases. The degree of albuminuria correlated with the viral load [26].

acute phase [16], with 1% of all paediatric patients developing renal insufficiency.

from all over the world.

male [24].

formation thus leading to less PSGN [18].

While global estimates of the burden of disease due to GAS infections are difficult to get, some estimates have been reported which are based on published population studies. The estimate of 500 000 deaths per year due to GAS makes it a major human Pathogen [2]. This minimal estimate places GAS infections as less common than HIV, *Mycobacterium tuberculosis*, *Plasmodium falciparum* and *Streptococcus pneumonia* but as common as rotavirus, measles, *Haemophilus influenzae* type b and hepatitis B as a cause of global mortality [2]. In addition there is the long-term morbidity associated with GAS infections.

The global burden of severe group A streptococcal disease is concentrated largely in developing countries and within disadvantaged populations living in developed countries such as Aboriginal Australians. The review of population based studies estimated the prevalence of severe GAS disease at a minimum of 18·1 million cases, with 1·78 million new cases each year [2]. The greatest burden was due to rheumatic heart disease, with a prevalence of at least 15·6 million cases, with 282 000 new cases and 233 000 deaths each year. The burden of invasive GAS diseases was found to be unexpectedly high, with at least 663 000 new cases and 163 000 deaths each year. In addition, there were more than 111 million prevalent cases of GAS pyoderma, and over 616 million incident cases per year of GAS pharyngitis [2]. The review estimated that over 470 000 cases of acute poststreptococcal glomerulonephritis occur annually, with approximately 5000 deaths (1% of total cases), 97% of which were in less developed countries [2].

The global incidence of acute PSGN was estimated at 472,000 cases per year, of which 456,000 ( 96.6%) occurred in less developed countries [3, 4]. A similar distribution of higher incident cases in less developed countries is also reported in a review of population based studies [2]. A review of 11 population-based studies documenting the incidence of acute PSGN in children from less developed countries or those that included substantial minority populations in more developed countries, estimated 24·3 cases per 100,000 person as the median PSGN incident rate [2]. The same review estimated an incidence in adults of 2 cases per 100,000 person-years for developing countries and 0.3 per 100,000 person-years in developed countries [2]. Due to the paucity of data in adults with PSGN, the estimate for developing countries was based on data from Kuwait and for developed countries on data from Italian Biopsy Registry and the most conservative estimates were reported [2]. Another recent study described a slightly higher incidence of 9.5-28.5 cases per 100,000 person-years in developing countries [5]. These rates represent only the clinical cases. When asymptomatic cases are screened for in household contacts and family members, asymptomatic disease is reported to be 4-19 times greater [5-7].

PSGN can occur sporadically or epidemically. The changing pattern of PSGN over the last few decades has been described in studies from Florida [8] and Singapore [9]. The overall incidence of PSGN has decreased over the last few decades [10]. The reasons for this decline have not been clearly delineated but possible reasons are the widespread use of antibiotics, changes in etiological pathogens, altered susceptibility of the host, better health care delivery and improved socioeconomic and nutritional conditions [8-10]. Nevertheless, epidemics and clusters of cases continue to appear in several regions of the world and sporadic cases of PSGN account for 21% (4.6–51.6%) of children admitted to the hospital with acute renal failure in developing countries [5]. Although epidemic PSGN has decreased dramatically and is almost unknown in the developed world, epidemics of PSGN continue to occur in the developing world, mainly in Africa, West Indies and the Middle East, as well as in Indigenous people living in the developed world [11]. Epidemics are described mainly in "closed" communities, clusters of densely populated

While global estimates of the burden of disease due to GAS infections are difficult to get, some estimates have been reported which are based on published population studies. The estimate of 500 000 deaths per year due to GAS makes it a major human Pathogen [2]. This minimal estimate places GAS infections as less common than HIV, *Mycobacterium tuberculosis*, *Plasmodium falciparum* and *Streptococcus pneumonia* but as common as rotavirus, measles, *Haemophilus influenzae* type b and hepatitis B as a cause of global mortality [2]. In

The global burden of severe group A streptococcal disease is concentrated largely in developing countries and within disadvantaged populations living in developed countries such as Aboriginal Australians. The review of population based studies estimated the prevalence of severe GAS disease at a minimum of 18·1 million cases, with 1·78 million new cases each year [2]. The greatest burden was due to rheumatic heart disease, with a prevalence of at least 15·6 million cases, with 282 000 new cases and 233 000 deaths each year. The burden of invasive GAS diseases was found to be unexpectedly high, with at least 663 000 new cases and 163 000 deaths each year. In addition, there were more than 111 million prevalent cases of GAS pyoderma, and over 616 million incident cases per year of GAS pharyngitis [2]. The review estimated that over 470 000 cases of acute poststreptococcal glomerulonephritis occur annually, with approximately 5000 deaths (1% of

The global incidence of acute PSGN was estimated at 472,000 cases per year, of which 456,000 ( 96.6%) occurred in less developed countries [3, 4]. A similar distribution of higher incident cases in less developed countries is also reported in a review of population based studies [2]. A review of 11 population-based studies documenting the incidence of acute PSGN in children from less developed countries or those that included substantial minority populations in more developed countries, estimated 24·3 cases per 100,000 person as the median PSGN incident rate [2]. The same review estimated an incidence in adults of 2 cases per 100,000 person-years for developing countries and 0.3 per 100,000 person-years in developed countries [2]. Due to the paucity of data in adults with PSGN, the estimate for developing countries was based on data from Kuwait and for developed countries on data from Italian Biopsy Registry and the most conservative estimates were reported [2]. Another recent study described a slightly higher incidence of 9.5-28.5 cases per 100,000 person-years in developing countries [5]. These rates represent only the clinical cases. When asymptomatic cases are screened for in household contacts and family members,

PSGN can occur sporadically or epidemically. The changing pattern of PSGN over the last few decades has been described in studies from Florida [8] and Singapore [9]. The overall incidence of PSGN has decreased over the last few decades [10]. The reasons for this decline have not been clearly delineated but possible reasons are the widespread use of antibiotics, changes in etiological pathogens, altered susceptibility of the host, better health care delivery and improved socioeconomic and nutritional conditions [8-10]. Nevertheless, epidemics and clusters of cases continue to appear in several regions of the world and sporadic cases of PSGN account for 21% (4.6–51.6%) of children admitted to the hospital with acute renal failure in developing countries [5]. Although epidemic PSGN has decreased dramatically and is almost unknown in the developed world, epidemics of PSGN continue to occur in the developing world, mainly in Africa, West Indies and the Middle East, as well as in Indigenous people living in the developed world [11]. Epidemics are described mainly in "closed" communities, clusters of densely populated

addition there is the long-term morbidity associated with GAS infections.

total cases), 97% of which were in less developed countries [2].

asymptomatic disease is reported to be 4-19 times greater [5-7].

dwellings or areas with poor hygienic conditions, both urban and rural. These conditions are especially prevalent in Aboriginal peoples of Australia living in remote communities, in settings with a high burden of infectious disease and overcrowding [11, 12]. Sporadic cases of PSGN occur in the Northern Territory of Australia each year with outbreaks every 5-7 years [13]. PSGN in New Zealand occurs mostly in children of Pacific Island and Maori heritage (>85% of cases) [14]. Sporadic cases of PSGN also continue to be reported from all over the world.

The rates are higher in children than in adults, and PANDAS is described solely in the paediatric age group. PSGN primarily affects children, aged 2-12 years, with clinically detectable cases estimated to be 10% of children with pharyngitis and up to 25% of children with impetigo during epidemics [15, 16]. Children account for 50-90% of epidemic cases, with 5-10% occurring in people > 40 years and 10% in those below 2 years of age [5]. PSGN is uncommon below 3 years of age and rarely seen below 2 years [17]. This low incidence of PSGN is likely to be due to the decreased immunogenicity of children below 2 years of age, for although GAS pharyngitis is uncommon in children of this age, GAS skin infections are common. Decreased immunogenicity likely results in less robust immune complex formation thus leading to less PSGN [18].

Males have more symptomatic disease, but this difference is no longer present when symptomatic and asymptomatic cases are considered together [19]. Spontaneous recovery occurs in almost all patients, including those who develop renal insufficiency during the acute phase [16], with 1% of all paediatric patients developing renal insufficiency.

There are no large scale published studies of bacterial infections associated with GN other than streptococcal infection. These are limited to small cases series and individual cases reports. The most common of these are related to staphylococcal infections, both methicillin sensitive [20] and methicillin resistant [21, 22]. A case series of 10 cases, age range 21-65 years, of MRSA-associated glomerulonephritis reported polyclonal increases of IgA and IgG and massive T cell activation and suggested the role of the enterotoxin as a bacterial superantigen initiating the immunological response leading to the glomerulonephritis [22]. The histopathologic findings on immunofluorescence in the patients with MRSA infection with nephritis resemble those seen in IgA nephropathy [22]. Nephritis associated with endocarditis and ventricular shunts is associated with staphylococcal infection [22]. A number of infections can cause nephritis as listed in table 1. Most have been reported as case reports, such as a report of nephritis following malaria due to *falciparum vivax* infection in a 7 year old girl [23], and a report of nephritis following pneumococcal pneumonia in an adult male [24].

Hepatitis-B-associated glomerulonephritis (HBGN) is a distinct entity occurring frequently in hepatitis-B-prevalent areas of the world. The disease affects both adults and children who are chronic hepatitis-B-virus (HBV) carriers with or without a history of overt liver disease. The diagnosis is established by serologic evidence of HBV antigens/antibodies, presence of an immune complex glomerulonephritis, immunohistochemical localization of 1 or more HBV antigens and pertinent clinical history [25]. With the high incidence of hepatitis B in Asia, this entity assumes a greater public health importance. A study from China reported 205 cases from a single hospital from September 1995 to November 2008 [26]. In this series, the peak incidence of HBV-GN was between 20 -40 years of age, with a 3:1 predominance of males. The most common clinic manifestation was nephrotic syndrome and the most common pathology was membranous nephropathy. Decreased renal function was present in 10% of cases. The degree of albuminuria correlated with the viral load [26].

