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## Meet the editor

Dr. Hülya Çiçek received her medical degree from the Çukurova University School of Medicine, Türkiye, in 1991. She completed her residency and training in biochemistry and clinical biochemistry in 2004 at the Gaziantep University, Türkiye. She became an associate professor in 2013. She is currently a full professor in the Department of Medical Biochemistry at Gaziantep University School of Medicine. Dr. Çiçek also con-

ducts academic studies in various disciplines. She worked as a thesis supervisor for numerous master's and doctoral students. She took on various assignments in many international congresses and is a member of journal editorial and reviewer boards. She is currently Head of the Department of Medical Biochemistry and Central Laboratory Coordinator at the University Hospital in Türkiye.

### Contents


Preface

Structural or functional disorders of the kidneys, which are organs with very important functions in the human body, lead to many clinical problems. This book provides a comprehensive overview of current approaches to the diagnosis, treatment, and monitoring of common kidney diseases, including nephritis, nephrotic syndrome, and nephrosis. It also discusses novel developments in subjects such as proteasuria in nephrotic syndrome, diabetic nephropathy, membranous nephropathy, complementmediated kidney disease, and sickle cell nephropathy. I would like to thank our authors and publishing team, who supported our book in the hope that it would shed light on the scientists who work devotedly on scientific platforms for humanity.

> **Hülya Çiçek**  School of Medicine,

Gaziantep University, Gaziantep, Turkey

Medical Biochemistry Department,

## Preface

Structural or functional disorders of the kidneys, which are organs with very important functions in the human body, lead to many clinical problems. This book provides a comprehensive overview of current approaches to the diagnosis, treatment, and monitoring of common kidney diseases, including nephritis, nephrotic syndrome, and nephrosis. It also discusses novel developments in subjects such as proteasuria in nephrotic syndrome, diabetic nephropathy, membranous nephropathy, complementmediated kidney disease, and sickle cell nephropathy. I would like to thank our authors and publishing team, who supported our book in the hope that it would shed light on the scientists who work devotedly on scientific platforms for humanity.

> **Hülya Çiçek**  School of Medicine, Medical Biochemistry Department, Gaziantep University, Gaziantep, Turkey

**1**

disease [4].

**Chapter 1**

*Hülya Çiçek*

**1. Introduction**

readers.

**2. Nephritis**

kidney function [2].

attributed to nephritis:

**2.1 Interstitial nephritis**

Introductory Chapter: An

Syndrome, and Nephrosis

Overview of Nephritis, Nephrotic

The prevalence of kidney diseases has increased globally and continues to rise. However, kidneys are organs that play an important role in metabolic activities. The absorption of minerals and water, acid-base balance, and the functionality of buffer systems are regulated by the kidney and are of vital importance [1]. The proteasuria, membranous nephropathy, diabetic nephropathy, complement-mediated-kidneydisease, and other kidney pathologies examined by eminent authors in this book with the aim of opening new horizons. I hope the content of our book will be useful to dear

Nephritis is a condition in which the nephrons, the functional units of the kidneys, become inflamed. This inflammation, or glomerulonephritis, can negatively affect

When the kidneys function normally, the rest of the body is constantly supplied with oxygen-rich blood. However, when the kidneys become inflamed, they cannot filter the blood effectively. The kidneys are the filters of the body. These two bean-shaped organs are a special evacuation system. It processes almost 150 L of blood every day and disposes of up to 2 L of useless residue and water. Acute nephritis occurs when the kidneys become rapidly inflamed, this inflammation causes kidney dysfunction. Acute nephritis

Although the disease has an acute and chronic course, there are several forms

Spaces between kidney tubules to inflammation are called interstitial nephritis. It can be acute or chronic, acute interstitial nephritis is considered a disease characterized by a decline in renal functions, inflammatory infiltrate in the interstitium, and inflammatory edema. It is the second most common cause of intrinsic kidney

has many causes and if left untreated, kidney failure occurs [3].

#### **Chapter 1**

## Introductory Chapter: An Overview of Nephritis, Nephrotic Syndrome, and Nephrosis

*Hülya Çiçek*

#### **1. Introduction**

The prevalence of kidney diseases has increased globally and continues to rise. However, kidneys are organs that play an important role in metabolic activities. The absorption of minerals and water, acid-base balance, and the functionality of buffer systems are regulated by the kidney and are of vital importance [1]. The proteasuria, membranous nephropathy, diabetic nephropathy, complement-mediated-kidneydisease, and other kidney pathologies examined by eminent authors in this book with the aim of opening new horizons. I hope the content of our book will be useful to dear readers.

#### **2. Nephritis**

Nephritis is a condition in which the nephrons, the functional units of the kidneys, become inflamed. This inflammation, or glomerulonephritis, can negatively affect kidney function [2].

When the kidneys function normally, the rest of the body is constantly supplied with oxygen-rich blood. However, when the kidneys become inflamed, they cannot filter the blood effectively. The kidneys are the filters of the body. These two bean-shaped organs are a special evacuation system. It processes almost 150 L of blood every day and disposes of up to 2 L of useless residue and water. Acute nephritis occurs when the kidneys become rapidly inflamed, this inflammation causes kidney dysfunction. Acute nephritis has many causes and if left untreated, kidney failure occurs [3].

Although the disease has an acute and chronic course, there are several forms attributed to nephritis:

#### **2.1 Interstitial nephritis**

Spaces between kidney tubules to inflammation are called interstitial nephritis. It can be acute or chronic, acute interstitial nephritis is considered a disease characterized by a decline in renal functions, inflammatory infiltrate in the interstitium, and inflammatory edema. It is the second most common cause of intrinsic kidney disease [4].

It is now thought that almost all drugs can cause acute interstitial nephritis, and drugs are responsible for 60–70% of acute interstitial nephritis cases. The disease can be idiopathic, as well as infections, and systemic lupus, except for drugs. Systemic diseases such as erythematosus and sarcoidosis may also be associated with malignancy. Antibiotics are the group of drugs that most commonly cause acute interstitial nephritis. However, it has been understood that proton pump inhibitors are the second most common drug group in patients over 65 years of age, and nonsteroidal anti-inflammatory drugs are the second most common drug group in patients under 65 years of age [5].

#### **2.2 Pyelonephritis**

Pyelonephritis usually occurs as a result of inflammation of the kidney by bacterial infections. In most cases, the infection begins in the bladder, spreads to the ureters, and finally reaches the kidneys [6].

Individuals with recurrent febrile urinary tract infections develop a kidney injury, high pulse, extreme changes in renal function, and then renal failure. The frequency of urinary tract diseases changes depending on age, gender, and diagnostic methods used. According to the American Academy of Pediatrics, the prevalence in children under two years of age with fever is over 5% [7]. For all age groups, it occurs at a rate of 3–5% in girls and 1% in boys. Although all kinds of microorganisms (fungi, parasites, and viruses) that can colonize the urinary system can cause urinary tract infections, the most common factor is gram (−) enteric bacteria. In this group, *E. coli* is the most common (75–90%) and the most common gram (+) agents are staphylococci and enterococci [8].

#### **2.3 Glomerulonephritis**

Glomerulonephritis is one of the most common renal disorders. Recently, it has been the third cause of kidney diseases in our country after diabetes mellitus (DM) and hypertension in 2010 records [9].

Glomerulonephritis (GN) is defined as an acute or chronic inflammation of the glomerulus. The capillary endothelium, mesenchyme and basement membrane compartments that form the basis of the glomerulus may be affected by this inflammation [10].

Acute glomerulonephritis (AGN) is a clinical picture and nephritic disorder characterized by the sudden onset of macroscopic hematuria, proteinuria at different levels, azotemia, oliguria, edema, and hypertension-like symptoms and findings [2–4]. Mostly antigen-antibody reaction is the factor initiating the immunological period. The causes of tissue destruction, increased vascular permeability, and glomerular inflammation that occur in this period are the activation of one or more of the inflammatory mediator systems such as complement system, cytokines, coagulation, and growth factors. Glomerular filtration rate (GFR) decreases abruptly due to inflammation and edema in the glomeruli with a change in tissue blood supply. Oliguria, edema, azotemia, hypertension, and damage to endothelial cells result in the retention of water and metabolic products in the urine, resulting in proteinuria and hematuria. The clinical picture varies according to the degree of volume excess. Life-threatening clinical findings such as severe pulmonary edema and hypertension may occur in case of severe volume overload [11].

*Introductory Chapter: An Overview of Nephritis, Nephrotic Syndrome, and Nephrosis DOI: http://dx.doi.org/10.5772/intechopen.113372*

#### **3. Diagnosis**

The most common symptoms are pain in the pelvic area, on urination or burning sensation in the penis, need to urinate more frequently, cloudy urine, presence of blood or pus in the urine, swelling of the face, legs, and feet, vomiting, fever, and high blood pressure [12].

In addition to the aforementioned physical findings, the presence of an infection in laboratory tests, and the presence of bacteria and white blood cells (WBC) in urinalysis are important. Elevated blood urea nitrogen (BUN) and creatinine levels in blood tests are important parameters used in the detection of the disease. Imaging techniques such as a CT scan or kidney ultrasound can show blockage or inflammation of the kidneys or urinary tract. A kidney biopsy is also one of the best ways to diagnose nephritis. The metabolic effects of impaired kidney function are monitored by changes in blood biochemistry.

Possible biochemical differences in the case of AGN in the patient are decreased serum sodium and calcium levels, metabolic acidosis, and increased levels of phosphorus, potassium, magnesium, uric acid, urea, and creatinine in blood serum [13].

There may be anemia of dilutional character. It is understood that plasma renin activity is suppressed in proportion to the severity of fluid retention [14].

Macroscopic hematuria, proteinuria, hypertension, and acute renal failure are common in hemolytic uremic syndrome, but thrombocytopenia and anemia are also seen. Diarrhea may be seen in HUS. Proteinuria and macroscopic hematuria occur; hearing loss, family history and renal biopsy will enable the diagnosis in some patients with Alport syndrome [11, 12].

#### **4. Nephrotic syndrome and nephrosis**

Nephrotic syndrome (NS) is a clinical picture characterized by massive proteinuria resulting from increased permeability of the glomerular filtration barrier and resulting in hypoproteinemia, edema, and hyperlipidemia. The annual incidence of NS is reported to be 2.0–2.7 per 100.000 in children under 16 years of age, with a male-to-female ratio of 2/1 in childhood, equal in adulthood and adolescence, and more common in blacks than whites.

In childhood, 2/3 of NSs start before the age of 5 years and 80–85% are caused by minimal change disease(MCD) [15].

 is classified according to clinical appearance, histopathological lesion, and response to steroid treatment.

#### **4.1 Clinical classification**

(NS) is analyzed in two main groups primary and secondary NS according to its formation.

In primary NS, the event is isolated in the kidney and the most common type is MCD, which responds well to steroids. Non-MCD glomerulonephritis also responds to steroids at a lower rate than MCD [16].

Primary NS consists of idiopathic NS [MCD, focal segmental glomerulosclerosis (FSGS), mesengioproliferative glomerulonephritis (MesPGN)], MGN, immunocomplex glomerulonephritis [acute poststreptoxic glomerulonephritis (APSGN),

membranoproliferative glomerulonephritis (MPGN)], and congenital NS. Secondary NS develops secondary to a systemic disease or event [16, 17].

Clinical Classification of (NS):

	- 1.Idiopathic NS
	- Minimal change disease (MCD)
	- Mesangial proliferative glomerulonephritis (MesPGN)
	- Focal glomerulosclerosis (FGS)
	- 2.Immunocomplex glomerulonephritis
	- Membranous glomerulonephritis (MGN)
	- Membranoproliferative glomerulonephritis (MPGN)
	- Acute post-streptococcal glomerulonephritis (APSGN)
	- 3.Congenital NS

#### *4.1.2 Secondary NS*


#### **4.2 Histopathological classification**

It is made according to the glomerular changes seen in light microscopy. This distinction was later supported by immunofluorescence and electron microscopic examinations [18].