Post-Infectious Glomerulonephritis 117

In the absence of evidence of a recent group A beta-hemolytic streptococcal infection, infections with Cytomegalovirus (CMV), coxsackievirus, Epstein-Barr virus (EBV), hepatitis B virus (HBV) [30], rubella, rickettsiae (as in scrub typhus), and mumps virus may be accepted as causal organisms. Similarly, attributing glomerulonephritis to a parasitic or fungal etiology requires the exclusion of a streptococcal infection. Possible organisms are *Coccidioides immiti, Plasmodium malariae, Plasmodium falciparum*, *Schistosoma mansoni*, *Toxoplasma gondii*, filariasis, trichinosis, and trypanosomes. While hepatitis B infection has been well documented as a cause of renal involvement and glomerulonephritis [25, 26],

Noninfectious causes of acute GN may be divided into primary renal diseases, systemic diseases, and miscellaneous conditions or agents. The primary renal diseases are membranoproliferative glomerulonephritis (MPGN), IgA nephropathy and mesangial proliferative glomerulonephritis. Multisystem systemic diseases that can cause acute GN are vasculitis such as Wegener granulomatosis, polyarteritis nodosa and hypersensitivity vasculitis, collagen-vascular diseases like systemic lupus erythematosus (SLE) which causes glomerulonephritis through renal deposition of immune complexes, Henoch-Schönlein purpura and Goodpasture syndrome. Miscellaneous noninfectious causes are Guillain-Barré syndrome, Diphtheria-pertussis-tetanus (DPT) vaccine and serum sickness. These are

It is important to identify the exact aetiology of the glomerulonephritis as the prognosis differs widely depending on the underlying cause. There are a number of clinical and laboratory features that may help to differentiate between or even point to a particular cause of glomerulonephritis. The latent period between infection and nephritis is helpful in differentiating PSGN from IgA nephropathy. In contrast to the latent period of 2-3 weeks seen in PSGN, the nephritis of IGA nephropathy may occur either at the same time or just 1- 2 days after an upper respiratory infection. Similarly, patients with nephritis of chronic infection have an active infection at the time nephritis becomes evident. Despite the chronic nature of the underlying infection, the associated nephritis can present acutely. Circulating immune complexes play an important role in the pathogenesis of acute GN in these

The failure of the C3 levels to return to normal should prompt consideration of the possibility of MPGN or SLE as the underlying cause. MPGN is a chronic disease which can manifest with an acute nephritic picture. Gross haematuria is unusual in lupus nephritis. Other associated systemic findings may identify the underlying systemic disease, for example, vasculitic lesions of the lower extremities point to an underlying vasculitis as the

The typical presentation is the abrupt onset of acute nephritis occurring 1-3 weeks after a streptococcal throat infection and 3-6 weeks after skin infection [28]. The nephritis is characterised by the triad of oedema, gross haematuria, and hypertension. The classical presenting feature is the presence of "coca-cola" coloured urine which is characteristic of homogenous gross haematuria [32]. Other common features are facial puffiness and hypertension secondary to fluid overload and urinary abnormalities such as albuminuria and the presence of red cell casts. General features like malaise, weakness, and anorexia may occur in about half the patients and a minority complain of nausea and vomiting. Within a

acute GN is as a rare complication of hepatitis A [31].

summarised in table 1.

cause of glomerulonephritis.

**4. Clinical presentation** 

diseases.

Renal disease is not uncommon in those infected with HIV. The most common manifestation of HIV in the kidney is HIV-associated nephropathy (HIVAN). Immunotactoid glomerulonephritis is a rare disorder found in 0.06% of renal biopsies characterized by organized tubular immune complex deposits. This is seen more commonly Caucasians and tends to occur in an older age group. There are 6 reported cases of HIV associated immunotactoid glomerulonephritis [27].

### **3. Pathogenesis**

The kidney has a limited number of ways of responding to injury. Similar pathological signs may be the end result of different processes, produced by different initiating mechanisms and different molecular pathways may perpetuate the injury process. The initiation and development of the inflammatory response of the kidney to infection are still poorly understood.

The pathognomonic feature of PSGN is the deposition of immune complexes in the glomerular basement membrane. A proposed sequence of events is that a nephritogenic antigen(s) leads to the activation of the complement pathway and/or activates plasmin or production of the circulating immune-complexes. These then lead to increased permeability of the glomerular basement membrane, which allows deposition of the immune complexes, and leakage of the protein and red blood cells. The nephritogenic antigen is responsible for the C3 deposition, the recruitment of immune cells, tissue destruction and IgG deposition which further aggravates tissue injury. Complement activation leads to the release of cytokines, such as C5a, which attracts phagocytes, and proliferation of intrinsic cells and formation of a membrane attack complex which also aggravate the process. The definitive nephritogenic antigen has not yet been defined, although a large number of streptococcal factors (M proteins) have been proposed as the triggering factor. M proteins are present on the pili of the organism and more than 100 have been identified so far. Nephritogenic M proteins are types 1, 2, 4, 3, 25, 49, and 12 following skin infections and types 47, 49, 55, 2, 60, and 57 following throat infections [28]. Infections with nephritogenic streptococci have considerable variability in their ability to cause nephritis. The reason for this variability is not known.

Pathology shows typical glomerular changes which include proliferation of mesangial, endothelial and epithelial cells, inflammatory exudate and deposition of C3 early in the disease process followed by deposition of IgG. This immune deposition has been classified into 3 patterns [29]. The "starry sky" pattern represents an irregular and finely granular deposit of C3 and IgG along the glomerular capillary walls and in the mesangium. This occurs early in the course of the disease and is also seen in subclinical cases [28, 29]. The "mesangial pattern" has mainly C3 and some IgG in the mesangium. The "garland pattern" shows dense deposits along the capillary walls, is commonly associated with severe proteinuria and a poor prognosis [28, 29].

The immunological response of the kidney to an insult results in glomerulonephritis. The causal factors that underlie acute GN can be broadly divided into infectious and noninfectious groups. The most common infectious cause of acute GN is infection by *Streptococcus* species (ie, group A, beta-hemolytic). Nonstreptococcal postinfectious GN may also result from infection by other bacteria, viruses, parasites, or fungi. Bacteria besides group A streptococci that can cause acute GN include diplococci, other streptococci, staphylococci, and mycobacteria. *Salmonella typhosa*, *Brucella suis*, *Treponema pallidum*, *Corynebacterium bovis*, and actinobacilli have also been identified.

Renal disease is not uncommon in those infected with HIV. The most common manifestation of HIV in the kidney is HIV-associated nephropathy (HIVAN). Immunotactoid glomerulonephritis is a rare disorder found in 0.06% of renal biopsies characterized by organized tubular immune complex deposits. This is seen more commonly Caucasians and tends to occur in an older age group. There are 6 reported cases of HIV

The kidney has a limited number of ways of responding to injury. Similar pathological signs may be the end result of different processes, produced by different initiating mechanisms and different molecular pathways may perpetuate the injury process. The initiation and development of the inflammatory response of the kidney to infection are still poorly

The pathognomonic feature of PSGN is the deposition of immune complexes in the glomerular basement membrane. A proposed sequence of events is that a nephritogenic antigen(s) leads to the activation of the complement pathway and/or activates plasmin or production of the circulating immune-complexes. These then lead to increased permeability of the glomerular basement membrane, which allows deposition of the immune complexes, and leakage of the protein and red blood cells. The nephritogenic antigen is responsible for the C3 deposition, the recruitment of immune cells, tissue destruction and IgG deposition which further aggravates tissue injury. Complement activation leads to the release of cytokines, such as C5a, which attracts phagocytes, and proliferation of intrinsic cells and formation of a membrane attack complex which also aggravate the process. The definitive nephritogenic antigen has not yet been defined, although a large number of streptococcal factors (M proteins) have been proposed as the triggering factor. M proteins are present on the pili of the organism and more than 100 have been identified so far. Nephritogenic M proteins are types 1, 2, 4, 3, 25, 49, and 12 following skin infections and types 47, 49, 55, 2, 60, and 57 following throat infections [28]. Infections with nephritogenic streptococci have considerable variability in their ability to cause nephritis. The reason for this variability is

Pathology shows typical glomerular changes which include proliferation of mesangial, endothelial and epithelial cells, inflammatory exudate and deposition of C3 early in the disease process followed by deposition of IgG. This immune deposition has been classified into 3 patterns [29]. The "starry sky" pattern represents an irregular and finely granular deposit of C3 and IgG along the glomerular capillary walls and in the mesangium. This occurs early in the course of the disease and is also seen in subclinical cases [28, 29]. The "mesangial pattern" has mainly C3 and some IgG in the mesangium. The "garland pattern" shows dense deposits along the capillary walls, is commonly associated with severe

The immunological response of the kidney to an insult results in glomerulonephritis. The causal factors that underlie acute GN can be broadly divided into infectious and noninfectious groups. The most common infectious cause of acute GN is infection by *Streptococcus* species (ie, group A, beta-hemolytic). Nonstreptococcal postinfectious GN may also result from infection by other bacteria, viruses, parasites, or fungi. Bacteria besides group A streptococci that can cause acute GN include diplococci, other streptococci, staphylococci, and mycobacteria. *Salmonella typhosa*, *Brucella suis*, *Treponema pallidum*,

associated immunotactoid glomerulonephritis [27].

**3. Pathogenesis** 

understood.

not known.

proteinuria and a poor prognosis [28, 29].

*Corynebacterium bovis*, and actinobacilli have also been identified.

In the absence of evidence of a recent group A beta-hemolytic streptococcal infection, infections with Cytomegalovirus (CMV), coxsackievirus, Epstein-Barr virus (EBV), hepatitis B virus (HBV) [30], rubella, rickettsiae (as in scrub typhus), and mumps virus may be accepted as causal organisms. Similarly, attributing glomerulonephritis to a parasitic or fungal etiology requires the exclusion of a streptococcal infection. Possible organisms are *Coccidioides immiti, Plasmodium malariae, Plasmodium falciparum*, *Schistosoma mansoni*, *Toxoplasma gondii*, filariasis, trichinosis, and trypanosomes. While hepatitis B infection has been well documented as a cause of renal involvement and glomerulonephritis [25, 26], acute GN is as a rare complication of hepatitis A [31].