*Introductory Chapter: An Overview of Nephritis, Nephrotic Syndrome, and Nephrosis DOI: http://dx.doi.org/10.5772/intechopen.113372*

Histopathological Classification of Glomerular Lesions


#### *4.2.1 Minimal change disease*

In minimal lesion disease, it is accepted that there is no histological change in glomeruli. In some cases, minimal mesangial thickening, focal mesangial cell increase, and thickening of the basement membrane may be seen. Immunofluorescence microscopy usually shows no immuno-deposit deposition. However, mesangial deposition consisting of IgM and complement may be found, although rarely. Electron microscopy shows hypertrophy of podocytes and enlargement of foot processes. In childhood, 2/3 of NSs start before the age of 5 years and 80–85% are caused by MCD [15, 19].

(NS) is classified according to clinical appearance, histopathological lesion, and response to steroid treatment. The annual incidence of NS is 2.0–2.7 per 100,000 in children under the age of 16, the male-female ratio is 2/1 in childhood, it is equal in adults and adolescents, and it is more common in the black race than white [20, 21].

#### *4.2.2 Focal glomerulosclerosis*

In focal glomerulosclerosis (FGS), some glomeruli show increased matrix and hyaline deposition with segmental areas of capillary collapse and obliteration (FSGS). There may be widespread involvement [focal global glomerulosclerosis (FGGS)]. In most lesions, podocyte hyperplasia is accompanied by sclerotic areas. The affected glomeruli are often in the juxtamedullary region but are not limited there. FGGS may

be an advanced form of FSGS or an independent entity unrelated to NS. As a rule, FSGS is accompanied by tubular atrophy and responds to steroids worse than MCD but better than FGGS. It has a progressive course [18, 22, 23].

#### *4.2.3 Mesengial proliferative glomerulonephritis*

Mesengioproliferative glomerulonephritis is characterized by a moderate to a marked increase in the number of mesengial cells (mesengial proliferation), leukocyte infiltration (exudation), and increased mesengial matrix (sclerosis) with obliteration of capillary loops and fibroepithelial proliferation (crescent and adhesion) on the inner surface of Bowman's capsule. Immunofluorescence microscopy is usually negative. However, NS findings secondary to postinfectious glomerulonephritis, Berger's disease (IgA nephropathy), and systemic disease may be present [18, 22].

#### *4.2.4 Membrano-proliferative glomerulonephritis*

Three histological subgroups have been defined under the title of membranoproliferative glomerulonephritis (MPGN). In Type-I MPGN, the main lesion is subendothelial IgG and complement deposition. In Type-II MPGN there is basement membrane thickening with intramembranous dense storage. Type-III MPGN is morphologically characterized by transmembranous deposition. Mesangial proliferation, crescent formation, hyperlobulation, and epimembranous deposition are observed in all types [18, 22, 24].

#### *4.2.5 Membranous glomerulonephritis*

In membranous glomerulonephritis, subendothelial deposits are usually regularly, sometimes irregularly distributed in the basement membrane. In light microscopy, they are seen as basement membrane protrusions indenting the lamina densa. This appearance is described as a lace-like appearance. Deposits are usually associated with only mild mesangial proliferation [25, 26].

#### *4.2.6 Chronic glomerulonephritis*

Most types of acute glomerulonephritis are susceptible to transformation into chronic glomerulonephritis. This condition causes progressive glomerular and tubulointerstitial fibrosis. There is an irreversible progressive decrease in glomerular filtration rate. Uremic toxins accumulate in the blood and lead to disease progression, resulting in chronic kidney disease (CKD), end-stage renal disease (ESRD), and cardiovascular disease. Chronic glomerulonephritis is a leading cause of chronic kidney disease and accounts for approximately 10% of all patients on dialysis [27].

#### **4.3 Pathophysiology of (NS)**

Proteinuria, which occurs with increased permeability in the filtration barrier, is the primary pathophysiological mechanism responsible for the development of NS. The protein most lost in urine is albumin, but other plasma proteins such as immunoglobulins, various coagulation factors, vitamin-D binding proteins, and

metalloproteins are also lost in urine [15, 19]. Albuminuria causes edema, hypoalbuminemia and hypercholesterolemia in patients [28].

#### *4.3.1 Proteinuria*

Protein excretion of more than 40 mg/m2/st in children and more than 3.5 g/day in adults is considered nephrotic proteinuria [18].

Normally, large molecular weight proteins are not seen in the urine due to the selective permeability of the glomerular filtration barrier and reabsorption of proteins from the proximal tubulus. In glomerular diseases, increased glomerular permeability to proteins results in proteinuria. Despite extensive research, the pathogenesis of proteinuria is still not fully explained. In filtration function, glomerular basement membrane (GBM) functions as a barrier based on both molar size and electrical charge, and epithelial cells, the outermost layer of the glomerular capillary network, acts as a selective barrier for the filtration of micromolecules. Visceral epithelial cells (podocytes) form the outermost part of the glomerular filtration barrier. Podocytes surround the glomerular capillaries in the form of scallop teeth. Each comb tooth is formed by neighboring epithelial cell peduncles. Peduncular outgrowths are associated with GBM. Between the neighboring adaxial processes on the GBM is the slit diaphragm (SD), a bridge containing filtration holes. The slit diaphragm is a very thin membrane closely associated with the GBM. Glycoproteins, predominantly podocalyxin, cover the apical and lateral surfaces of the peduncular processes. These structures cause the foot processes to carry a strong negative electric charge. The negative electric charge also prevents the adhesion of neighboring peduncles to each other [18].

#### *4.3.2 Definition of normal and nephrotic proteinuria*

#### 1.Qualitative


#### *4.3.3 Hypoalbuminemia*

Hypoalbuminemia resulting from massive proteinuria is a constant laboratory finding of NS1–3. There is an inverse relationship between urinary protein loss and serum albumin level. However, this is not always valid. In children with prolonged proteinuria unresponsive to treatment, serum albumin levels may be normal or near normal without any change in protein excretion rate. The hepatic albumin synthesis rate may be normal or increased in NS [29].

The severity of hypoalbuminemia varies from patient to patient. The serum albumin level during relapse varies between 0.5 g/dl and 2.5 g/dl [18, 22].

Other protein abnormalities in plasma include decreased gamma-globulin, normal or low alpha1-globulin, alpha2-globulin, and increased fibrinogen levels. The increase in alpha2-globulin level is thought to be due to accumulation. In patients with NS, especially MCD, IgG level decreases while IgM level increases [28].

#### *4.3.4 Edema*

It is the main clinical finding of NS is classic, edema seen in NS is caused by a decrease in plasma oncotic pressure as a result of hypoalbuminemia, and secondary to this, water and solutes pass into the interstitial space. This results in a decrease in intravascular volume, which activates the renin-angiotensin-aldosterone system. As a result, water and salt retention increases (underfilling theory) [18, 22]. The observation that intravascular volume is normal or increased and plasma renin activity is not increased in some NS patients suggests that other factors may also play a role in the formation of NS edema. In these cases, sodium retention is thought to occur by intrarenal mechanisms (overfilling theory).

Sodium accumulation originating from the kidneys is caused by increased sodium absorption in the cortical collecting ducts and collecting ducts as a result of Na -K-ATPase production in the basolateral membrane and increased permeability in the epithelial sodium channels (ENaC) in the apical membrane. In addition, the lack of response to the natriuretic response of atrial natriuretic peptide (ANP) in the medullary collecting ducts is also effective, and it is reported that this is not fully related to aldosterone [30, 31].

#### *4.3.5 Hyperlipidemia and hyperlipoproteinemia*

Plasma concentrations of cholesterol, triglycerides, phospholipids, and fatty acids are increased in NS. Generally, there is an inverse relationship between serum albumin level and cholesterol level 3. The level of triglycerides is more variable and may be within normal limits in mild hypoalbuminemia [29, 32].

Like hypoalbuminemia, hyperlipidemia results from increased synthesis or decreased degradation. Increased synthesis is usually associated with increased albumin synthesis [33]. Because lipoproteins and albumin are synthesized in the liver by metabolic pathways that are very close to each other. Decreased lipoprotein catabolism, accompanied by a decrease in lipase activity, also contributes to hyperlipidemia [34].

#### **4.4 Laboratory findings**

Laboratory evaluation in patients with (NS); diagnosis and determination of severity of NS are performed to determine possible etiological factors and to make a definitive histological diagnosis with renal biopsy [18].

#### *Introductory Chapter: An Overview of Nephritis, Nephrotic Syndrome, and Nephrosis DOI: http://dx.doi.org/10.5772/intechopen.113372*

Urinary stick shows proteinuria and hematuria, and microscopy shows hyaline casts and fat bodies. The presence of erythrocytes, granular, waxy, and wide casts is a finding in favor of glomerulonephritis other than MCD [17, 18].

quantitative proteinuria. In children, normal values are defined as <4 mg/m2/ hr. and the nephrotic border is defined as >40 mg/m2/ hr. While the urine protein/ creatinine ratio is 0.2 in normal, this ratio rises to 2.0 in NS. The serum albumin value of patients with NS is below 2.5 mg/dl. Alpha 2 and beta-globulin levels increased due to increased hepatic synthesis [17, 18]. While the serum IgG level decreases. IgM and IgE levels increase. Serum cholesterol and triglyceride levels increased. Evaluation of BUN and serum creatinine is necessary for kidney functions [17, 18, 22].

Serum complement level, hypertension, and macroscopic should be measured in patients with hematuria or reduced renal function. While complement level is normal in MCD, it is low in APSGN, MPGN, and SLE. Serological evaluation for hepatitis and syphilis should be performed. It is also necessary to conduct research on HIV in patients in the risk group. Secondary for SLE, ANA should be investigated in anti-DNA as it causes NS [18].

For years, scientific institutions and communities dealing with the kidney have debated whether hemodynamic changes alone are responsible for glomerular and tubulointerstitial damage and whether other mechanisms are involved in this process [35].

Recent studies have shown that proteinuria itself, which is a determinant of the severity of some kidney diseases, is harmful. In this context, the amount of protein detected in the urine appears to be the most important factor determining the progression to end-stage renal failure, sometimes to a greater extent than the original disease [36].

Increased proteinuria leads to a progression of renal disease by causing excessive protein uptake by the proximal tubule cells with an inflammatory phenotype and ultimately progressive renal interstitial damage [37]. Proteinuria is associated with the proliferation and apoptosis of proximal tubular cells and interstitial inflammation [38].

#### **5. Conclusion**

As a conclusion, starting treatment as early as possible to prevent proteinuria that occurs in cases of nephritis, (NS), and nephrosis will prevent the development of hypoalbuminemia and hyperlipidemia. Proteinuria can be reduced by suppressing the factors that cause increased permeability in the filtration barrier. It is also known that reactive oxygen radicals play a role in (NS), so avoiding oxidative stress may also slow the progression of the disease. Inflammatory kidney diseases such as chronic glomerulonephritis and hereditary polycystic kidney disease are among the kidney diseases that can lead to chronic kidney failure. People at risk for chronic kidney failure; Those who are overweight, have high blood pressure, are diabetic, and have a family history of kidney disease. Lifestyle changes are of great importance in delaying the progression of chronic kidney diseases. Since the kidneys are very rich in blood vessels, all measures that protect our cardiovascular system also have a protective effect on the kidneys.

#### **Conflict of interest**

The author declares no conflict of interest.

*Novel Topics in the Diagnosis, Treatment, and Follow-Up of Nephritis, Nephrotic Syndrome...*

### **Author details**

Hülya Çiçek Department of Medical Biochemistry, Gaziantep University School of Medicine, Gaziantep, Turkey

\*Address all correspondence to: drhulyacicek@hotmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Introductory Chapter: An Overview of Nephritis, Nephrotic Syndrome, and Nephrosis DOI: http://dx.doi.org/10.5772/intechopen.113372*

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[35] Remuzzi G, Ruggenenti P, Benigni A. Understanding the nature of renal disease progression. Kidney International. 1997;**51**(1):2-15

[36] Luyckx VA, Brenner BM. Clinical consequences of developmental programming of low nephron number. Anatomy Record (Hoboken). 2020 Oct;**303**(10):2613-2631

[37] Kameda Y, Hanai K, Uchigata Y, Babazono T, Kitano S. Vitreous hemorrhage in diabetes patients with proliferative diabetic retinopathy undergoing hemodialysis. Journal of Diabetes Investigation. 2020 May;**11**(3):688-692

[38] Saadatagah S, Pasha AK, Alhalabi L, Sandhyavenu H, Farwati M, Smith CY, et al. Coronary heart disease risk associated with primary isolated hypertriglyceridemia: A Population-Based Study. Journal of the American Heart Association. 2021;**10**(11):e019343

### **Chapter 2**

## Proteasuria: The Link between Physiopathogenesis and Edema Management in Nephrotic Syndrome?

*Mario Alamilla-Sanchez, Miguel Alcala Salgado, Gandhy Fonseca González, Carlos Chavez Mendoza, Cecilia Acosta Peña, Pamela Prado Lozano, Daniel Diaz Garcia and Julio Nieto Gutiérrez*

#### **Abstract**

Nephrotic syndrome is a pathology characterized by severe proteinuria, hypoalbuminemia, dyslipidemia, and edema. Edema has classically been associated with an alteration of the forces that govern Starling's principle. However, some proteins eliminated in excess in the urine (proteasuria) can activate the epithelial sodium channel (ENaC), favoring sodium retention and edema. The α- and γ-ENaC subunits are activated by releasing inhibitory segments that favor the probability of channel opening. Some proteases that have been investigated include plasmin, prostasin, transmembrane protease serine 4, cathepsin, and neutrophil elastases. Therapeutic strategies include water and sodium restriction in the diet, appropriate dosing of diuretics (loop, thiazides, or acetazolamide), and in severe cases, mechanical ultrafiltration. Due to the continuous activation of ENaC in nephrotic syndrome, amiloride is an attractive diuretic strategy that has been shown to be effective in some patients with an acceptable safety profile.

**Keywords:** nephrotic syndrome, proteasuria, epithelial sodium channel, edema, amiloride

#### **1. Introduction**

Peripheral edema and extracellular volume expansion are features commonly observed in patients with nephrotic syndrome (NS), this is consequential from sodium retention. Two different theories have been suggested putting forward the links between protein loss in NS and enhanced sodium reabsorption by the epithelial sodium channel (ENaC). Since 1917 the underfill theory was proposed to explain that hypoalbuminemia due to proteinuria leads to intravascular volume depletion through the loss of oncotic pressure and this led the way to fluid accumulation in the interstitial space but an underfilled intravascular compartment. This in turn would activate the renin-angiotensin-aldosterone system (RASS). On the other hand, the overfill hypothesis, formulated in 1979 suggest that proteinuria-induced sodium retention is an intrinsic renal event that is independent of the RASS activation. Despite these theories mechanisms leading to primary sodium retention in nephrotic kidneys are not yet well understood. But recently intraluminal activation of the ENaC by abnormally filtered proteases has been proposed as a mechanism that could explain avid sodium reabsorption, which would be consistent with the overfill theory. In 2018 the term "proteasuria" was first introduced to indicate the increased excretion of active plasma proteases found in the urine of patients with NS.

#### **2. Underfill theory in nephrotic syndrome**

This theory was first proposed more than 100 years ago by Epstein. In the original article published in 1917 by the American Journal of Medicine he proposed that the loss of protein incurred by the blood serum through the continuous albuminuria causes a decrease in the osmotic pressure of the blood, which favors the absorption and retention of fluid by the tissues.

To understand the exact nature of the processes which leads to the production of edema we must consider for a moment the mechanisms in which the exchange of fluid between the blood and the tissues is regulated. In normal conditions the loss of fluid from the blood is rapidly restored by the passage of fluid from the tissue spaces back into the blood capillaries.

Under normal conditions the hydrostatic pressure within capillaries and the oncotic pressure determine the movement of fluid from the vascular compartment to the interstitium. The oncotic pressure is determined by the proteins in the blood. The Starling equation was created in 1896 by Ernest Starling, he expressed the flux of water across capillaries. Oncotic pressure moves fluid out, while hydrostatic pressure moves fluid in [1]. According to Starling, during the production and absorption of tissue fluid, there is normally a balance between the filtration pressure in the capillaries and the osmotic pressure of the colloids of the blood and tissue fluid. Plasma and tissue fluids are practically identical in everything except protein. Plasma exceeds tissue protein concentrations.

Thus, Epstein suggested that in patients with NS this loss of proteins represented a pressure equal to 20–24 mmHg, a factor strong enough to disturb the equilibrium in the exchange of fluid between the blood and the tissues. Leading to a fall in the osmotic pressure of the blood and giving to the tissues the controlling power to absorb and retain fluid.

The imbalance of Starling's forces leads to interstitial leakage of fluid and decreased circulating volume that in the end will decrease circulating volume and renal perfusion, activation of the RAAS will occur trying to compensate this imparity [2]. The activation of the sympathetic system and the renin angiotensin axis can be observed both in renal parenchymal involvement and in hypovolemia, but it is the increase in plasma vasopressin of non-osmotic origin what supports that the main trigger is hypovolemia [3].

Extravasation of fluid decreases intravascular volume and increases neurohormonal markers of intravascular depletion, such as vasopressin and aldosterone, resulting in a highly concentrated urine with very low sodium content. Therefore, renal sodium retention in this scenario is a secondary phenomenon, a physiological consequence of intravascular underfilling [4].

#### **2.1 Clinical evidence**

Through studies where capillary permeability with albumin marked with TC99m is made, it was shown that the permeability percentage was significantly increased in glomerular diseases compared to healthy controls. The percentage of permeability did not vary from a glomerular disease to another nor was it correlated with the degree of albuminemia or proteinuria [5].

Vande Walleet et al., compared children with NS secondary to minimum change disease versus those without minimal changes and subsequently divided them by the presence or absence of signs and symptoms of hypovolemia. Patients with hypovolemia symptoms in both groups had significantly high levels of norepinephrine, renin, and aldosterone unlike their non -symptomatic counterpart [6].

Usberti et al., studied 16 pediatric and adult patients with NS but normal renal function. They found a decreased plasma sodium concentration, increased plasma vasopressin (AVP), impaired excretion of an acute water load, and elevation of plasma renin activity (PRA) and urinary norepinephrine levels as compared to controls. When they were treated with isotonic albumin infusion they decreased plasma AVP concentration and increased water diuresis [7].

Based on the above, a volume load would decrease the activation of all mechanisms that influence sodium and water reabsorption, however the mechanisms of compensation of the edema at the capillary level are found exhausted so that when administering fluids these are quickly spread to the interstitium, making almost impossible the restoring of intravascular volume.

#### **2.2 Critique to the underfill hypothesis in nephrotic syndrome**

Several clinical and laboratory observations have disputed about the accuracy of underfill hypothesis. Historically, the description of edema formation in NS was simple: an unknown trigger leads to proteinuria, as the plasma protein level falls, the intravascular oncotic pressure decreases with consequent leakage of plasma water into the interstice, thus generating edema. Owing to the extravasation of the fluid, the intravascular volume is decreased and neurohormonal markers of intravascular depletion are increased, such as vasopressin and aldosterone, resulting in highly concentrated urine with very low sodium concentrations [8].

The therapeutic consequences of this hypothesis for the treatment of edema are clear, expansion of intravascular volume and restoration of plasma oncotic pressure by administration of albumin [4].

#### **2.3 Treatments based on the underfill hypothesis**

Albumin infusion is used to correct hypoalbuminemia since it is the principal cause for edema [3, 4]. Treatment by albumin alone reduces plasma renin activity, but do not reflect adequate diuresis response [9]. Some of these patients require high doses of oral or intravenous diuretics for edema management. Patients with diuretic resistance edema might benefit from coadministration of albumin and furosemide. Several mechanisms are proposed to explain the improved efficacy of combined therapy, the most accepted being that intravenous albumin enhances the secretion of loop diuretics in proximal tubules, with increased delivery at its primary site of action [10, 11].

It's recognized that two groups of patients with NS benefit form albumin infusions: patients with intravascular hypovolemia, and those with severe refractory edema. Patients with severe hypoalbuminemia, especially those with refractory and resistant steroid illness, require repeated courses of albumin infusion [10]. Once the patient goes into remission, the first symptom (besides the disappearance of proteinuria) is a large diuresis, before plasma protein levels (and thus oncotic pressure) have normalized [12, 13].

Generally, loop diuretics were administered as bolus dose during or at end of the albumin infusion. The rate of albumin infusion must be monitored to avoid risk of overload and pulmonary edema, as it has associated in some patients [14]. The effect of albumin is transient, and during relapse most patients would excrete the amount infused over next 24–48 hours [10]. It is recommended that adequate urine output be measured before initiating albumin infusion, and being careful in patients with hypertension, pulmonary edema, respiratory distress, or congestive heart failure.

Several clinical studies have sought the improvement of refractory edema with albumin infusion in the treatment of NS with partial efficacy and opposite results. A recent Cochrane review failed to draw any conclusion due lack of studies as they excluded cross-over studies [15]. Only one study met their inclusion criteria (26 children with minimal change disease) and compared albumin infusion plus furosemide with and equal volume of dextrose. The authors reported clinical improvement as a greater weight loss difference overall at 10 days; however, they also identified changes in serum sodium and blood pressure but data in text are inconsistent and reflects low certainty evidence.

Meena J, et al. [10], performed a meta-analysis that included six studies, involving 69 patients, only one study included children, and most of them had small sample size and high risk of bias. This meta-analysis showed that combination therapy was more effective in increasing diuresis than furosemide therapy alone.

#### **2.4 Analbuminemia and edema**

Inherited albumin abnormalities include bisalbuminemia and analbuminemia. Analbuminemia, also called idiopathic hypoalbuminemia, is a rare congenital disorder of albumin characterized by very low levels of this protein inherited as an autosomal recessive trait [16]. The diagnosis is realized with electrophoresis who shown a markedly decreased levels of albumin and elevation of other proteins.

An exponential increase in "non-albumin" plasma proteins (globulins and proteins of higher molecular weight) compensates intravascular colloid osmotic pressure [17]. With the increase of other plasma proteins, a decrease in hydrostatic pressure has been found, which explains the absence of edema in these patients, or slight edema. Also, patients with analbuminemia had slightly decreased renal plasma flow, slightly diminished glomerular filtration and markedly augmented filtration fraction compared to healthy subjects [16].

There is very little correlation among the markedly decreased levels of serum albumin and the reported signs and symptoms of the patients. Clinically, 50% of patients are completely asymptomatic and the rest have fatigue, asthenia, and orthostatism. One third of the patients presents without edema [4], while the others have slight edema of legs and ankles. Lipid metabolism abnormalities are observed in some patients [16, 17].

*Proteasuria: The Link between Physiopathogenesis and Edema Management in Nephrotic… DOI: http://dx.doi.org/10.5772/intechopen.108591*

Finally, other observations that do not fit well with underfill hypothesis includes that reducing the activity of the renin-aldosterone axis by mineralocorticoid receptor antagonists, such as spironolactone, or angiotensin-converting enzyme inhibitors does not result in a marked increase in sodium excretion in most patients with NS [9, 18]. Also, attempts at trying to measure blood volume and/or neurohumoral markers of volume depletion, such as renin/aldosterone, do not show a consistent observation, but suggest volume depletion in some subjects with NS, and normal or excess volume in others [4, 9, 13]. Not all nephrotic states are equal.

#### **3. Overfill theory in nephrotic syndrome**

There are two paradigms of edema formation in nephrosis: the so-called underfill and overfill models; it is thought that these can be present in the same individual at different times over the course of their disease. Both result in sodium and water retention and increased interstitial fluid volume presenting as oedema.

The overfill hypothesis postulates that sodium retention is a primary renal phenomenon and would be produced by an intrinsic renal defect in sodium excretion, which in turn would produce expansion of plasma volume (hence the term overfill) [19]. The overfill theory, first formulated by Meltzer et al., in 1979, states that sodium retention is mainly caused by the diseased kidney due to a tubular defect leading to sodium avidity without any signs of volume depletion or a stimulated RAAS [20].

The molecular mechanisms of sodium retention in the NS have derived from the use of the animal model of NS induced by the action of the aminonucleoside puromycin (PAN), which, when administered to rats, produces proteinuria mass and sodium retention [21, 22].