Noninfectious causes of acute GN may be divided into primary renal diseases, systemic diseases, and miscellaneous conditions or agents. The primary renal diseases are membranoproliferative glomerulonephritis (MPGN), IgA nephropathy and mesangial proliferative glomerulonephritis. Multisystem systemic diseases that can cause acute GN are vasculitis such as Wegener granulomatosis, polyarteritis nodosa and hypersensitivity vasculitis, collagen-vascular diseases like systemic lupus erythematosus (SLE) which causes glomerulonephritis through renal deposition of immune complexes, Henoch-Schönlein purpura and Goodpasture syndrome. Miscellaneous noninfectious causes are Guillain-Barré syndrome, Diphtheria-pertussis-tetanus (DPT) vaccine and serum sickness. These are summarised in table 1.

It is important to identify the exact aetiology of the glomerulonephritis as the prognosis differs widely depending on the underlying cause. There are a number of clinical and laboratory features that may help to differentiate between or even point to a particular cause of glomerulonephritis. The latent period between infection and nephritis is helpful in differentiating PSGN from IgA nephropathy. In contrast to the latent period of 2-3 weeks seen in PSGN, the nephritis of IGA nephropathy may occur either at the same time or just 1- 2 days after an upper respiratory infection. Similarly, patients with nephritis of chronic infection have an active infection at the time nephritis becomes evident. Despite the chronic nature of the underlying infection, the associated nephritis can present acutely. Circulating immune complexes play an important role in the pathogenesis of acute GN in these diseases.

The failure of the C3 levels to return to normal should prompt consideration of the possibility of MPGN or SLE as the underlying cause. MPGN is a chronic disease which can manifest with an acute nephritic picture. Gross haematuria is unusual in lupus nephritis. Other associated systemic findings may identify the underlying systemic disease, for example, vasculitic lesions of the lower extremities point to an underlying vasculitis as the cause of glomerulonephritis.

### **4. Clinical presentation**

The typical presentation is the abrupt onset of acute nephritis occurring 1-3 weeks after a streptococcal throat infection and 3-6 weeks after skin infection [28]. The nephritis is characterised by the triad of oedema, gross haematuria, and hypertension. The classical presenting feature is the presence of "coca-cola" coloured urine which is characteristic of homogenous gross haematuria [32]. Other common features are facial puffiness and hypertension secondary to fluid overload and urinary abnormalities such as albuminuria and the presence of red cell casts. General features like malaise, weakness, and anorexia may occur in about half the patients and a minority complain of nausea and vomiting. Within a

Post-Infectious Glomerulonephritis 119

associated with more severe disease. Red blood cell casts are pathognomonic of acute

Serological evidence of an antecedent streptococcal infection is present in the form of raised ASOT (> 200 IU/ml) and increased anti-DNAse B (which is a better serological marker of preceding streptococcal skin infection). Bacteriological evidence of streptococcal disease

Complement levels especially C3 are low at the onset of symptoms. C4 is usually within normal limits in post-streptococcal GN. Causes of nephritis with low complement besides PSGN are MPGN, SLE, cryoglobulinemia, Diabetes Mellitus and Hepatitis C Virus. These diseases should be screened for in case of either an atypical presentation or atypical clinical

Renal function tests, blood urea, sodium, potassium and serum creatinine may be elevated in the acute phase and reflect the decrease in the glomerular filtration rate that occurs at this time. These elevations are usually transient. Failure of renal function tests to normalize within several weeks or months suggests that the patient may not have PSGN and indicates the need to seek an alternative diagnosis by further investigation. The full blood count may show anaemia which is usually dilutional and will return to normal once the fluid overload

Renal ultrasound is not required to make a diagnosis of glomerulonephririts. Renal ultrasound images usually reveal normal-sized kidneys bilaterally. Renal imaging is often done to confirm that there are two kidneys and that they are structurally normal. It may be done as a prelude to a renal biopsy. PSGN is a clinical diagnosis and requires the detection of glomerulonephritis and evidence of preceding streptococcal infection. A renal biopsy is indicated in cases with an atypical presentation, an atypical course, persistence of clinical features, or a persisting low level of complement (C3) or abnormal renal function tests. Features that suggest a diagnosis other than PSGN in the early stages may also indicate a need for a biopsy. These include the absence of the latent period between streptococcal infection and acute glomerulonephritis, anuria, rapidly deteriorating renal function, normal serum complement levels, lack of rise in antistreptococcal antibodies, general symptoms of systemic disease and either persistent hypertension or lack of improvement in glomerular filtration rate for more than 2 weeks. In the recovery phase, persistent low C3 beyond 8 and definitely beyond 12 weeks from the onset of illness would indicate a need to look for

Treatment of PSGN remains largely supportive. Complete recovery occurs in over 90% of children, but only 60% of adults fully recover. The rest develop hypertension or renal

Treatment is directed towards monitoring the signs and symptoms, in particular facial puffiness and hypertension, the main cause of which is fluid overload. Treatment when it is required, is mainly directed towards managing the fluid overload, which is responsive to diuresis and sodium restriction. Effective diuresis reduces cardiac congestion and controls hypertension and in most cases no further treatment is required. Strict input-output

As the hypertension is caused by fluid overload, loop diuretics are the first line treatment and may be adequate for control of hypertension. Furosemide in a dose of 40 mg either

glomerulonephritis. Occasionally other cellular casts and pyuria are present.

may be present in throat or skin swabs.

alternate causes and for a renal biospsy.

monitoring is recommended during the acute phase.

course of the glomerulonephritis.

resolves.

**6. Treatment** 

impairment.

week or so from onset of symptoms, most patients with PSGN begin to experience spontaneous resolution of fluid retention and hypertension and the urine abnormalities begin to subside. The low C3 levels begin to rise and normalise by 8 weeks. Normal urine findings are found by 12 weeks.

Microscopic haematuria is universally present. Of the triad of features, oedema is seen in 85% of cases and is often the presenting symptom, gross haematuria in 40% of cases (range 30-50%) and hypertension in 50-95% of hospitalised cases. Approximately 95% of clinical cases have at least 2 manifestations, and 40% have the full-blown acute nephritic syndrome. The puffiness of the face or eyelids is sudden, usually prominent upon awakening and tends to subside at the end of the day if the patient is active. The oedema is a result of a defect in renal excretion of salt and water leading to fluid overload. The severity of edema does not correlate well with the degree of renal impairment.

In most cases, urinary abnormalities clear by 12 weeks, although proteinuria may persist for 6 months to 3 years and microscopic haematuria from 1 year to 4 years after the onset of nephritis [33]. In some cases, generalized edema and other features of circulatory congestion, such as dyspnea, may be present.

Accompanying this clinical picture is laboratory evidence of streptococcal infection, typically increasing antistreptolysin-O titers (ASOT) or streptozyme titres following throat infections and anti-DNase B titers following skin infections. Complement levels are decreased; low C3 levels are found in almost all patients with acute PSGN and C4 levels may be slightly low. These low levels of C3 usually normalize within 8 weeks after the first sign of PSGN [34], although up to 12 weeks has been reported [33]. The typical accompanying histopathology is one of diffuse cellular proliferation in the glomerulus, an exudate containing neutrophils and monocytes and variable degrees of complement and immunoglobulin deposition. In most cases, hypertension subsides, renal function returns to normal and all urinary abnormalities eventually disappear [33].

There is, however, a wide variation in the clinical presentation as well in the histopathology associated with PSGN. At the severe end of the spectrum is a rapidly rising azotemia with a rapidly progressive nephritic picture associated with severe cell proliferation, massive exudates and crescent formation in biopsy specimens. This is seen in <5% of PSGN cases [19]. The severity of renal failure tends to be directly related to the degree of proliferation and crescent formation, and about 50% of these patients recover renal function [35]. This type of presentation is more common in the elderly. The mild end of the spectrum, represented by subclinical or asymptomatic glomerulonephritis, is more common. Diligent examination of people with acute, trivial or self-limited infections caused by a range of organisms including various bacteria, parasites or viruses reveal subclinical infection in the form of microscopic hematuria, proteinuria and pyuria. Histopathology reveals mesangial proliferation with mesangial deposits of C3 and IgG deposits [19]. Asymptomatic household contacts of PSGN cases show sub-clinical disease 4-5 times more commonly than the acute classical presentation [6, 36]. An older study puts the ratio of asymptomatic cases as high as 19:1 [7].

#### **5. Typical findings on investigations**

Urinalysis reveals haematuria in all patients and proteinuria (from trace to 2+ on dipstick testing) is usually present. Proteinuria may be in the nephrotic range and is usually

week or so from onset of symptoms, most patients with PSGN begin to experience spontaneous resolution of fluid retention and hypertension and the urine abnormalities begin to subside. The low C3 levels begin to rise and normalise by 8 weeks. Normal urine

Microscopic haematuria is universally present. Of the triad of features, oedema is seen in 85% of cases and is often the presenting symptom, gross haematuria in 40% of cases (range 30-50%) and hypertension in 50-95% of hospitalised cases. Approximately 95% of clinical cases have at least 2 manifestations, and 40% have the full-blown acute nephritic syndrome. The puffiness of the face or eyelids is sudden, usually prominent upon awakening and tends to subside at the end of the day if the patient is active. The oedema is a result of a defect in renal excretion of salt and water leading to fluid overload. The severity of edema does not

In most cases, urinary abnormalities clear by 12 weeks, although proteinuria may persist for 6 months to 3 years and microscopic haematuria from 1 year to 4 years after the onset of nephritis [33]. In some cases, generalized edema and other features of circulatory

Accompanying this clinical picture is laboratory evidence of streptococcal infection, typically increasing antistreptolysin-O titers (ASOT) or streptozyme titres following throat infections and anti-DNase B titers following skin infections. Complement levels are decreased; low C3 levels are found in almost all patients with acute PSGN and C4 levels may be slightly low. These low levels of C3 usually normalize within 8 weeks after the first sign of PSGN [34], although up to 12 weeks has been reported [33]. The typical accompanying histopathology is one of diffuse cellular proliferation in the glomerulus, an exudate containing neutrophils and monocytes and variable degrees of complement and immunoglobulin deposition. In most cases, hypertension subsides, renal function returns to

There is, however, a wide variation in the clinical presentation as well in the histopathology associated with PSGN. At the severe end of the spectrum is a rapidly rising azotemia with a rapidly progressive nephritic picture associated with severe cell proliferation, massive exudates and crescent formation in biopsy specimens. This is seen in <5% of PSGN cases [19]. The severity of renal failure tends to be directly related to the degree of proliferation and crescent formation, and about 50% of these patients recover renal function [35]. This type of presentation is more common in the elderly. The mild end of the spectrum, represented by subclinical or asymptomatic glomerulonephritis, is more common. Diligent examination of people with acute, trivial or self-limited infections caused by a range of organisms including various bacteria, parasites or viruses reveal subclinical infection in the form of microscopic hematuria, proteinuria and pyuria. Histopathology reveals mesangial proliferation with mesangial deposits of C3 and IgG deposits [19]. Asymptomatic household contacts of PSGN cases show sub-clinical disease 4-5 times more commonly than the acute classical presentation [6, 36]. An older study puts the ratio of asymptomatic cases as high as

Urinalysis reveals haematuria in all patients and proteinuria (from trace to 2+ on dipstick testing) is usually present. Proteinuria may be in the nephrotic range and is usually

findings are found by 12 weeks.

correlate well with the degree of renal impairment.

normal and all urinary abnormalities eventually disappear [33].

congestion, such as dyspnea, may be present.

**5. Typical findings on investigations** 

19:1 [7].

associated with more severe disease. Red blood cell casts are pathognomonic of acute glomerulonephritis. Occasionally other cellular casts and pyuria are present.

Serological evidence of an antecedent streptococcal infection is present in the form of raised ASOT (> 200 IU/ml) and increased anti-DNAse B (which is a better serological marker of preceding streptococcal skin infection). Bacteriological evidence of streptococcal disease may be present in throat or skin swabs.

Complement levels especially C3 are low at the onset of symptoms. C4 is usually within normal limits in post-streptococcal GN. Causes of nephritis with low complement besides PSGN are MPGN, SLE, cryoglobulinemia, Diabetes Mellitus and Hepatitis C Virus. These diseases should be screened for in case of either an atypical presentation or atypical clinical course of the glomerulonephritis.

Renal function tests, blood urea, sodium, potassium and serum creatinine may be elevated in the acute phase and reflect the decrease in the glomerular filtration rate that occurs at this time. These elevations are usually transient. Failure of renal function tests to normalize within several weeks or months suggests that the patient may not have PSGN and indicates the need to seek an alternative diagnosis by further investigation. The full blood count may show anaemia which is usually dilutional and will return to normal once the fluid overload resolves.

Renal ultrasound is not required to make a diagnosis of glomerulonephririts. Renal ultrasound images usually reveal normal-sized kidneys bilaterally. Renal imaging is often done to confirm that there are two kidneys and that they are structurally normal. It may be done as a prelude to a renal biopsy. PSGN is a clinical diagnosis and requires the detection of glomerulonephritis and evidence of preceding streptococcal infection. A renal biopsy is indicated in cases with an atypical presentation, an atypical course, persistence of clinical features, or a persisting low level of complement (C3) or abnormal renal function tests. Features that suggest a diagnosis other than PSGN in the early stages may also indicate a need for a biopsy. These include the absence of the latent period between streptococcal infection and acute glomerulonephritis, anuria, rapidly deteriorating renal function, normal serum complement levels, lack of rise in antistreptococcal antibodies, general symptoms of systemic disease and either persistent hypertension or lack of improvement in glomerular filtration rate for more than 2 weeks. In the recovery phase, persistent low C3 beyond 8 and definitely beyond 12 weeks from the onset of illness would indicate a need to look for alternate causes and for a renal biospsy.

### **6. Treatment**

Treatment of PSGN remains largely supportive. Complete recovery occurs in over 90% of children, but only 60% of adults fully recover. The rest develop hypertension or renal impairment.

Treatment is directed towards monitoring the signs and symptoms, in particular facial puffiness and hypertension, the main cause of which is fluid overload. Treatment when it is required, is mainly directed towards managing the fluid overload, which is responsive to diuresis and sodium restriction. Effective diuresis reduces cardiac congestion and controls hypertension and in most cases no further treatment is required. Strict input-output monitoring is recommended during the acute phase.

As the hypertension is caused by fluid overload, loop diuretics are the first line treatment and may be adequate for control of hypertension. Furosemide in a dose of 40 mg either

Post-Infectious Glomerulonephritis 121

Improvement in housing, especially reduction in overcrowding, will hinder spread of infectious disease. The significant decline in PSGN in Singapore children is attributed to an improvement in the socioeconomic status, the health care system and urbanization of the

Although research into the development of a vaccine is advanced and 3 GAS vaccines have been approved for phase 1 human trials [45], it is unlikely to be available in the near future.

In keeping with the clinico-pathological picture, the prognosis of PSGN is also extremely variable, and largely influenced by clinical presentation and histopathology. An episode of PSGN may result in complete recovery, progression of symptoms or progression to renal failure. Persistence of symptoms may represent either a slow recovery, limited injury

The immediate prognosis is generally good. In general, children are believed to have an excellent prognosis with the majority showing complete recovery [5]. Fewer than 1% of children have elevated serum creatinine values after 10-15 years of follow-up. Adults have a poorer prognosis overall. Early mortality can be as high as 25% in the elderly who have congestive cardiac failure or azotemia in the early phase. In adults approximately 25% will progress to chronic renal failure. These are usually those with massive proteinuria which suggests a worse prognosis and often signifies the garland pattern of immune deposits on

It is difficult to predict the prognosis in an individual case particularly early in the disease. While a typical presentation and clinical course indicates a good prognosis and an atypical presentation, severe persistent hypertension and abnormal renal function tests, massive proteinuria and older age group suggest a poor prognosis, there is a lack of a clinical or biochemical marker that might differentiate those with a good prognosis from those with a poorer outcome. Neutrophil gelatinase-associated lipocalin (NGAL), is emerging as a promising biomarker of acute kidney injury [46, 47], but has not yet been evaluated in PSGN. However, some studies have reported persistent urinary abnormalities [8, 11, 19, 48, 49] and subtle abnormalities in renal function, as defined by reduction in renal functional reserve, in

Epidemic cases have a better prognosis than sporadic cases [14, 51, 52], but not always. An outbreak of PSGN in Brazil following an epidemic of *Streptococcus equi zooepidemicus*  resulted in a high prevalence of renal abnormalities at a mean follow-up of 5.4 years [53, 54]. These were, however, mainly adult patients. A study of Iranian children has shown that even mild PSGN may result in impaired renal function and that a rising diastolic blood

Elderly people have poorer outcomes as do those with co-morbidities, including diabetes, cardiovascular and liver diseases [5, 28]. In an Aboriginal population with high rates of ESRD, follow-up of children 6-18 years (mean 14.6 years) after epidemic PSGN showed that risk of overt proteinuria was 6 times ( 95% CI 2.2-16.9) greater than in healthy controls after adjustment of age, sex and birth weight [11, 56]. The Australian Aboriginal population is at considerably higher risk than the general Australian population of developing chronic diseases such as diabetes mellitus, cardiovascular and renal diseases. There is also a greater burden of infectious disease and adverse early life factors such as low birth and infant weights. It is proposed that in this high risk population with multiple adverse renal

patients who had recovered from PSGN without apparent sequelae [50]

pressure may be an early sign of worsening renal function [55].

without further progression or progression to renal failure.

country [9].

**8. Prognosis** 

pathology.

orally or intravenously is given 12 hourly. Usually treatment is required for less than 48 hours. Sometimes other anti-hypertensive agents may be needed, especially when the blood pressure is very high and it is unsafe to wait for the effect of diuretic therapy. Nifedipine is then given every 4-6 hours in doses of 5-10 mg. Rarely parenteral hydralazine may be required. Captopril has shown to be effective [37, 38] but should be used in caution in the presence of renal failure and hyperkalemia. Occasionally acute renal failure requires dialysis. Pulmonary oedema may be complication of severe fluid overload and needs urgent treatment.

It is important to check serum complement levels 6-8 weeks after initial testing to make sure they have returned to normal. Blood pressure should be monitored every month for 6 months and then 6 monthly. Renal function tests and serum creatinine levels repeated every 3 months after the acute phase for 1 year and then yearly after that. Urine should be checked for hematuria and proteinuria every 3-6 months.

Aggressive therapy using pulse methyprednisolone has been used in adults with poor prognostic factors such as nephritic range proteinuria, cellular crescents on biopsy and renal insufficiency [39]. Plasmapharesis and pulse methyprednisolone was successfully used in a 6 year old girl with garland pattern PSGN [40]. Whether this would benefit all patients with poor prognosis has not been studied.

Penicillin treatment is given to treat any persisting streptococcal infection [41]. Methicillin resistant staphylococcus should be treated with appropriate antibiotics. Specific infections require treatment with specific antibiotics or antiviral agents. Treatment of the underlying infection may resolve the glomerulonephritis as well. A review of six trials (a total of 159 patients) of which five were specified as hepatitis B virus-associated membranous glomerulonephritis (HBV-MN) showed that antiviral therapy for hepatitis B infections including IFN and lamivudine is effective in leading to remission of proteinuria, HBeAg clearance, and HBV-DNA reduction in both children and adults [42].

### **7. Preventing spread**

A case of PSGN has 2 or more of the following clinical manifestations: oedema, macroscopic hematuria or dipstick hematuria of ≥ 2, or diastolic blood pressure of >80mmHg if ≤ 13 years of age and >90 mmHg if >13 years of age, in the presence of a reduced complement level (C3) and evidence of streptococcal infection by either elevated ASO or anti-DNAse B titres or positive cultures of GAS from skin, if sores are present, or from the throat in the absence of skin sores.

There is evidence that outbreaks can be halted by treating all children with any evidence of skin sores with intra-muscular (IM) benzathine penicillin to stop the transmission of the bacteria in the community [43]. In experimental PSGN, the nephritic process is prevented if penicillin is given within 3 days of the streptococcal infection [41]. Prevention of epidemics requires the control of spread of skin sores and infected scabies [44]. Following the identification of a case(s), family and household members are screened for the presence of skin sores and scabies and tested for urinary abnormalities. Those with skin manifestations are treated with penicillin. Those with urinary abnormalities undergo complete investigation for PSGN including urea, electrolytes, C3, ASO, anti-DNAse B and cultures for streptococcal infection.

Prevention of epidemics of PSGN requires a community level control of skin sores and infected scabies. Promotion of regular washing, especially of children, will prevent spread. Improvement in housing, especially reduction in overcrowding, will hinder spread of infectious disease. The significant decline in PSGN in Singapore children is attributed to an improvement in the socioeconomic status, the health care system and urbanization of the country [9].

Although research into the development of a vaccine is advanced and 3 GAS vaccines have been approved for phase 1 human trials [45], it is unlikely to be available in the near future.