Regulation of ENaC occurs through two main mechanisms: regulation of channel density at the apical membrane and regulation of channel opening probability [23]. ENaC receptor density is regulated by both aldosterone and vasopressin. The open channel probability is regulated by proteolytic processing and by anionic phospholipids present in the inner cell membrane [24, 25]. In the rat PAN nephrosis model, they showed that amiloride increases urine sodium excretion and reduces ascites volume. This effect was attributed both to the ability of amiloride to inhibit ENaC and the ability of amiloride to inhibit urokinase-type plasminogen activator and thus reduce the amount of active plasmin present [1]. Subsequently, plasmin would activate ENaC, and sodium retention would occur with the consequent appearance of edema. In many patients with NS, the pathophysiology of edema would be caused by an intrinsic renal defect in sodium excretion, due to retention in the cortical collector duct (CCD) [26].

In the clinical assessment it should be noted that because Na + retention occurs both in underfill and overfill states, it is not possible to use the FeNa+ to clinically differentiate primary from secondary Na + retention in NS. An increase in RAAS and circulating aldosterone effect can be inferred based on more readily measured values such as an increased transtubular potassium gradient (TTKG) index, uK+/uCr, uNa+, which is observed in hypovolemic patients and not when blood volume is preserved [3]. This also suggests a primary role of aldosterone in the intrinsic activation of Na+/K+ ATPase in CCD. Such patients may benefit from diuretic use. By contrast, diuretic use in patients with NS and secondary Na + retention triggered by hypovolemia or circulatory insufficiency may have serious deleterious consequences.

### **4. Epithelial sodium channel (ENaC)**

The epithelial sodium channel (ENaC) was first cloned in 1993 and its subunits were detected in 1994 by Canessa and Rossier's research group [27, 28]. At the renal level, it is found in the distal portion of the distal convoluted tubule, connecting duct, and collecting duct (aldosterone-sensitive distal nephron-ASDIN). It promotes an electrogenic gradient by the diffusion of sodium (Na+) from the tubular lumen, which facilitates the renal secretion of potassium (K+).

#### **4.1 Biophysical properties and chemical structure**

ENaC channels (also called SCNN1 and amiloride-sensitive epithelial sodium channel) are Na+ and Li+ permeable channels with very little conductance for K+ (4 pS) and no conductance for divalent cations; it can have a long opening time, up to 10 seconds, depending on the action of proteases on the channel [29–31]. They are members of the ENaC/degenerin family of cation-selective channels, related to acid-sensitive ion channels (ASICs) [32].

ENaC has 4 subunits (α, β, γ, δ, encoded as SCNN1 A, B, G, and D, respectively) and requires the assembly of 3 subunits (α- β- γ- or δ- β- γ-) to get maximum capacity, although there is partial activity with αENaC, αβENaC or αγENaC [33].

The αβγENaC channel is sensitive to low concentrations of amiloride (IC50 0.1 μM) or benzamil (IC50 0.01 μM), a Li+ /Na+ permeability ratio of 1.6, and has low conductance at physiological Na<sup>+</sup> concentrations (5 pS); while δβγENaC has higher Na+ conductance (12 pS), lower Li+ /Na+ permeability ratio (0.6) and requires higher inhibitory concentrations of amiloride and benzamil (IC50 2.6 μM and 0.27 μM, respectively) [34, 35], in addition, the δ subunit reduces sensitivity to proteasemediated activation [36, 37]. ENaC has a long extracellular region connected to two transmembrane domains (TM1 and TM2) that form the pore and where the channel gate is, as well as a short cytoplasmic portion of the NH2- and COOH- termini.

The topology of each individual ENaC subunit resembles a hand: "central palm" linking TM1 and TM2 by "wrist" and squeezes a "ball" together with domains: "finger", "thumb" and "knuckle" [38]. Among these domains is GRIP (gating relief inhibition by proteolysis) that harbors inhibitory peptides. The GRIP domain has been found in αENaC or γENaC. βENaC also has GRIP domains, but they are not cleaved by proteases [39]. The removal of GRIP causes a conformational change in the molecule that alters the wrist domain, affecting the conductance of the channel (**Figure 1**).

The extracellular portion has regions of high relevance for ENaC function: Na + binding site in the α subunit, with inhibition capacity in response to extracellular concentrations of Na<sup>+</sup> [40]; protease binding sites that favor the removal of inhibitory sites on the α or γ subunit, which activates the channel [38]; glycosylation sites relevant to channel maturation; and cysteine-rich domains (CRDs), involved in the interaction between the "finger" and "thumb" domains within the same α or γ subunit [41].

ENaC expression is increased especially with stimulation of aldosterone, arginine vasopressin, and cortisol. In the ASDIN region, the presence of 11βHSD2 converts cortisol to 11-dehydrocorticosterone, allowing aldosterone to stimulate the mineralocorticoid receptor (MR) [42].

ENaC represents the only member of the ENaC/Degenerin ion channel family that constitutively opens in the absence of an activating stimulus, that is, it has *Proteasuria: The Link between Physiopathogenesis and Edema Management in Nephrotic… DOI: http://dx.doi.org/10.5772/intechopen.108591*

#### **Figure 1.** *Function and regulation of the epithelial Na+ channel (α−/γ-subunit).*

a high probability of spontaneous opening (Po) [43]. Therefore, detailed control over the expression of ENaC in the cell membrane, as well as the regulation of Po, is essential. Defects in specific sequences that reduce Po led to a risk of developing Pseudohypoaldosteronism type 1 (PHA1) [44], the opposite effect leading to an increase in Po produces Liddle syndrome [45]. Factors that can modify Po include intra- and extracellular pH, Na + ion concentration, temperature, laminar shear stress, and GRIP domain cleavage by proteases [43].

#### **4.2 Nephrotic syndrome and regulation of ENaC by urinary proteases**

The first observations that suggested the regulation of ENaC by proteases were published by Orce et al. [46], who showed that aprotinin (serine protease inhibitor) reduced Na + transport in toad bladder epithelium. It was confirmed by the studies by Bohnert et al., using aprotinin in mice with NS and showing improvement in natriuresis [47]. For their part, Vallet et al. demonstrated that ENaC could be activated by the protease trypsin [31], and subsequently identified prostasin as a serine protease capable of activating ENaC. Furin, another serine protease, can activate the α- and γ-subunits [48]. Cleavage of the α-subunit requires the binding of furin to two specific sites, releasing an inhibitory tract of 26 residues [49]. Furin, on the other hand, binds only once to the γ subunit, releasing a 40-residue inhibitory tract [49]. Other proteases that can activate ENaC include transmembrane protease serine 4 (TMPRSS 4), urokinase, plasmin, pancreatic elastase, cathepsin B, neutrophil elastase, kallikrein, and bacterial proteases [50–54].

In NS there is clearance of many proteins, some of which retain their enzymatic function. Several studies have detected plasminogen and plasmin in urine and their urinary concentrations have been correlated with albuminuria [55–58]. In the long term (25 years of follow-up), the urinary plasminogen/plasmin ratio correlated with the incidence of arterial hypertension and cardiovascular mortality, although

independently of albuminuria [58]. A case of membranous nephropathy associated with a Liddle syndrome phenotype with ENaC hyperactivation without direct evidence of mutation of any subunit was recently reported [45].

Experiments in Xenopus laevis oocytes have shown the activation of γENaC by plasminogen and urokinase-type plasminogen activator (uPA); however, in knockout models (uPA −/−) with NS, sodium retention was not different compared to uPA +/+ models. However, amiloride prevented sodium retention in uPA −/− nephrotic syndrome mice; this suggests that uPA has an important but not essential role for γENaC-mediated sodium retention [59].

In a recent doxorubicin-induced nephrotic syndrome model, two types of genetically modified mice were produced, one group of knock-in mice with inactivating mutations of the prostasin protein and another group with activating mutations of the prostasin protein. Cleavage of αENaC and γENaC was observed in the same proportion, demonstrating that ENaC activation in nephrotic syndrome occurred independently of prostasin activity [60].

In addition, Artunc et al. evaluated Factor VII activating protease (FASP) in a study involving murine models and humans with nephrotic syndrome. In humans, high levels of FSAP in both active and zymogen forms were detected in urine. Mutation of the prostasin activation site at γENaC in mice prevented stimulation of the channel by FSAP. However, the absence of FSAP did not prevent cleavage of the α- and γ-ENaC subunits [61].

These data suggest that cleavage of ENaC subunits probably requires the action of multiple proteases rather than the action of a single protease [62]. Likewise, the incomplete activation of ENaC by proteases in the zymogen state can produce intolerance to treatment with distal blockers such as Triamterene, and potentially cause a state of kidney injury due to severe salt loss, due to the absolute blockade of the channel [63].

#### **5. Edema**

Edema is one of the defining characteristics of nephrotic syndrome, as well as being the symptom that most frequently requires medical intervention. This can be severe, representing an increase in body weight of up to 30%. Likewise, it is associated with complications such as movement restriction, increased skin tension with consequent skin denudation, increased risk of soft tissue infection and pulmonary edema [2].

There is currently no consensus regarding the treatment of edema in nephrotic syndrome. In general, we can divide the treatment of edema into non-pharmacological and pharmacological management.

#### **5.1 Conservative management**

An essential part of treatment is to determine intravascular volume status since the initiation of high doses of diuretics and fluid restriction in a patient with effective volume depletion can precipitate renal function deterioration. Some authors recommend an initial goal of weight loss of 0.5–1 kg/day, to prevent complications such as acute kidney injury, hydroelectrolytic imbalance and thromboembolism secondary to hemoconcentration [11].

*Proteasuria: The Link between Physiopathogenesis and Edema Management in Nephrotic… DOI: http://dx.doi.org/10.5772/intechopen.108591*

Another key aspect of treatment is achieving a negative sodium balance. Strict sodium restriction of less than 100 mEq/day or 2.3 g/day has been shown to have an additive effect to pharmacological therapies. While fluid restriction can be reserved for those patients with hyponatremia, it is generally suggested to limit fluid intake accord to fluid overload [3, 11].

Regarding hygienic-dietary recommendations, in adults it is recommended to restrict protein intake to 0.8–1 g/kg/day and maintain blood pressure goals <125/75 mmHg if the patient has proteinuria >1 g/day and < 130/80 mmHg with proteinuria >1 g/day [3].

About 20% of patients have infectious complications. There are no current recommendations for prophylactic antibiotics, although immunization against *Streptococcus pneumoniae* is recommended, according to age and previous immunizations [11].

#### **5.2 Diuretics**

Loop diuretics are usually the first line of treatment. They act by inhibiting the NKCC2 cotransporter on the apical surface of the thick ascending limb of the loop of Henle, which is responsible for approximately 25% of total sodium reabsorption. They bind strongly to proteins and are secreted in the proximal tubule [2].

Furosemide has variable intra- and inter-individual bioavailability; this varies between 20 and 60% orally and can be reduced to 30% in edematous states. The presence of intestinal edema can limit its absorption and hypoalbuminemia decrease delivery to its site of action, requiring higher doses, with a greater probability of presenting adverse effects or its parenteral administration.

On the other hand, its chronic use causes hypertrophy and hyperplasia of the epithelial cells of the distal tubule, with increased expression of the NCC cotransporter, limiting the natriuretic effect. This phenomenon is usually counteracted by adding diuretics from different classes. For example, the combination with thiazides increases diuresis, however, it requires close monitoring to avoid severe hypokalemia and metabolic alkalosis.

A randomized clinical trial in patients with nephrotic syndrome and refractory edema comparing the use of acetazolamide + hydrochlorothiazide vs. furosemide + hydrochlorothiazide, both followed by the administration of furosemide; demonstrated greater weight loss and increased diuresis with the combination of acetazolamide + hydrochlorothiazide [64].

Although the administration of amiloride or mineralocorticoid receptor antagonists (MRA) reduce the risk of loop diuretic-induced hypokalemia, they have a minimal diuretic effect on their own; except for spironolactone at high doses (400 mg daily), which was associated with weight loss and increased urinary excretion of sodium in patients with nephrotic syndrome, compared to healthy controls [65].