### **8. Prognosis**

120 An Update on Glomerulopathies – Clinical and Treatment Aspects

orally or intravenously is given 12 hourly. Usually treatment is required for less than 48 hours. Sometimes other anti-hypertensive agents may be needed, especially when the blood pressure is very high and it is unsafe to wait for the effect of diuretic therapy. Nifedipine is then given every 4-6 hours in doses of 5-10 mg. Rarely parenteral hydralazine may be required. Captopril has shown to be effective [37, 38] but should be used in caution in the presence of renal failure and hyperkalemia. Occasionally acute renal failure requires dialysis. Pulmonary oedema may be complication of severe fluid overload and needs urgent

It is important to check serum complement levels 6-8 weeks after initial testing to make sure they have returned to normal. Blood pressure should be monitored every month for 6 months and then 6 monthly. Renal function tests and serum creatinine levels repeated every 3 months after the acute phase for 1 year and then yearly after that. Urine should be checked

Aggressive therapy using pulse methyprednisolone has been used in adults with poor prognostic factors such as nephritic range proteinuria, cellular crescents on biopsy and renal insufficiency [39]. Plasmapharesis and pulse methyprednisolone was successfully used in a 6 year old girl with garland pattern PSGN [40]. Whether this would benefit all patients with

Penicillin treatment is given to treat any persisting streptococcal infection [41]. Methicillin resistant staphylococcus should be treated with appropriate antibiotics. Specific infections require treatment with specific antibiotics or antiviral agents. Treatment of the underlying infection may resolve the glomerulonephritis as well. A review of six trials (a total of 159 patients) of which five were specified as hepatitis B virus-associated membranous glomerulonephritis (HBV-MN) showed that antiviral therapy for hepatitis B infections including IFN and lamivudine is effective in leading to remission of proteinuria, HBeAg

A case of PSGN has 2 or more of the following clinical manifestations: oedema, macroscopic hematuria or dipstick hematuria of ≥ 2, or diastolic blood pressure of >80mmHg if ≤ 13 years of age and >90 mmHg if >13 years of age, in the presence of a reduced complement level (C3) and evidence of streptococcal infection by either elevated ASO or anti-DNAse B titres or positive cultures of GAS from skin, if sores are present, or from the throat in the

There is evidence that outbreaks can be halted by treating all children with any evidence of skin sores with intra-muscular (IM) benzathine penicillin to stop the transmission of the bacteria in the community [43]. In experimental PSGN, the nephritic process is prevented if penicillin is given within 3 days of the streptococcal infection [41]. Prevention of epidemics requires the control of spread of skin sores and infected scabies [44]. Following the identification of a case(s), family and household members are screened for the presence of skin sores and scabies and tested for urinary abnormalities. Those with skin manifestations are treated with penicillin. Those with urinary abnormalities undergo complete investigation for PSGN including urea, electrolytes, C3, ASO, anti-DNAse B and cultures for

Prevention of epidemics of PSGN requires a community level control of skin sores and infected scabies. Promotion of regular washing, especially of children, will prevent spread.

clearance, and HBV-DNA reduction in both children and adults [42].

treatment.

for hematuria and proteinuria every 3-6 months.

poor prognosis has not been studied.

**7. Preventing spread** 

absence of skin sores.

streptococcal infection.

In keeping with the clinico-pathological picture, the prognosis of PSGN is also extremely variable, and largely influenced by clinical presentation and histopathology. An episode of PSGN may result in complete recovery, progression of symptoms or progression to renal failure. Persistence of symptoms may represent either a slow recovery, limited injury without further progression or progression to renal failure.

The immediate prognosis is generally good. In general, children are believed to have an excellent prognosis with the majority showing complete recovery [5]. Fewer than 1% of children have elevated serum creatinine values after 10-15 years of follow-up. Adults have a poorer prognosis overall. Early mortality can be as high as 25% in the elderly who have congestive cardiac failure or azotemia in the early phase. In adults approximately 25% will progress to chronic renal failure. These are usually those with massive proteinuria which suggests a worse prognosis and often signifies the garland pattern of immune deposits on pathology.

It is difficult to predict the prognosis in an individual case particularly early in the disease. While a typical presentation and clinical course indicates a good prognosis and an atypical presentation, severe persistent hypertension and abnormal renal function tests, massive proteinuria and older age group suggest a poor prognosis, there is a lack of a clinical or biochemical marker that might differentiate those with a good prognosis from those with a poorer outcome. Neutrophil gelatinase-associated lipocalin (NGAL), is emerging as a promising biomarker of acute kidney injury [46, 47], but has not yet been evaluated in PSGN.

However, some studies have reported persistent urinary abnormalities [8, 11, 19, 48, 49] and subtle abnormalities in renal function, as defined by reduction in renal functional reserve, in patients who had recovered from PSGN without apparent sequelae [50]

Epidemic cases have a better prognosis than sporadic cases [14, 51, 52], but not always. An outbreak of PSGN in Brazil following an epidemic of *Streptococcus equi zooepidemicus*  resulted in a high prevalence of renal abnormalities at a mean follow-up of 5.4 years [53, 54]. These were, however, mainly adult patients. A study of Iranian children has shown that even mild PSGN may result in impaired renal function and that a rising diastolic blood pressure may be an early sign of worsening renal function [55].

Elderly people have poorer outcomes as do those with co-morbidities, including diabetes, cardiovascular and liver diseases [5, 28]. In an Aboriginal population with high rates of ESRD, follow-up of children 6-18 years (mean 14.6 years) after epidemic PSGN showed that risk of overt proteinuria was 6 times ( 95% CI 2.2-16.9) greater than in healthy controls after adjustment of age, sex and birth weight [11, 56]. The Australian Aboriginal population is at considerably higher risk than the general Australian population of developing chronic diseases such as diabetes mellitus, cardiovascular and renal diseases. There is also a greater burden of infectious disease and adverse early life factors such as low birth and infant weights. It is proposed that in this high risk population with multiple adverse renal

Post-Infectious Glomerulonephritis 123

[20] Handa, T., et al., *Glomerulonephritis induced by methicillin-sensitive Staphylococcus aureus* 

[21] Kobayashi, M. and A. Koyama, *Methicillin-resistant Staphylococcus aureus (MRSA)* 

[22] Koyama, A., et al., *Glomerulonephritis associated with MRSA infection: a possible role of* 

[23] Zaki, S.A. and P. Shanbag, *Acute glomerulonephritis: an unusual manifestation of* 

[24] Kaehny, W.D., et al., *Acute nephritis and pulmonary alveolitis following pneumococcal* 

[25] Venkataseshan, V.S., et al., *Hepatitis-B-associated glomerulonephritis: pathology, pathogenesis, and clinical course.* Medicine (Baltimore), 1990. 69(4): p. 200-16. [26] Wei, R.B., et al., *[Clinicopathological analysis on hepatitis B virus-associated* 

[27] Chen, Y.M., et al., *An unusual cause of membranous glomerulonephritis in a patient with HIV.*

[28] Nordstrand, A., M. Norgren, and S.E. Holm, *Pathogenic Mechanism of Acute Post-*

[29] Sorger, K., et al., *Subtypes of acute postinfectious glomerulonephritis. Synopsis of clinical and* 

[30] Safadi, R., et al., *Glomerulonephritis associated with acute hepatitis B.* Am J Gastroenterol,

[31] Aggarwal, A., D. Kumar, and R. Kumar, *Acute glomerulonephritis in hepatitis A virus* 

[32] Pan, C.G., *Evaluation of gross hematuria*. Pediatr Clin North Am, 2006. 53(3): p. 401-12, vi.

[35] El-Husseini, A.A., et al., A*cute postinfectious crescentic glomerulonephritis: clinicopathologic* 

[36] Tasic, V. and M. Polenakovic, *Occurrence of subclinical post-streptococcal glomerulonephritis* 

[37] Morsi, M.R., et al., *Evaluation of captopril versus reserpine and frusemide in treating* 

[38] Parra, G., et al., *Short-term treatment with captopril in hypertension due to acute* 

[39] Raff, A., et al., *Crescentic post-streptococcal glomerulonephritis with nephrotic syndrome in the adult: is aggressive therapy warranted?* Clin Nephrol, 2005. 63(5): p. 375-80. [40] Suyama, K., Y. Kawasaki, and H. Suzuki, *Girl with garland-pattern poststreptococcal acute* 

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*glomerulonephritis presenting with renal failure and nephrotic syndrome.* Pediatr Int,

*glomerulonephritis in 205 patients].* Zhonghua Shi Yan He Lin Chuang Bing Du Xue

*Streptococcal Glomerulonephritis.* Scandinavian Journal of Infectious Diseases, 1999.

*Plasmodium vivax malaria.* Ann Trop Paediatr, 2011. 31(2): p. 181-4.

*infection in glomerulonephritis--a novel hazard emerging on the horizon.* Nephrol Dial

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*in family contacts.* J Paediatr Child Health, 2003. 39(3): p. 177-9.

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#### **9. References**


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[1] Kurlan, R., D. Johnson, and E.L. Kaplan, *Streptococcal infection and exacerbations of* 

[2] Carapetis, J.R., et al., *The global burden of group A streptococcal diseases.* Lancet Infect Dis,

[3] Steer, A.C., M.H. Danchin, and J.R. Carapetis, *Group A streptococcal infections in children.* J

[4] Jackson, S.J., A.C. Steer, and H. Campbell, *Systematic Review: Estimation of global burden of* 

[6] Rodriguez-Iturbe, B., L. Rubio, and R. Garcia, *Attack rate of poststreptococcal nephritis in* 

[7] Sagel, I., et al., *Occurrence and nature of glomerular lesions after group A streptococci infections* 

[8] Ilyas, M. and A. Tolaymat, *Changing epidemiology of acute post-streptococcal* 

[9] Yap, H.K., et al., *Acute glomerulonephritis--changing patterns in Singapore children.* Pediatr

[10] Markowitz, M., *Changing epidemiology of group A streptococcal infections.* Pediatr Infect Dis

[11] White, A.V., W.E. Hoy, and D.A. McCredie, *Childhood post-streptococcal glomerulonephritis as a risk factor for chronic renal disease in later life.* Med J Aust, 2001. 174(10): p. 492-6. [12] Streeton, C.L., et al., *An epidemic of acute post-streptococcal glomerulonephritis among* 

[13] *Communicable Diseases Surveillance System.* 2002, Centre for Communicable Diseases:

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[16] Tejani, A. and E. Ingulli, *Poststreptococcal glomerulonephritis. Current clinical and pathologic* 

[17] Bingler, M.A., D. Ellis, and M.L. Moritz, *Acute post-streptococcal glomerulonephritis in a 14 month-old boy: why is this uncommon?* Pediatr Nephrol, 2007. 22(3): p. 448-50. [18] Shet, A., et al., *Immune response to group A streptococcal C5a peptidase in children: implications for vaccine development.* J Infect Dis, 2003. 188(6): p. 809-17. [19] Kanjanabuch, T., W. Kittikowit, and S. Eiam-Ong, *An update on acute postinfectious* 

*glomerulonephritis worldwide.* Nat Rev Nephrol, 2009. 5(5): p. 259-69.