#### **5.3 Albumin**

The combined use of 25% albumin with loop diuretics is controversial. Although it is proposed that its administration improves the efficacy of these drugs by increasing their arrival at their site of action, their therapeutic effect is short, and their use is expensive.

The first reports of the concomitant use of albumin infusion with furosemide were promising, however, subsequent reports failed to demonstrate a significant increase in natriuresis or in a decrease in edema [15].

A recent meta-analysis, which included 13 studies with 422 patients, showed an increase of 31.45 ml/hour in urine output (95% CI, 19.30–43.59) and an increase in sodium excretion of 1.76 mEq/hour (95% CI, 0.83–2.69) with the coadministration of albumin and Furosemide, compared with the use of Furosemide alone; although with a high heterogeneity between the studies (I2 = 87%, p < 0.01) and (I2 = 92%, p < 0.01), respectively. It was also observed that the diuretic and natriuretic effect were more significant in those patients with serum albumin <2.5 g/dL, serum creatinine >1.2 mg/dL, estimated glomerular filtration rates (eGFR) < 60 ml/min/1.73 m2 and in those who received doses of albumin greater than 30 gr. In addition, these increases occurred mainly in the first 12 hours after the administration of the albumin infusion [66].

Therefore, we can conclude that, although this combination could increase diuresis and natriuresis in selected patients, the response to treatment is variable. In addition to this, care must be taken with the use of albumin, since there is a risk of increasing overload, precipitating hypertensive uncontrol and the development of acute pulmonary edema, particularly in oliguric patients [11].

#### **5.4 Vasopressin receptor antagonists (AVPr)**

These drugs, also called aquaretics, act by reducing the luminal expression of aquaporins, increasing the urinary excretion of sodium-free water. There are case reports that describe the successful reduction of refractory edema in nephrotic syndrome with the use of Tolvaptan, without being associated with significant adverse effects. However, randomized controlled trials are required to standardize its dosage, duration of treatment and evaluate its safety profile in nephrotic syndrome [67, 68].

#### **6. Amiloride**

Amiloride is a pyrazine-carbonyl-guanidine derivative [69]. It consists of a pyrazine ring substituted with an acylguanidine group attached to position 2 of the ring, where amino groups are attached to positions 3 and 5 of the ring, and a Cl group attached to position 6 (**Figure 2**). Amiloride is a weak base with a pKa of 8.7 in aqueous solution. The protonation of amiloride occurs at the guanidine group. Due to these acid-base properties, amiloride can effectively penetrate biological and artificial membranes [70]. Amiloride is rarely soluble in water.

#### **6.1 Pharmacodynamics**

Amiloride reaches the nephron largely by glomerular filtration and acts on the luminal membrane of the distal convoluted tubule and collecting ducts [69]. In the distal tubules and cortical collecting ducts, there are principal cells that have epithelial sodium channels (ENaC) in their luminal membranes. ENaC is a heterotrimer consisting of three subunits: alpha, beta, and gamma, such that maximum sodium permeability is induced when all three subunits are co-expressed in the same cell. ENaC are a highly regulated site for sodium reabsorption, entering the cell by passive diffusion down the electrochemical gradient created by the basolateral Na+ /K+ pump [71]. High sodium permeability depolarizes the luminal membrane but not the basolateral membrane. The transepithelial potential difference provides a driving force for potassium secretion into the lumen through apical potassium channels. *Proteasuria: The Link between Physiopathogenesis and Edema Management in Nephrotic… DOI: http://dx.doi.org/10.5772/intechopen.108591*

#### **Figure 2.**

*Amiloride chemical structure.*

Amiloride reversibly blocks principal cell luminal ENaC from binding to the channel pore, increasing Na + and Cl- excretion rates. Inhibition of ENaC hyperpolarizes the luminal membrane, reducing the negative transepithelial lumen voltage. Normally, the negative potential difference of the lumen facilitates cation secretion, their attenuation then decreases the excretion rates of K+ , H+ , Ca2+, and Mg2+ [69].

#### **6.2 Pharmacokinetics**

One study suggested that a single oral dose of 20 mg amiloride had a biological half-life of 9.6 ± 1.8 hours in humans. Its effects began approximately 2 hours after drug administration, and peak activity was reached within 4 to 8 hours [72].

Amiloride is absorbed from the gastrointestinal tract and has an oral bioavailability of about 50%. Co-administration with food may decrease absorption by up to 30%, but the absorption rate does not change. After administration, the onset of diuretic activity usually occurs within 2 hours. Maximum diuresis occurs within 6–10 hours. Diuretic effects persist for approximately 24 hours after administration. Amiloride is not metabolized in the liver. About 50% is excreted unchanged by the kidneys in the urine, about 40% is excreted in the feces. In patients with normal renal function, amiloride has a serum half-life of approximately 6–9 hours [73].

#### **6.3 Clinical evidence in nephrotic syndrome**

Nephrotic syndrome is associated with aberrant glomerular filtration of plasminogen and conversion to plasmin in the urine proteolytically activates ENaC and thus contributes to sodium retention and edema. Amiloride inhibits urokinase-type plasminogen activator (uPA) activity in urine, which attenuates plasminogen activation and protease activity in urine in vivo. Urinary uPA is a relevant target for amiloride in vivo [74].

Several lines of evidence support proteolytic activation of ENaC in nephrotic syndrome. In the experimental model of nephrotic syndrome (nephrotic syndrome induced by puromycin aminonucleoside (PAN) and adriamycin), amiloride blocks sodium retention. Three simultaneous patient case reports showed the therapeutic

effect of amiloride and triamterene [45, 75, 76] in patients with different morbidities but severe and resistant nephrotic edema.

Urinary plasmin has been shown to be the dominant serine protease in nephrotic urine that activates ENaC [26, 77]. Plasmin is generated from the proteolytic activation of plasminogen by urokinase-type plasminogen activator. Urinary plasmin excretion in serial samples from nephrotic patients was identified as an independent factor for edema remission [78]. In pediatric patients, volume-expanded acute nephrotic syndrome was associated with significantly higher urine plasmin compared with the remission phase [55]. In the urinary system, uPA is the dominant plasminogen activator [79]. Urinary urokinase is inhibited by amiloride in nephrotic rats [74], mice [59, 80], and diabetic patients [81] by preventing activation of plasmin and ENaC in proteinuria. Since amiloride is a uPA and ENaC blocker, these findings cannot be used to conclude that urinary plasmin is the causal link between proteinuria and ENaC activation.

Two studies have directly addressed the hypothesis that uPA-activated plasmin is involved in sodium retention during nephrotic syndrome [59], one of them using uPA-deficient mice rendered nephrotic using doxorubicin injection. With this approach, excretion of urinary plasmin, but not plasminogen, was blocked in uPA knockout mice with nephrotic syndrome. On the other hand, another study found that inhibition of uPA activity during nephrotic syndrome reduced sodium accumulation [80]. Nephrotic syndrome was induced using inducible inactivation of the podocyte slit diaphragm protein podocin, resulting in massive proteinuria and sodium retention. Amiloride blocked sodium retention and urinary plasmin excretion.

Although plasmin appears to be the dominant active serine protease in nephrotic urine [26], other urinary proteases such as cathepsin B, identified by mass spectrometry, are also present [82]. Thus, at present, several serine proteases might redundantly contribute to ENaC activation and sodium retention during nephrotic syndrome, and direct inhibition of specific proteases only partially attenuates proteinuria-induced sodium retention.

#### **7. Conclusion**

The pathophysiology of edema in nephrotic syndrome is probably related to several factors, essentially the alteration of the forces that govern Starling's principle and sodium retention due to hyperactivation of ENaC in the aldosterone-sensitive distal nephron. Therapeutic strategies that include knowledge of the basic mechanisms that generate edema may be useful to the clinician to make better decisions during decongestion treatment. The use of amiloride has a very intuitive approach on one of the pathways that promote sodium retention. Its efficacy has been proven in some case reports, but it is necessary to carry out controlled studies that can thoroughly evaluate its theoretical efficacy.

#### **Conflict of interest**

"The authors declare no conflict of interest."

*Proteasuria: The Link between Physiopathogenesis and Edema Management in Nephrotic… DOI: http://dx.doi.org/10.5772/intechopen.108591*

#### **Author details**

Mario Alamilla-Sanchez1 \*, Miguel Alcala Salgado2 , Gandhy Fonseca González1 , Carlos Chavez Mendoza3 , Cecilia Acosta Peña1 , Pamela Prado Lozano1 , Daniel Diaz Garcia1 and Julio Nieto Gutiérrez1

1 National Medical Center "20 de Noviembre", Mexico City, Mexico

2 "Christus Muguerza" Hospital, Saltillo Coahuila, Mexico

3 Medica Sur Clinic and Foundation, Mexico City, Mexico

\*Address all correspondence to: silenoz1@hotmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 3**

## The Risk for the Development of Diabetic Nephropathy: Interplay of Biochemical, Genetic, Nutritional and Lifestyle Factors

*Syed M. Shahid and Muhammad Jawed*

#### **Abstract**

The recent unprecedented pandemic COVID-19 has blatantly exposed the healthcare system globally. The increasing rate of mortality and morbidity/co-morbidity were observed due to an interplay of COVID-19 infection with chronic diseases like diabetes, cancers, CVDs, respiratory and mental illness. According to World Health Organization, diabetes kills 1.7 million people annually. The prevalence and incidence of diabetes mellitus, representing >90% of all cases of diabetes and its complications, are increasing rapidly. The International Diabetes Federation has estimated that the number of people with diabetes is expected to rise from 366 million in 2011 to 552 million by 2030 if no urgent action is taken. Diabetes is a leading cause of kidney disease. About one in three adult diabetic patients has kidney disease, that is, diabetic nephropathy. In search for the risk and causes of diabetes and its complications such as diabetic nephropathy, research has now advanced to the molecular level. Genetics, epigenetics, genomics, proteomics, and metabolomics are opening ways to a new and deeper understanding of bodily processes and are providing the tools for more precisely targeted interventions when their function is disturbed. Similarly, tobacco use, physical inactivity, the harmful use of alcohol, and unhealthy diets all increase the risk of developing diabetic nephropathy. This chapter will focus on analyzing recently researched and published biochemical, genetic, nutritional, and lifestyle factors in various populations to ascertain the interplay of a wide variety of modifiable and non-modifiable factors, which will help delay and/or prevent the development of kidney disease in diabetes.

**Keywords:** diabetes, nephropathy, genetic factors, biochemical factors, nutrition, lifestyle behavior, risk assessment

#### **1. Introduction**

#### **1.1 Diabetic mellitus (DM)**

Diabetes mellitus (DM) is a global health issue and is on the rise. About 463 million people worldwide have diabetes and 1.7 million deaths are directly attributed to diabetes each year. The International Diabetes Federation (IDF) estimates that as many as 212 million people, or half of all adults currently living with diabetes, are undiagnosed. Most of these have type 2 diabetes mellitus (T2DM) [1]. Both the number of cases and the prevalence of diabetes have been steadily increasing over the past few decades [2].

DM is a prevalent non-communicable disease worldwide. In most high-income countries as well as middle-income and low-income countries, DM is among the top causes of death [3]. DM is considered a group of metabolic disorders which have multiple etiologies. Chronic hyperglycemia is a characteristic of DM and is related to impaired carbohydrate, protein, and fat metabolism. The etiology of DM is a complex interaction of genetics, biochemical, environmental, nutritional factors, and lifestyle choices [4, 5]. The causes of abnormalities include impaired insulin action or inadequate insulin secretion or both. As a result of chronic hyperglycemia, extensive damage such as abnormal functions and failure of various body organs which especially include blood vessels, eyes, kidneys, nerves, and heart [6].

DM has been classified into four distinctive types [7]:


The risk factors which play a major role in the development of T2DM and the progression of diabetic complications such as diabetic nephropathy (DN) include modifiable behavior risk factors such as unhealthy diet and physical activity which alone is attributed to 1.6 million deaths annually. The leading metabolic risk factor globally is elevated blood pressure to which 19% of global deaths are attributed, followed by overweight and obesity and raised blood glucose [8]. The IDF estimates that as many as 212 million people (or half of all adults) currently living with diabetes mellitus, are undiagnosed. Most of these have T2DM, which is preventable and by knowing the risk score, the chances of developing T2DM can be minimized [9].

#### **1.2 Diabetic nephropathy (DN)**

Microvascular complications in DM involve effects on small vessels, arterioles, capillaries, and venules. These complications develop early in the pathogenesis of T2DM and account for morbidity in the form of neuropathy, retinopathy, and nephropathy [10, 11]. The major cause of chronic renal disorder in patients who starts renal replacement therapy is DN [12]. CVD mortality becomes increased in DN patients [13, 14]. The low glomerular filtration rate (GFR) without micro or

*The Risk for the Development of Diabetic Nephropathy: Interplay of Biochemical, Genetic... DOI: http://dx.doi.org/10.5772/intechopen.108800*

macroalbuminuria is observed in 10% of DM and also in type 1 DM and microalbuminuria [15]. A 24-hours urine collection or spot urine measurement of microalbumin may screen microalbuminuria or DN [16]. Urinary tract infections (UTI), hematuria, and exercise may produce falsely elevated urine protein levels. Prevention is the only initial treatment of DN. The development of DN is strongly associated with glucose control like other microvascular complications of DM.

#### **2. Risk factors for the development and progression of diabetic nephropathy**

There are various risk factors for developing DM and progression of DN, which include age, gender, obesity, ethnicity, genetics, diet and exercise, abnormal cholesterol level, malnutrition, maternal malnutrition, maternal hyperglycemia, lifestyle and development in childhood, lifestyle in adulthood, physical inactivity, and depression.

#### **2.1 Age**

It was generally considered that T2DM patients are above 40 years of age at diagnosis time, but an increased number of children and adolescents are being diagnosed due to increased obesity in childhood. For the incidence of T2DM age is one of the important factors universally. Compared to the western countries DN occurs at lower ages in low- and middle-income countries [17, 18]. An inverse relationship has been observed between potential harm from T2DM and age at diagnosis. The younger you are at the time of diagnosis, the bigger the possible complications such as diabetic nephropathy. As age progresses, it is likely to have multiple medical conditions, including high blood pressure and high cholesterol. That can make it harder to keep the development of T2DM under control. As a result, T2DM can lead to other health problems such as heart disease.

#### **2.2 Gender**

Men have double the likelihood of getting T2DM as compared to women. Studies suggested that T2DM is highly prevalent in males than females, while females show high prevalence rates for IGT than males [17, 18]. A noticeable female excess of T2DM was observed in the first half of the last century, but now T2DM prevalence is equal among male and female in most populations, with some evidence of male majority in early middle age [19].

#### **2.3 Ethnicity**

A number of sociocultural factors including family history, and environmental factors play a part in developing T2DM, but it is still not clear why people from certain ethnic backgrounds have an increased risk. Various ethnic groups with different lifestyles may account for some of the predisposition to T2DM. The more determinant role may be played by genetic factors. Compared with Individuals of European origin, African-Americans Native Hawaiians, Hispanic/Latino Americans, Asian Americans, American Indians, or other Pacific Islanders are at increased risk for T2DM [20].

#### **2.4 Obesity**

One of the thriving predictors of developing T2DM and its complications, such as DN is obesity. Majority of DM that go into the DN are overweight at the time of diagnosis, and they are more expected to have central obesity (fat concentrated around the waist). In different parts of the world, children and women showed higher obesity rates which is associated with the level of insulin and metabolic risk [21].

#### **2.5 Diet and physical inactivity**

Excessive susceptibility to DM, mainly through the development of obesity is associated with animal fats and carbohydrates. In the USA and other westernized societies, the main changes in the diet include increased consumption of processed carbohydrates and animal fats while decreased fiber intake [22].

#### **2.6 Malnutrition**

Diet should be nutritionally adequate for DM patients. There are various evidence of the strong association between micronutrient deficiencies and risk for DM [23]. The role of undernutrition in the occurrence of type 2 DM in different populations has been established [24].

#### **2.7 Maternal malnutrition**

Pregnant mothers are commonly malnourished, due to this reason offspring are exposed to intrauterine growth retardation (IUGR) and compromised metabolic potential during the prenatal period. Maternal malnutrition ultimately results in maternal mortality rate and low birth weight [25].

#### **2.8 Lifestyle and growth in childhood**

Growth of malnourished children may result in increased body fat than bone length and muscle mass, it may expose these children to higher risk of developing DM. One of the indications of early malnutrition is stunting and accumulation of fat in the child is indicated by stunting and overweight among children who have been malnourished in early life [26]. Central obesity and type 2 DM as a result of higher body fat percentage were found to be higher in stunted children even at body weight falling within the normal range [27, 28]. Higher frequency of various risk factors which include physical inactivity and overweight were observed in children with positive family history of DM, upper income group, and children living in urban areas. The risk of DM increases in children from lower income group because they have poor nutrient density of diets [29] and those low birth weights were also common in these children. Children from urban areas show low activity levels [30].

#### **2.9 Lifestyle in adulthood**

The risk of developing DM is increased in the population, who have a high intake of carbohydrates, as a result of which insulin resistance is developed. An increased oxidative stress is developed as a result of low intake of vitamins and minerals as well as decreased immunity. The dietary pattern of society, in general, favors diabetic

*The Risk for the Development of Diabetic Nephropathy: Interplay of Biochemical, Genetic... DOI: http://dx.doi.org/10.5772/intechopen.108800*

conditions, so-called pro-diabetic. The risk of diabetes would therefore be high in those who are taking lower than average proteins and micronutrients. The levels of toxicities caused by minerals are found higher among diabetic patients as compared to non-diabetics which will certainly cause higher levels of oxidative stress [31, 32]. The incidence of T2DM development decreases in people who are used to physical activity which includes 30 minutes walking or cycling regularly [33].

#### **2.10 Depression**

There is a significant association between newly diagnosed DM with depression [34]. Depression affects all aspects of a person's life being a common public health problem. It has been recognized as an important co-morbid condition in DM [35]. Depression frequently co-exists with DM and DM-related complications. Abnormal glucose level and increased rates of DM complications along with disabilities are observed in depressive patients [36].

#### **3. Genetic susceptibility**

The high risk of developing DN in T2DM is associated with a positive family history of T2DM and/or DN. Genetics is an important factor for the predisposition of South Asians to T2DM and its complications. Pathogenesis of DN depends on genetic factors as well and genetically susceptible individuals can develop DN after environmental interaction DN is a complex, polygenic disease. For the identification of associated genes for the development of DN, two main strategies have been used which include analysis of candidate genes and more recently genome-wide scan [37].

For the identification of these main genes efforts have been made, but due to different genes associated with small effects in the specific population, results are inconsistent. Detection of the individual at high risk of developing DN and understanding the pathophysiology of DN is possible after the identification of candidate genes. Identification of those genes associated will allow the recognition of individuals at high risk and an understanding of mechanism and progression of DN is also possible. Disease burden and mortality can be reduced by providing earlier and more aggressive therapies to the individual at high risk. Advances in pharmacogenetic research may help treatment choices by selecting renoprotective drugs according to individual haplotypes [38–40]. Candidate genes involved in different metabolic functions of the body, responsible for DN development in type 2 DM are illustrated in **Table 1**.

#### **3.1 Renin angiotensin aldosterone system**

Blood pressure and water (fluid) balance is being regulated by the hormonal system known as renin-angiotensin system (RAS) or the renin-angiotensin-aldosterone system (RAAS). Juxtaglomerular cells in the kidneys secrete renin directly into circulation when blood volume is small. The angiotensinogen which is being released by the liver is converted into angiotensin I by plasma renin. ACE which is found in the lungs converts angiotensin I into angiotensin II. Blood vessel constriction is caused by angiotensin II which is a potent vaso-active peptide, it results in high BP. It also stimulates the secretion of the hormone aldosterone from the adrenal cortex. Aldosterone causes the reabsorption of sodium and water into the blood by the kidney tubules. It increases the fluid volume in the body, which increases BP [41].


#### **Table 1.**

*Genes involved in the progression of T2DM complications such as DN.*

#### **3.2 Angiotensin converting enzyme gene**

ACE gene is localized on the long arm of chromosome 17 (17q23). It is 21 kb long and consists of 26 exons and 25 introns. In the National Center for Biotechnology Information (NCBI) records, greater than 160 ACE gene polymorphisms are enlisted, most of these polymorphisms are SNPs. Only 34 of those are located in coding regions of the gene while 18 are missense mutations. ACE is a zinc metallopeptidase, which is dispersed on the surface of endothelial and epithelial cell surfaces [42].

ACE gene has received substantial attention as a possible candidate for DM and its complications like hypertension, cardiovascular diseases, and nephropathy. The insertion–deletion (I/D) polymorphism of 287-bp alu repetitive sequences at intron 16 of the ACE gene is the frequently occurring variant that results in three different genotypes; II, ID, and DD. The II and DD are homozygotes and ID is heterozygote. This polymorphism is responsible for the variability in the activity of ACE in serum and various tissues as well as deletion is found to be associated with the raised activity of the enzyme. On the other hand, the low ACE activity increases insulin-stimulated hexose transport in adipocytes and insulin suppression of non-esterified fatty acid flux [43, 44].

*The Risk for the Development of Diabetic Nephropathy: Interplay of Biochemical, Genetic... DOI: http://dx.doi.org/10.5772/intechopen.108800*

#### **3.3 ACE insertion/deletion polymorphism**

The possible genetic factors for the development and progression of nephropathy in DM are the ACE gene insertion (I) and deletion (D) mutation or polymorphism (rs1799752). Based on the presence or absence of 287 bp alu-repetitive sequences in intron 16, three genotypes II (homozygous for I), and ID (heterozygous for ID) and DD (homozygous for D) are found. ACE mediates the regulation of blood volume, arterial pressure, cardiac and vascular functions, and electrolyte metabolism [45].

Conflicting findings in different populations on the association between ACE I/D gene polymorphism and DN development led us to investigate the ACE I/D gene polymorphism as an important risk factor for the development of hypertension and nephropathy in DM [46]. Wide inter-ethnic allelic variations of the ACE, I/D gene polymorphism were thought to be responsible for the conflicting gene-diabetic nephropathy disease association worldwide. The I/D polymorphism of the ACE gene and association of diabetic nephropathy was positively identified in Japan, South India [47, 48].

#### **3.4 ACE G2350A polymorphism**

In the ACE gene, the exonic polymorphism G2350A (rs4343) is located in exon 17. It has been revealed to exert the most significant effect on plasma ACE levels. Dimorphism was found to be significantly associated with SBP with an average increase of 3.2 mmHg. There are three genotypes on the basis of such polymorphism which include AA (homozygous for adenine), AG (heterozygous for adenine and guanine), and GG (homozygous for guanine) [49].

ACE G2350A polymorphism association with hypertension showed inconclusive results of either null or positive associations [50]. Another study revealed that in Muslims from the Arab Gulf and Pakistan the ACE 2350A allele is associated with significantly reduced hypertension while elevated risk among Chinese [51]. The allele A of the G2350A polymorphism is considered to be an independent risk factor for susceptibility to ESRD among Malays [52]. Another study has also shown the positive association of I/D and G2350A polymorphism with the development of ESRD [53].

Several factors are responsible for the lack of reproducibility which might include sample size, study design, power issues, and true variability among different populations [54].

#### **3.5 Angiotensinogen gene**

The AGT gene is localized on the long arm of chromosome 1(1q42–43), which consists of five exons. More than 23 variants of the AGT gene have been linked with hypertension [55]. However, whether or not the genetic variations of the AGT gene contribute to the risk of developing T2DM remains to be confirmed.

#### **3.6 AGT M268T polymorphism**

M268T polymorphism of the AGT gene was previously recognized as M235T. About 78 molecular variants of the human ACE gene have been reported, since the identification of its gene sequence [56]. The association between AGT M268T (M235T) gene polymorphisms and CVD has been identified in various studies [57]. Increase in levels of angiotensinogen in T268 homozygous variant leads to an increase in BP. The AGT M268T polymorphism was found to be in linkage disequilibrium with T207M and promoter region A-6 G polymorphisms [58]. Haplotype analysis of AGT T207M and M268T revealed a significant association with hypertension among the Caucasian and Taiwan Chinese population [59]. The response of different variants of the AGT and other members of RAAS is different to different antihypertensive treatments. The role of AGT variants has been studied in different ethnic groups and variable results were identified [60].