*aboriginal children.* J Paediatr Child Health, 1995. 31(3): p. 245-8.

*prevention.* Medicine (Baltimore), 1955. 34(4): p. 431-50.

*concepts.* Nephron, 1990. 55(1): p. 1-5.

*glomerulonephritis in Northeast Florida: a comparative study*. Pediatr Nephrol, 2008.

*streptococcal glomerulonephritis.* Trop Med Int Health, 2011. 16(1): p. 2-11. [5] Rodriguez-Iturbe, B. and J.M. Musser, *The current state of poststreptococcal* 

*glomerulonephritis.* J Am Soc Nephrol, 2008. 19(10): p. 1855-64.

*families. A prospective study.* Lancet, 1981. 1(8217): p. 401-3.

*in children.* Ann Intern Med, 1973. 79(4): p. 492-9.

*childhood tics and obsessive-compulsive symptoms: a prospective blinded cohort study.*

*non-suppurative sequelae of upper respiratory tract infection: rheumatic fever and post-*

be in lower risk populations [11, 44, 57].

2005. 5(11): p. 685-94.

23(7): p. 1101-6.

Darwin, NT.

Nephrol, 1990. 4(5): p. 482-4.

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Pediatrics, 2008. 121(6): p. 1188-97.

Paediatr Child Health, 2007. 43(4): p. 203-13.

**9. References** 


**8** 

*Brazil* 

*S. pyogenes* **Infections and Its Sequelae** 

*3Immunology Investigation Institute, National Institute for Science and Technology,* 

Suppurative streptococcal infections of the throat and the skin generate stimuli that lead Rheumatic fever (RF) in 1 to 5% of susceptible children. The disease manifests initially as polyarthritis, carditis/valvulitis, Sydenham`s chorea, erythema marginatum and/or

RF occurs at an early phase of life (3 to 19 years of age); thus, heart damage (carditis) can appear in very young children. Rheumatic carditis usually presents as pancarditis, affecting the endocardium, myocardium and pericardium. Recurrent acute cardiac lesions frequently evolve into chronic rheumatic heart disease (RHD), of which valvular deformities are the most important sequelae; these deformities lead to mitral and aortic regurgitation and/or stenosis. Valve replacement surgery is usually the only treatment for chronic RHD patients

Here, we will present three cases of young RHD patients who underwent valve replacement

Post-streptococcal glomerulonephritis (PSGN) is another immune sequelae that presents a latency period of one to three weeks after scarlet fever, streptococcal pharyngitis and

PSGN has become a rare disease, especially in adults in developed countries, due to an improved standard of living, earlier treatment of pharyngeal infections and widespread use of antibiotics (Rodriguez-Iturbe & Musser, 2008). Despite decades of research, the pathogenesis of PSGN remains obscure. It is still unclear whether or to what extent autoimmune reactions are involved, but several studies have shown that different streptococcal antigens are detectable by immunohistology in the diseased kidneys (Rodriguez-Iturbe & Batsford, 2007). These data are in favor of direct contributions of streptococcal nephritogenic factors to PSGN pathogenesis, although intact bacteria have

and the autoimmune reactivity that triggered the heart-tissue rheumatic lesions.

never been found in affected kidneys. Two PSGN cases will also be presented.

subcutaneous nodules. Chronic renal disease can also occur.

and incurs high costs for both public and private health systems.

**1. Introduction** 

purulent skin infections.

L. Guilherme1,3, S. Freschi de Barros1,3

*1Heart Institute (InCor), School of Medicine,* 

*University of São Paulo, São Paulo,* 

*University of São Paulo, São Paulo,* 

*University of São Paulo, São Paulo, 4Nephrology Division, School of Medicine, University of São Paulo, São Paulo,* 

A.C. Tanaka1, M.C. Ribeiro Castro4 and J. Kalil1,2,3

*2Clinical Immunology and Allergy Division, School of Medicine,* 


## *S. pyogenes* **Infections and Its Sequelae**

L. Guilherme1,3, S. Freschi de Barros1,3 A.C. Tanaka1, M.C. Ribeiro Castro4 and J. Kalil1,2,3 *1Heart Institute (InCor), School of Medicine, University of São Paulo, São Paulo, 2Clinical Immunology and Allergy Division, School of Medicine, University of São Paulo, São Paulo, 3Immunology Investigation Institute, National Institute for Science and Technology, University of São Paulo, São Paulo, 4Nephrology Division, School of Medicine, University of São Paulo, São Paulo, Brazil* 

#### **1. Introduction**

124 An Update on Glomerulopathies – Clinical and Treatment Aspects

[41] Bergholm, A.M. and S.E. Holm, *Effect of early penicillin treatment on the development of* 

[42] Yi, Z., Y.W. Jie, and Z. Nan, *The efficacy of anti-viral therapy on hepatitis B virus-associated* 

[43] Johnston, F., et al., *Evaluating the use of penicillin to control outbreaks of acute poststreptococcal glomerulonephritis.* Pediatr Infect Dis J, 1999. 18(4): p. 327-32. [44] Van Buynder, P.G., et al., *Streptococcal infection and renal disease markers in Australian* 

[45] Georgousakis, M.M., et al., *Moving forward: a mucosal vaccine against group A* 

[46] Haase, M., et al., *Accuracy of neutrophil gelatinase-associated lipocalin (NGAL) in diagnosis* 

[47] Devarajan, P., *Neutrophil gelatinase-associated lipocalin (NGAL): a new marker of kidney* 

[48] Wong, W., M. Morris, and J. Zwi, *Outcome of severe acute post-streptococcal* 

[49] Buzio, C., et al., *Significance of albuminuria in the follow-up of acute poststreptococcal* 

[50] Cleper, R., et al., *Renal functional reserve after acute poststreptococcal glomerulonephritis.*

[51] Blyth, C.C., P.W. Robertson, and A.R. Rosenberg, *Post-streptococcal glomerulonephritis in Sydney: a 16-year retrospective review.* J Paediatr Child Health, 2007. 43(6): p. 446-50. [52] Eison, T.M., et al., *Post-streptococcal acute glomerulonephritis in children: clinical features and* 

[53] Sesso, R. and S.W. Pinto, *Five-year follow-up of patients with epidemic glomerulonephritis due to Streptococcus zooepidemicus*. Nephrol Dial Transplant, 2005. 20(9): p. 1808-12. [54] Sesso, R., S. Wyton, and L. Pinto, *Epidemic glomerulonephritis due to Streptococcus zooepidemicus in Nova Serrana, Brazil.* Kidney Int Suppl, 2005(97): p. S132-6. [55] Gheissari, A., et al., *Outcome of Iranian children with mild post streptococcal* 

[56] Yamagata, K., et al., *Chronic kidney disease perspectives in Japan and the importance of* 

[57] Hoy, W., et al., *Stemming the tide: reducing cardiovascular disease and renal failure in* 

*glomerulonephritis.* Saudi J Kidney Dis Transpl, 2010. 21(3): p. 571-4.

*urinalysis screening.* Clin Exp Nephrol, 2008. 12(1): p. 1-8.

*Australian Aborigines.* Aust N Z J Med, 1999. 29(3): p. 480-3.

*and prognosis in acute kidney injury: a systematic review and meta-analysis.* Am J Kidney

*glomerulonephritis in New Zealand children.* Pediatric Nephrology, 2009. 24(5): p.

*aboriginal children.* Med J Aust, 1992. 156(8): p. 537-40.

*streptococcus.* Expert Rev Vaccines, 2009. 8(6): p. 747-60.

*disease.* Scand J Clin Lab Invest Suppl, 2008. 241: p. 89-94.

*glomerulonephritis.* Clin Nephrol, 1994. 41(5): p. 259-64.

*pathogenesis.* Pediatr Nephrol, 2011. 26(2): p. 165-80.

Pediatr Nephrol, 1997. 11(4): p. 473-6.

Scand C, 1983. 91(4): p. 271-81.

Dis, 2009. 54(6): p. 1012-24.

165-73.

1021-1026.

*experimental poststreptococcal glomerulonephritis.* Acta Pathol Microbiol Immunol

*glomerulonephritis: A systematic review and meta-analysis.* Ann Hepatol, 2011. 10(2): p.

Suppurative streptococcal infections of the throat and the skin generate stimuli that lead Rheumatic fever (RF) in 1 to 5% of susceptible children. The disease manifests initially as polyarthritis, carditis/valvulitis, Sydenham`s chorea, erythema marginatum and/or subcutaneous nodules. Chronic renal disease can also occur.

RF occurs at an early phase of life (3 to 19 years of age); thus, heart damage (carditis) can appear in very young children. Rheumatic carditis usually presents as pancarditis, affecting the endocardium, myocardium and pericardium. Recurrent acute cardiac lesions frequently evolve into chronic rheumatic heart disease (RHD), of which valvular deformities are the most important sequelae; these deformities lead to mitral and aortic regurgitation and/or stenosis. Valve replacement surgery is usually the only treatment for chronic RHD patients and incurs high costs for both public and private health systems.

Here, we will present three cases of young RHD patients who underwent valve replacement and the autoimmune reactivity that triggered the heart-tissue rheumatic lesions.

Post-streptococcal glomerulonephritis (PSGN) is another immune sequelae that presents a latency period of one to three weeks after scarlet fever, streptococcal pharyngitis and purulent skin infections.

PSGN has become a rare disease, especially in adults in developed countries, due to an improved standard of living, earlier treatment of pharyngeal infections and widespread use of antibiotics (Rodriguez-Iturbe & Musser, 2008). Despite decades of research, the pathogenesis of PSGN remains obscure. It is still unclear whether or to what extent autoimmune reactions are involved, but several studies have shown that different streptococcal antigens are detectable by immunohistology in the diseased kidneys (Rodriguez-Iturbe & Batsford, 2007). These data are in favor of direct contributions of streptococcal nephritogenic factors to PSGN pathogenesis, although intact bacteria have never been found in affected kidneys. Two PSGN cases will also be presented.

*S. pyogenes* Infections and Its Sequelae 127

reviewed. Briefly, antibodies against N-acetyl β-D-glucosamine, a polysaccharide present in both the streptococcal cell wall and heart valvular tissue displayed cross reactivity against laminin, an extracellular matrix alpha-helical coiled-coil protein that surrounds heart cells

Among human proteins, cardiac myosin and vimentin seem to be the major target antigens. By using affinity-purified anti-myosin antibodies, Cunningham´s group identified a five amino acid residue (Gln-Lys-Ser-Lys-Gln) epitope of the N-terminal M5 and M6 proteins as

Cunningham´s group found that streptococcal and human cross-reactive antibodies upregulate the adhesion molecule VCAM-1 after binding to the endothelial surface, leading to inflammation, cellular infiltration and valve scarring (Gavin et al., 2000, Roberts et al, 2001). These data established the role of the heart–tissue cross-reactive antibodies (anti-cardiac myosin and laminin) in the early stages of inflammation and T cell infiltration in RHD lesions. Studies performed in the last 25 years showed that CD4+ cells are the major effectors of autoimmune reactions in the heart tissue in RHD patients (Raizada et al., 1984; Kemeny et al.,1989; Guilherme et al., 1995). However, the role of T cells in the pathogenesis of RF and RHD was demonstrated through the analysis of heart-tissue infiltrating T cell clones (Guilherme et al., 1995). Immunodominant peptides of the M5 protein (residues 81-96 and 83-103) displayed cross-reactivity with valvular proteins and cardiac myosin peptides by molecular mimicry (Faé et al., 2006; Yoshinaga et al., 1995; Guilherme et al, 1995). These M5 epitopes were also preferentially recognized by peripheral T lymphocytes from RHD patients when compared with normal individuals, mainly in the context of HLA-DR7 (Guilherme et al., 2001). Analysis of the T cell receptors (TCR) of peripheral and intralesional T cells from RHD patients showed several antigen-driven oligoclonal T cell expansions at the site of heart-tissue lesions (Guilherme et al, 2000). These autoreactive cells are CD4+ and produce inflammatory cytokines (TNF and IFNγ). IL-4+ cells are found in the myocardium; however, these cells are very scarce in the valve lesions of RHD patients. IL-4 is a Th2-type cytokine and plays a regulatory role in the inflammatory response mediated by Th1 cytokines. These findings indicate that the Th1/Th2 cytokine balance has a role in healing myocarditis, while the low numbers of IL-4-producing cells in the valves probably

and is also present in the valves (Cunningham, 2000; Guilherme et al., 2005).

induced progressive and permanent valve damage (Guilherme et al, 2004).

**Inflammation Rheumatic**

**Case 1** (++) AB-PR(+)

**Activity** 

Table 1. Histological data of Rheumatic Heart Disease patients

**Patients** 

**Case 3**- 1st surgery

(++) moderate

Three cases of RHD patients (clinical, surgical data) will be presented. Histological and immunological data obtained from peripheral blood and T-cell lines and T cell clones derived

**Mitral Valve Myocardium (LA)** 

VER(+) (+) (+) () AB-PR (+) (+)

(+) AB()

**Neovasc Fibrosis Calcification Inflammation Rheumatic** 

**Activity** 

from heart-tissue infiltrating T cells of these patients are summarized in Tables 1 and 2.

**Case 2-** (+) () () (++) () () ()

2nd surgery (+) (+) () LA-left atrium; AB-PR- Achoff Bodies in proliferative phase; Ver- verrucae, (-) negative;(+) mild;

cross-reactive with cardiac myosin (Cunningham et al., 1989).

### **2. Epidemiology**

### **2.1 Acute rheumatic fever**

The incidence of ARF in some developing countries exceeds 50 cases per 100,000 children (Carapetis et al., 2005). The worldwide incidence of RHD is at least 15.6 million cases and is responsible for around 233,000 deaths / year. However, these estimates are based on conservative assumptions, so the true disease burden is probably substantially higher (Carapetis et al., 2005). The incidence of ARF can vary from 0.7 to 508 per 100,000 children per year in different populations from several countries (Carapetis et al., 2005). In Brazil, according to the WHO epidemiological model and data from IBGE (Brazilian Institute of Geography and Statistics), the number of Streptococcal pharyngitis infections is around 10 million cases, which could lead to 30,000 new cases of RF, of which around 15,000 could develop cardiac lesions (Barbosa et al.,2009).

### **2.2 Post-streptococcal glomerulonephritis (PSGN)**

PSGN has become a rare disease, especially in adults in developed countries, due to an improved standard of living, earlier treatment of pharyngeal infections and widespread use of antibiotics (Rodriguez-Iturbe & Musser, 2008). However, the occurrence of acute postinfection glomerulonephritis (APIGN) has emerged as a major risk in diabetic patients all over the world (Nars et al., 2008).

The global incidence of acute PSGN was estimated at 472,000 cases per year, of which 456,000 occurred in less-developed countries (Carapetis, 2005). In agreement with these data, the incidence of PSGN ranges from 9.5 to 28.5 new cases per 100,000 individuals per year in developing countries (Rodríguez-Iturbe, 2008).

### **3. Autoimmunity is the major mechanism leading to both diseases**

### **3.1 Rheumatic fever and rheumatic heart disease**

The autoimmune reactions in RF and RHD are controlled by several genes related to both the innate and adaptive immune responses (Guilherme et al., 2011). Briefly, in the last 50 years, several genetic markers from different populations have been studied, and the susceptibility of developing RF/RHD was first associated with some alleles of HLA (human leukocytes antigens) class II genes (DRB1, DQB and DQA), which are located on human chromosome 6. HLA alleles are involved in antigen recognition by T lymphocytes through the T cell receptor (TCR). Later, some studies showed that the TNF-α gene, located in the same region of this chromosome was also associated with the disease. The TNF-α gene encodes the inflammatory TNF alpha protein, which is involved in the inflammatory process mediating heart-tissue lesions in RHD. Several other associations have been established based on gene variability by studying single nucleotide polymorphisms (SNPs). These genes code for other proteins also involved with the immune response (innate and adaptive pathways) (see Diagram 1) (Guilherme et al., 2011).

#### **3.1.1 Molecular mimicry**

Molecular mimicry mediates cross-reactivity between streptococcal antigens and human proteins. Several autoantigens have been identified, including cardiac myosin epitopes, vimentin and other intracellular proteins.

Several streptococcal and human cross-reactive antibodies have been found in the sera of RF patients and immunized rabbits and mice over the last 50 years and have been recently

The incidence of ARF in some developing countries exceeds 50 cases per 100,000 children (Carapetis et al., 2005). The worldwide incidence of RHD is at least 15.6 million cases and is responsible for around 233,000 deaths / year. However, these estimates are based on conservative assumptions, so the true disease burden is probably substantially higher (Carapetis et al., 2005). The incidence of ARF can vary from 0.7 to 508 per 100,000 children per year in different populations from several countries (Carapetis et al., 2005). In Brazil, according to the WHO epidemiological model and data from IBGE (Brazilian Institute of Geography and Statistics), the number of Streptococcal pharyngitis infections is around 10 million cases, which could lead to 30,000 new cases of RF, of which around 15,000 could

PSGN has become a rare disease, especially in adults in developed countries, due to an improved standard of living, earlier treatment of pharyngeal infections and widespread use of antibiotics (Rodriguez-Iturbe & Musser, 2008). However, the occurrence of acute postinfection glomerulonephritis (APIGN) has emerged as a major risk in diabetic patients all

The global incidence of acute PSGN was estimated at 472,000 cases per year, of which 456,000 occurred in less-developed countries (Carapetis, 2005). In agreement with these data, the incidence of PSGN ranges from 9.5 to 28.5 new cases per 100,000 individuals per

The autoimmune reactions in RF and RHD are controlled by several genes related to both the innate and adaptive immune responses (Guilherme et al., 2011). Briefly, in the last 50 years, several genetic markers from different populations have been studied, and the susceptibility of developing RF/RHD was first associated with some alleles of HLA (human leukocytes antigens) class II genes (DRB1, DQB and DQA), which are located on human chromosome 6. HLA alleles are involved in antigen recognition by T lymphocytes through the T cell receptor (TCR). Later, some studies showed that the TNF-α gene, located in the same region of this chromosome was also associated with the disease. The TNF-α gene encodes the inflammatory TNF alpha protein, which is involved in the inflammatory process mediating heart-tissue lesions in RHD. Several other associations have been established based on gene variability by studying single nucleotide polymorphisms (SNPs). These genes code for other proteins also involved with the immune response (innate and adaptive pathways) (see Diagram 1)

Molecular mimicry mediates cross-reactivity between streptococcal antigens and human proteins. Several autoantigens have been identified, including cardiac myosin epitopes,

Several streptococcal and human cross-reactive antibodies have been found in the sera of RF patients and immunized rabbits and mice over the last 50 years and have been recently

**3. Autoimmunity is the major mechanism leading to both diseases** 

**2. Epidemiology** 

**2.1 Acute rheumatic fever** 

develop cardiac lesions (Barbosa et al.,2009).

over the world (Nars et al., 2008).

(Guilherme et al., 2011).

**3.1.1 Molecular mimicry** 

vimentin and other intracellular proteins.

**2.2 Post-streptococcal glomerulonephritis (PSGN)** 

year in developing countries (Rodríguez-Iturbe, 2008).

**3.1 Rheumatic fever and rheumatic heart disease** 

reviewed. Briefly, antibodies against N-acetyl β-D-glucosamine, a polysaccharide present in both the streptococcal cell wall and heart valvular tissue displayed cross reactivity against laminin, an extracellular matrix alpha-helical coiled-coil protein that surrounds heart cells and is also present in the valves (Cunningham, 2000; Guilherme et al., 2005).