#### **3.7 Fat mass and obesity (FTO) gene**

FTO-Gene variants are found to be associated with food intake, obesity, and metabolic risks. This study explored the differences in food intake and a possible association between diet and metabolic risk markers in T2DM having different FTO gene alleles. Metabolism of AA variants appears to be more sensitive to dietary components and supports the application of nutrigenomics in the management of T2DM. A linear correlation was observed between diet and metabolic profile markers such as BMI, waist circumference, blood pressure, and lipid profile among high-risk alleles AA. The associations of energy intake and percent level of carbohydrate and protein intake with metabolic syndrome were significantly higher among risk alleles AA. Studies suggest that the genetic profile is likely to affect both dietary habits as well as the association between diet and metabolic syndrome markers. Therefore, it can be concluded that the diet-disease associations are more prominent in individuals having risk alleles AA as compared to protective alleles TT and heterozygous alleles AT as far as FTO gene is concerned [61, 62].

#### **4. Conclusions**

A number of genetic, biochemical, nutritional, and lifestyle factors are found to be involved in the progression of type 2 diabetes mellitus and its complications such as diabetic nephropathy. Many genes including ACE, AGT, and FTO polymorphisms are significantly associated with the development and progression of diabetes nephropathy. ACE gene D-allele and DD-genotype are significantly correlated with abnormal metabolism and progression of diabetic nephropathy. Studies show that the distribution of T-allele and TT-genotype in the AGT gene were significantly different in diabetic patients with and without complications, nephropathy, for example.

Similarly, FTO rs9939609 SNP was found to be associated with an increased risk of metabolic syndrome in diabetic patients. AA-allele and A-genotype appear to be sensitive to dietary components and support the application of nutrigenomics in the management of diabetes and its complications. Future studies with a larger number of population and a variety of samples are recommended to confirm the findings and susceptibility to inform and progress the prevention and better management of the disease.

*The Risk for the Development of Diabetic Nephropathy: Interplay of Biochemical, Genetic... DOI: http://dx.doi.org/10.5772/intechopen.108800*

#### **Author details**

Syed M. Shahid1 \* and Muhammad Jawed<sup>2</sup>

1 School of Health and Sport Science, Eastern Institute of Technology (EIT), Auckland Campus, New Zealand

2 Department of Biochemistry, Fazaia Ruth Pfau Medical College, Karachi, Pakistan

\*Address all correspondence to: sshahid@eit.ac.nz

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[62] Hakeem R, Fawwad A, Shaheen F, Waris N, Nawab S, Shahid S, et al. Fat mass and obesity associated gene (FTO) and differences in food intake and diet-disease relationships. Journal of the Academy of Nutrition and Dietetics. 2017;**117**(9):A57. DOI: 10.1016/j. jand.2017.06.169

#### **Chapter 4**

## Recent Advances in Clinical Diagnosis and Pharmacotherapy Options of Membranous Nephropathy in Iraqi Patients

*Ali Lateif Al Geboury, Maha Hameed Al-Bahrani and Nawar Mohameed Alsayhood*

#### **Abstract**

Membranous nephropathy (MN) is one of the various glomerular diseases causing nephrotic syndrome, also referred to as membranous glomerulopathy. It can be diagnosed at any age in general, and males are more often affected than females (with the sex ratio being 2–3:1). Membranous nephropathy is a relatively rare disease in adults (approximately half of all cases are common in older White adults). Statistical analysis shows that 80% of patients with MN have high creatinine level, dyslipidemia, hypoalbuminemia, proteinuria more than (3.5 g/day), and fluid retention (edema), while 20% with asymptomatic with non-nephrotic levels of proteinuria (< 3.5 gram/ day) involves the reaction of an inflammatory process in the basement membrane. It can be distinguished from nephritic syndromes by the absence of active sediments, hematuria, and red cell casts in urine microscopy. The two main causes of nephrotic range proteinuria are the loss of the anionic charge barrier in the membrane and podocyte destruction, which results in albuminuria. The field has advanced greatly and quickly over the past 10 years thanks to the development of cutting-edge instruments for disease diagnosis, classification, monitoring, and treatment. This core curriculum aims to serve as both a broad guide for the clinical management of disease and an overview of recent developments in the field. In the review, we critically summarized different diagnosis biomarker therapies used for the treatment of MN patients in Iraq. These groundbreaking discoveries were swiftly applied to clinical diagnosis and management. The diagnosis and treatment monitoring processes benefited significantly from significant advancements in detection techniques.

**Keywords:** idiopathic membranous nephropathy, membranous nephropathy, PLA2R, THSD7A, membranous glomerulopathy

#### **1. Introduction**

Membranous nephropathy (MN), a glomerular disorder, is the most common cause of nephrotic syndrome in Caucasian individuals who do not have diabetes. The majority of MN cases are primary (idiopathic), while the remainder are related to systemic illness, pathogen exposure, or medications use [1]. Membranous nephropathy develops when the glomeruli, which are small blood capillaries in the kidney that are responsible for ultrafiltration of waste products and other polymers from the blood, suffer injury and thicken. As a result, a large amount of protein infiltration into the urine (proteinuria) with an increase in single nephron glomerular filtration rate (SNGFR) and increase in glomerular capillary hydraulic pressure (GCHP) [2]. The loss of these proteins eventually results in the nephrotic syndrome's signs and symptoms. However, the kidney has a huge number of filtering cells or units known as glomeruli. There were only three layers of capillary walls in these glomeruli:


### **2. Signs and symptoms of MN**

Membrane nephropathy occurs gradually, so you may not notice any abnormal signs. Some patients may not exhibit any symptoms until they develop renal disease, despite the fact that many patients experience mild symptoms at the outset of the disease [7]. MN symptoms and signs include:


*Recent Advances in Clinical Diagnosis and Pharmacotherapy Options of Membranous… DOI: http://dx.doi.org/10.5772/intechopen.109186*


Or symptoms and signs due to complications include:


A small percentage of MN patients report no symptoms; in these cases, clinicians may discover symptoms either during routine office visits or after evaluating certain biochemical indicators. Most individuals (60–70%) will have symptoms of MN, and 1–3% having proteinuria in the subnephrotic range less than 3.5 g/day. One to three percent of MN patients also experience microscopic hematuria. Only a small percentage of patients experience hypertension or renal failure, and older people are more likely to experience these conditions. About 7% prevalence of venous thrombophlebitis and dyslipidemia has been found in MN. The rate of MN is high, and it is challenging to predict how the condition will develop naturally. Up to 30% of cases are known to have spontaneous remission. The percentage of patients who enter spontaneous remission is significantly lower. 2 to 3% of patients either have ongoing proteinuria with stable kidney functions or will eventually experience renal failure. The risk of fatal thromboembolic and cardiovascular events rises even in patients who do not develop nephrotic disease. If kidney function rapidly decreases, a secondary disorder such as interstitial nephritis or renal vein disease may be present [8].

#### **3. Causes of MN**

Frequently, some types of autoimmune activity cause membranous nephropathy (primary membranous nephropathy). Immune complexes accumulate on the glomerular basement membrane (GBM) and cause GBM thickening, which is known as membranous nephropathy. Primary MN is typically unknown, although secondary MN causes the following:


### **4. The possibility of developing MN**


### **5. Complications**

Membranous nephropathy has a number of complications that include the following:


*Recent Advances in Clinical Diagnosis and Pharmacotherapy Options of Membranous… DOI: http://dx.doi.org/10.5772/intechopen.109186*


#### **6. Epidemiology**

Adults with MN have a higher frequency of nephropathy than those with focal segmental glomerulosclerosis (FSGS) [15]. In White people over 40 years, it is the most frequent cause of primary nephrotic syndrome. In 2002, a report showed patterns of glomerular diseases in Iraq and other Arabic countries as shown in **Table 1**. The most frequently diagnosed lesion in our patients with primary glomerular disease is FSGS [16]. This Iraqi study indicates that the histology of the patients' biopsies with proteinuria exhibits certain patterns that might reflect glomerular pattern illnesses in Iraq and might not be distinct compared to those in the other Arab nations and Middle Eastern nations in particular. In the USA, the peak incidence occurs between 50 and 60 years [13, 17]. MN is more prevalent in men than in women, and the latter often has a favorable outcome in women. When MN is diagnosed in young women, lupus nephritis should be suspected. Approximately 75% of the MN cases occur as a primary disease and about 25% as secondary MN that is associated with autoimmune diseases (e.g., systemic lupus erythematosus (SLE)), infection with hepatitis B or hepatitis C, HIV, and less commonly Epstein–Barr virus (EBV) that can also cause acute or chronic kidney disease. Parasitic agents which cause malaria or schistosomiasis are potential causes of secondary MN, cancer (breast cancer), drugs, and toxin as aforementioned in paragraph [18].

In children, MN is less common due to secondary causes, accounting less than 7% of biopsies and is associated with other diseases such as hepatitis B, autoimmune, or thyroid diseases. It may also develop in conjunction with other types of glomerulonephritis (focal segmental glomerulosclerosis (FSGS), IgA nephropathy (Berger's


*\*PGN: primary glomerulonephritis, FSGS: focal segmental glomerulosclerosis, MCD: minimal change disease, MPGN: membranoproliferative glomerulonephritis, MGN: membranous glomerulonephritis.*

#### **Table 1.**

*Relative frequency of various types of glomerular lesions among adults in different countries.*

disease), and lupus nephritis) [12, 19]. According to an Iraqi study, Denovo Iraqi patients with MN are at the last stage of renal disorders as a result of a distinct main renal ailment. Children having transplanted kidneys are more likely to experience it [20]. It was discovered in 48 of 530 pediatric allograft biopsies in one study. It typically happens many years after a kidney transplant, as in this instance. The interim period for de novo MN ranged from 63 to 102 months, in two significant retrospective studies. Despite this infestation, other reports described the de novo MN at an early onset [13]. Recently, it was discovered that children with early-onset MN had circulating antibodies reactive with bovine serum albumin (BSA) in their blood and also had immunological deposits in the kidneys. It is suggested that ingestion of modified albumin (BSA) will lead to the development of anti-BSA AB and will attach to anionic residues in the BM, where it will act as a planted antigen for the development of immunological accumulations in situ [21, 22].

#### **7. Pathogenesis**

Membranous nephropathy, also known as membranous glomerulonephritis (MGN) (**Figure 1**) [22], was first described as a type of glomerular disease by Bell in 1946. In adults, it is accounts 20–30% of idiopathic nephrotic syndrome and about 1–9% in children. In large majority of cases, this condition occurs in a primary ("idiopathic") form, but the disease has been related to a wide variety of conditions (secondary) in approximately 20–25% of adults and 80% of children with MGN. Circulating autoantibodies bind to an autoantigen on the surface of the podocytes, resulting in in situ immune complex formation that activates the lectin complement pathway and causes podocyte injury and proteinuria. Two mechanisms presumed responsible for the development and localization of the deposits along the GBM are those of in situ immune complex formation in the subepithelial capillary wall or that of circulating immune complexes being deposited in that location [6].

According to experimental findings, most immune complexes are formed in situ by circulating antibodies binding to antigens normally found in the glomerulus or to extrinsic antigens previously planted as free antigens in the subepithelial area. Primary human MGN appears to be mostly an autoimmune disease characterized by the

#### **Figure 1.**

*Shows a glomerulus, compared to normal on the left and one affected by MN on the right. On the right, the dark spots are collections of Ag-Ab complexes that accumulate between layers of filters and then lead to thick. The green color is podocytes caused by damaged part of the filter by the immune system and stopped working properly.*

*Recent Advances in Clinical Diagnosis and Pharmacotherapy Options of Membranous… DOI: http://dx.doi.org/10.5772/intechopen.109186*

formation of glomerular immune complexes in situ. Over 70–80% of primary MGN patients have autoantibodies directed against PLA2R expressed in podocytes and proximal tubules. These circulating PLA2R autoantibodies are of the IgG4 subtype [14].