Among human proteins, cardiac myosin and vimentin seem to be the major target antigens. By using affinity-purified anti-myosin antibodies, Cunningham´s group identified a five amino acid residue (Gln-Lys-Ser-Lys-Gln) epitope of the N-terminal M5 and M6 proteins as cross-reactive with cardiac myosin (Cunningham et al., 1989).

Cunningham´s group found that streptococcal and human cross-reactive antibodies upregulate the adhesion molecule VCAM-1 after binding to the endothelial surface, leading to inflammation, cellular infiltration and valve scarring (Gavin et al., 2000, Roberts et al, 2001). These data established the role of the heart–tissue cross-reactive antibodies (anti-cardiac myosin and laminin) in the early stages of inflammation and T cell infiltration in RHD lesions.

Studies performed in the last 25 years showed that CD4+ cells are the major effectors of autoimmune reactions in the heart tissue in RHD patients (Raizada et al., 1984; Kemeny et al.,1989; Guilherme et al., 1995). However, the role of T cells in the pathogenesis of RF and RHD was demonstrated through the analysis of heart-tissue infiltrating T cell clones (Guilherme et al., 1995). Immunodominant peptides of the M5 protein (residues 81-96 and 83-103) displayed cross-reactivity with valvular proteins and cardiac myosin peptides by molecular mimicry (Faé et al., 2006; Yoshinaga et al., 1995; Guilherme et al, 1995). These M5 epitopes were also preferentially recognized by peripheral T lymphocytes from RHD patients when compared with normal individuals, mainly in the context of HLA-DR7 (Guilherme et al., 2001). Analysis of the T cell receptors (TCR) of peripheral and intralesional T cells from RHD patients showed several antigen-driven oligoclonal T cell expansions at the site of heart-tissue lesions (Guilherme et al, 2000). These autoreactive cells are CD4+ and produce inflammatory cytokines (TNF and IFNγ). IL-4+ cells are found in the myocardium; however, these cells are very scarce in the valve lesions of RHD patients. IL-4 is a Th2-type cytokine and plays a regulatory role in the inflammatory response mediated by Th1 cytokines. These findings indicate that the Th1/Th2 cytokine balance has a role in healing myocarditis, while the low numbers of IL-4-producing cells in the valves probably induced progressive and permanent valve damage (Guilherme et al, 2004).

Three cases of RHD patients (clinical, surgical data) will be presented. Histological and immunological data obtained from peripheral blood and T-cell lines and T cell clones derived from heart-tissue infiltrating T cells of these patients are summarized in Tables 1 and 2.


LA-left atrium; AB-PR- Achoff Bodies in proliferative phase; Ver- verrucae, (-) negative;(+) mild; (++) moderate

Table 1. Histological data of Rheumatic Heart Disease patients

*S. pyogenes* Infections and Its Sequelae 129

left ventricular diastolic diameter (67/43 mm) and left atrial diameter of 62 mm, significant mitral regurgitation, aortic insufficiency and moderate impact tricuspid regurgitation with mild rebound were found. Subjected to two surgeries, first for mitral valve repair and prosthetic aortic and tricuspid valves, then for exchange of the mitral and aortic

Acute glomerulonephritis can occur sporadically or endemically as a result of infections of

Genetic susceptibility factors are likely involved with the development of the disease. HLA class II alleles (DR4 and DRB1\* 03011) have been found to be associated with PSGN compared to healthy controls. Genetic association with endothelial nitric oxide synthase intron 4 a/b (eNOSa/b) defined by variable numbers of tandem repeats (VNTR)

The disease is mediated by immune complexes and complement pathway activation. Several theories seek to explain the formation of immune complexes in glomeruli. The most accepted one is that a streptococcal antigen, with affinity for the glomerular structures, can be deposited in the glomerulus, activating the host immune response and initiating

Apparently, molecular mimicry between streptococcal antigens and glomerular proteins leads to tissue damage. Two antigens have been investigated as potential causes of PSGN: the plasmin receptor linked to nephritis (NAPlr), identified as glyceraldehyde 3-phosphate dehydrogenase, and a protein known as streptococcal pyrogenic exotoxin B (SpeB). Both are present in renal biopsies of patients with PSGN and are capable of activating the alternative pathway of the complement system. In addition, they are capable of promoting enhanced expression of adhesion molecules, facilitating inflammatory reactions mediated by cytokines (IL-6, TNF α, IL-8 and TGF ß). It seems that the nephritogenic properties of NAPlr and SpeB are related to the binding ability of plasmin, which facilitates the deposition of immune complexes (IgG and C3, properdin and C5) in the glomeruli and subsequent inflammation

Molecular mimicry, as mentioned above, leads to the recognition of streptococcal antigens and laminin, collagen and glomerular basement membrane (GBM). Sub-epithelial localization of immune complexes and complement factors in the injured glomeruli points

As mentioned before, renal inflammation may result from a myriad of insults and is often characterized by the presence of infiltrating inflammatory leukocytes within the glomerulus or tubular interstitium. Accumulating evidence indicates that infiltrating leukocytes are the

Two cases of PSGN are presented in which anti-streptolysin O (ASO) was positive,

Male 6 years old, presented swollen eyes followed by bilateral periorbital edema followed by progression of lower limb edema and increased abdominal size, decreased urine volume and urine darkness. Lab tests detected hematuria, increased serum levels of urea (63.0 mg/dl) and creatinine (1.2 mg/dl). Decreased levels of complement (16.1 mg/dl) and fractions C3 and C4 (both 11.7 mg/dl) were found. The patient also presented increased levels of ASO (1055 IU).

development of immune complexes *in situ* (Rodriguez-Iturbe & Bastford, 2007).

(Rodriguez-Iturbe & Bastford, 2007; Rodriguez-Iturbe & Musser, 2008).

towards a crucial role of the host immune system in tissue destruction.

key to the induction of renal injury.

**Case #1**

indicating a previous infection by *S. pyogenes.*

bioprostheses, and tricuspid valve repair.

**3.2 Post Streptococcal Glomerulonephritis (PSGN)** 

polymorphism was also described (Ahn & Ingulli, 2008).

both the upper airways and skin by group A streptococcus strains.


Amino acid sequences of M5 protein were based on sequence published by Philips et al, 1981 and Manjula et al, 1985. M5 (81-96)-DKLKQQRDTLSTQKET; M5 (83-103)-LKQQRDTLSTQKETLEREVQN; M5-(163-177) ETIGTLKKILDETVK; cardiac myosin beta chain sequences published by Diederich et al, 1989: LMM 10 (1413-1430) CSSLEKTKHRLQNEIEDL; LMM12 (1439-1456) AAAAALDKKRNFDKILA; LMM25 (1607-1624) RSRNEALRVKKKMEGDLN, (Guilherme et al, 1995, Faé et al, 2006).

Table 2. T cells from heart-tissue of Rheumatic Heart Disease patients recognize streptococcal peptides and cardiac proteins.

### **Case # 1**

Male 4 years old, presented mitral, aortic and tricuspid regurgitation, left ventricular diastolic diameter of 51 mm and systolic diameter of 34 mm, ejection fraction (LVEF) of 78%, left atrium (LA) of 40 mm, thickened pericardium.

At surgery, mitral valve prolapse was observed with very long strings and small tears of rope. Mitral annulus was dilated. A mitral valve replacement was done. Heart biopsy showed chronic valvulitis with areas of mucoid collagen degeneration and papillary muscles with Aschoff nodules in the granulomatous stage (Table 1).

#### **Case # 2**

Male 6 years old, presented clinical features of fever, polyarthritis and carditis with mitral valve involvement. On this occasion, patient showed evidence of inflammatory activity; Gallium 67 positive scintigraphy; endomyocardial biopsy suggestive of rheumatic carditis. A left ventricular diastolic diameter of 59 mm, left ventricular systolic diameter of 39 mm, ejection fraction (LVEF) of 71% and left atrium (LA) of 52 mm were observed. Two valve correction surgeries were performed. Pathological examination of the mitral valve showed sequelae of chronic valvulitis with intense fibrosis and mucoid degeneration.

#### **Case #3**

Male 10 years old, presented clinical features of fever, polyarthritis and carditis with progressive cardiac heart failure and mitral and aortic shortcomings as well as relapses of acute outbreak by irregular use of secondary prophylaxis with benzathine penicillin and progression to chronic atrial fibrillation, culminating in death at 18 years of life. Increased

M5 (163-177) 30-44 kDa;

M5 (83-103) >150 kDa

Male 4 years old, presented mitral, aortic and tricuspid regurgitation, left ventricular diastolic diameter of 51 mm and systolic diameter of 34 mm, ejection fraction (LVEF) of 78%,

At surgery, mitral valve prolapse was observed with very long strings and small tears of rope. Mitral annulus was dilated. A mitral valve replacement was done. Heart biopsy showed chronic valvulitis with areas of mucoid collagen degeneration and papillary

Male 6 years old, presented clinical features of fever, polyarthritis and carditis with mitral valve involvement. On this occasion, patient showed evidence of inflammatory activity; Gallium 67 positive scintigraphy; endomyocardial biopsy suggestive of rheumatic carditis. A left ventricular diastolic diameter of 59 mm, left ventricular systolic diameter of 39 mm, ejection fraction (LVEF) of 71% and left atrium (LA) of 52 mm were observed. Two valve correction surgeries were performed. Pathological examination of the mitral valve showed

Male 10 years old, presented clinical features of fever, polyarthritis and carditis with progressive cardiac heart failure and mitral and aortic shortcomings as well as relapses of acute outbreak by irregular use of secondary prophylaxis with benzathine penicillin and progression to chronic atrial fibrillation, culminating in death at 18 years of life. Increased

Amino acid sequences of M5 protein were based on sequence published by Philips et al, 1981 and Manjula et al, 1985. M5 (81-96)-DKLKQQRDTLSTQKET; M5 (83-103)-LKQQRDTLSTQKETLEREVQN; M5-(163-177) ETIGTLKKILDETVK; cardiac myosin beta chain sequences published by Diederich et al, 1989: LMM 10 (1413-1430) CSSLEKTKHRLQNEIEDL; LMM12 (1439-1456) AAAAALDKKRNFDKILA;

LMM25 (1607-1624) RSRNEALRVKKKMEGDLN, (Guilherme et al, 1995, Faé et al, 2006). Table 2. T cells from heart-tissue of Rheumatic Heart Disease patients recognize

M5 protein peptides

M5 (81-96)

M5 (83-103)

M5(11-25) M5 (81-96) M5 (83-103)

HLA Class II