• plasma levels correlate with:

\*Disease activity.

\*Therapeutic response.

\*The prediction of the risk of posttransplantation recurrence.

\*\*2018 7A (THSD7A), further expanding the role of autoimmunity in these patients.

\*\*\*In a subset of patients with MGN, all-immune response appears to play a role in developing MGN. Neutral endopeptidase (NEP), expressed in podocytes and proximal tubular brush border, has been set as the target antigen (Ag) of antibodies accumulated in the epithelial space in patients with antenatal MN. In these cases, it is likely that the anti-NEP antibodies produced by the mother are transplacentally transferred to her child with genetic deficiency of NEP [13, 21].

\*Some patients with Pompe disease and mucopolysaccharidosis IV receiving recombinant α glucosidase and aryl sulfatase B, respectively, also develop alloimmune responses resulting in MGN. De novo MGN in allograft also appears to be an alloimmune response. Conditions associated with secondary MGN include as those in **Table 2** [23].

Secondary forms of MGN typically lack circulating PLA2R, but a small subset of patients with hepatitis B, hepatitis C, malignant neoplasm, and sarcoidosis have circulating serum PLA2R. While this may be coincidental, it also raises the possibility that "primary" MGN is triggered by an underlying disease. The underlying disease and how it is treated can have a significant impact on the natural history and overall prognosis of MGN. When secondary MN is caused by drugs, toxic substances, or infections, removing the etiologic agent often results in the clinical symptoms disappearing and the renal lesion healing. The nephrotic syndrome may abate and the glomerular changes regress after resection and treatment of a malignant tumor. In patients with membranous lupus nephritis, the course is indolent, whereas those who develop a superimposed anti-GBM antibody disease undergo a rapid progression to renal failure.


#### **Table 2.**

*Condition associated with membrane nephropathy.*

### **8. Diagnosis of MN**

Membranous nephropathy may not manifest any clinical symptoms. A routine urine test for another health issue may reveal that the patient has high levels of protein in the urine in this case. If the patient has no signs or symptoms of protein in the urine, a medical physical exam should be done, and blood pressure should be measured. The following tests may be performed:

#### **8.1 A urine test (urinalysis)**


#### **8.2 Blood tests**

Blood test measures either plasma or serum in order to give a numeric value that directly indicates renal function. These tests include


#### **8.3 Imaging test**

• The noninvasive diagnostic imaging process is known as computed tomography (CT). This test may be done with or without contrast. This test can offer a lot of detail about the kidneys compared with normal kidneys, such as injuries, various kidney diseases, and an accumulation of fluid and stones in the kidney [29, 30].

*Recent Advances in Clinical Diagnosis and Pharmacotherapy Options of Membranous… DOI: http://dx.doi.org/10.5772/intechopen.109186*

#### **8.4 Renal biopsy**

• A small fragment of the kidney is removed by the histopathologist. This test is required to confirm the details of the information for diagnosis. It can reveal the kind of kidney illness, the degree of renal damage, and the potential effectiveness of various treatments.

#### *8.4.1 Microscopic (histological) description*


### *8.4.2 Immunofluorescence description*

• In all stages of glomeruli, IgG4 staining may be seen in a subepithelial distribution, as shown in **Figure 4**.

**Figure 2.**

#### **Figure 3.**

*Silver stain shows a pattern of subepithelial spikes of glomerular membrane basement in a primary membranous nephropathy [32].*

#### **Figure 4.**

*Immunofluorescence microscopic examination for IgG under 400x power demonstrates a good diffuse granular staining along the capillaries of glomerular in a primary membranous nephropathy [17, 32].*

#### *8.4.3 Electron microscopy description*

	- 1.Scattered on the epithelial cells of the glomerular basement membrane
	- 2. Subepithelial deposits with dark silver staining with thickened basement membrane material as shown in **Figure 5**.

*Recent Advances in Clinical Diagnosis and Pharmacotherapy Options of Membranous… DOI: http://dx.doi.org/10.5772/intechopen.109186*

**Figure 5.** *Subepithelial deposits with thicker basement membrane material and dark silver staining [33].*

**Figure 6.** *Basement membrane material between and surrounding subepithelial deposits.*


#### **8.5 New antibodies tests**

Renal biopsy is frequently used to establish a new diagnosis of membranous nephropathy. The gold standard for analyzing and identifying the damage pattern of MN is renal biopsy. Standard light and electron microscopic investigation, however, was unable to capture the true nature of MN. When kidney tissue is examined histologically, immunological deposits can be seen along the glomerulus' basal membrane if the analysis is successful. Future diagnostics will greatly benefit from the

**Figure 7.** *Detailed foot process effacement and subepithelial immune complex deposits.*

identification of the M-type antiphospholipase A2 receptor 1 (PLA2R) and antithrombospondin type 1 domain-containing 7A (THSD7A) antibodies as specific biomarkers for primary membranous nephropathy because it enables noninvasive diagnosis using straightforward serological testing.

#### *8.5.1 Anti-PLA2R antibodies*

In 2009, the M-type phospholipase A2 receptor (PLA2R) was discovered as the primary target in membranous nephropathy (MN). Anti-PLA2R antibodies have a high specificity and sensitivity of around 70–80% for primary membranous nephropathy (pMN) primary with various ethnic groups [35], but the prevalence of autoantibodies against PLA2R is unknown among Iraqi patients with MN [15]. Human podocytes express PLA2R, a 180-kDa transmembrane glycoprotein that is a member of the MR family of mannose receptors. Autoantibodies against M-type phospholipase A2 receptor (PLA2R) serve as specific diagnostic and tracking biomarkers for idiopathic membranous nephropathy (IMN), and their quantification helps monitor disease activity [36, 37]. The MN related to PLA2R defines patients with elevated levels of anti-PLA2R antibodies circulating in the serum as well as those with the presence of the PLA2R antigen in the kidney biopsy specimen stained with special staining. Patients with the presence of autoantigen but no autoantibodies account for approximately 10–15% of pMN cases. Iraqi study described a renal transplant patient with recurrent allograft membranous glomerulopathy who had a great rituximab response and outstanding graft function. Both the staining of the biopsies for the Ag of both PLA2.R and THSDA.7 and the serum testing for anti-PLA2R and anti-THSA7 were negative. IgG4 was confirmed by IgG subclass staining. He took two doses of rituximab once more and continued to respond quite well with no protein and normal renal function. Circulating anti-Ab of PLA2R1 is thought to be a particular biomarker of immunological activity in primary membranous nephropathy that correlates with indicators of disease activity (degree of proteinuria), and it is useful in predicting clinical outcomes like response to treatment and disease recurrence [9, 17, 38].

*Recent Advances in Clinical Diagnosis and Pharmacotherapy Options of Membranous… DOI: http://dx.doi.org/10.5772/intechopen.109186*

#### **8.6 Anti-THSD7A antibodies**

Thrombospondin type-I domain-containing 7A is a multi-domain transmembrane protein expressed on podocytes with molecular weight of 250 kDa [1, 39]. Its function is to increase the adhesion of cells. Therefore, anti-THSD7A antibodies lead to changes in the structure and function of the permeability of the slit diaphragm to plasma proteins. Scientific reports show a relationship between the THSD7A antigens and their antibodies with neoplasms [40]. They have linked proliferative conditions including rectal cancer, gallbladder cancer, or ALHE-angiolymphoid hyperplasia with eosinophilia with secondary MN related to the presence of anti-THSD7A as a result of the expression of THSD7A antigens within the tissues of some tumors. Allograft membranous glomerulopathy might be a recurring or de novo condition, according to an Iraqi study published in 2015. Since THSD7A antibodies seem to be uncommon in the serum of individuals with cancer-related membranous nephropathy, it was unlikely to have a diagnosis of malignancy-associated MN with 100% accuracy in this report [41, 42].

In pMN patients, autoantibodies against THSD7A are prevalent at a rate of 2.5 to 5%. This prevalence corresponded to 8–14% of the patients not having anti-PLA2R antibodies. It is noteworthy to notice that none of the individuals who tested positive for anti-THSD7A also tested negative for anti-PLA2R. Therefore, to identify a greater proportion of potential pMN patients, it is advantageous to preform simultaneous detection of anti-PLA2R and anti-THSD7A [20, 27].

THSD7A is expressed by some types of cancer, and many studies have been suggested that anti-THSD7A antibodies are associated with an increased risk of cancerassociated MN and screening for cancer was recommended in patients with anti-THSD7A positive MN. Anti-Ab of THSD7A can be present in primary MN patients and is not found in healthy people or people who have renal autoimmune disorders. The detection of PLA2R1 antibodies and anti-THSD7A antibodies enables the diagnosis of roughly 75–85% of cases, as PLA2R antibodies are present in about 70% of primary MN patients. Therefore, the simultaneous detection of anti-THSD7A and anti-PLA2R antibodies offers a comprehensive method for pMN detection [12, 21].

#### **9. Response measurements**

Improved kidney function, CR, and PR are the best recognized responses. CR is defined as a urine protein excretion of less than 0.3 g/24 hours together with normal serum albumin and creatinine levels. PR is defined as a urine protein excretion of less than 3.5 gram/day (24 hrs) together with improved or normalized serum albumin concentration and steady serum creatinine.

Following a CR, 30% or more of MN cases will relapse. However, most of them will return to subnephrotic level proteinuria and maintain acceptable long-term renal function. Clinical remission in patients with anti-PLA2R antibody positivity precedes a decline in circulating antibody titer, and a clinical resistance is linked to antibody persistence despite treatment.

#### **9.1 Treatment**

Patients can be provided with either nonimmune suppressive or immunosuppressive therapy based on the predicted criteria mentioned above.

In patients with MN, treating proteinuria with PR/CR is unmistakably related to a low rate of kidney disease development. There are potential newer markers like anti-PLA2R and THS D7A to distinguish between primary and secondary MN, predict prognosis, and evaluate therapy response [6].

#### **9.2 Conventional treatment**

This treatment includes managing edema, dietary protein intake, blood pressure, and hyperlipidemia. Loop diuretics are the cornerstone of treatment for edema control, along with a low-salt diet. It is typically advised to consume between 0.75 and 1.0 g of protein per kilogram each day. First-line antihypertensive medications should be antiprotein uric medicines such angiotensin-converting enzyme inhibitors (ACEi) or angiotensin receptor blockers (ARBs). Treatment with an (ACEi) or an (ARBs) may be adequate to diminish concentration levels of proteinuria to a level of subnephritic in patients with low proteinuria levels of less than 4 grams per 24 hours, significantly reducing renal and cardiovascular risk. However, the use of these drugs alone is unlikely to result in a significant decrease in the level of proteinuria or the preservation of kidney function in individuals with greater levels of proteinuria.

About 7% of individuals with MN have severe nephrotic syndrome, and their risk of developing thromboembolic consequences is higher if their serum albumin level is lower than 2.8 g/dl. People with MN who are severely nephrotic (proteinuria >10 g/ 24 h and serum albumin 2.5 g/dl) should generally be considered for anticoagulation; prophylactic anticoagulation has been demonstrated to be more effective in nephrotic patients with range proteinuria.

#### **9.3 Immunosuppressive agent therapy**

It has been shown that a variety of immunosuppressive drug-based therapeutic modalities are effective in reducing the levels of proteinuria. According to the risk factor profiles, patients with MN can be categorized into three levels: low, medium, and high risk.

#### **9.4 Treatment of those with a low risk factor**

This group had a risk of developing kidney disease of less than 5% over a 5-year period following notification. Over a 6-month monitoring period, the patient at low risk had normal kidney function with a level of proteinuria less than 4 grams per 24 hour period. Therefore, a cautious course of treatment is effective for these patients.

#### **9.5 Treatment of those with a moderate risk**

This group still exhibits sustained daily excretion of urine protein at rates ranging from 4 to 8 g per 24 hours and intact renal function after 6 months of such therapy. Patients in this case utilized more than one of below approaches.

#### **9.6 Corticosteroid immunotherapy**

An early study was done on 72 patients with idiopathic nephrotic syndrome who took a course of high dose alternate day prednisolone for 2–3 months, and the results significantly reduced the progression to kidney failure, but this course had no effect

*Recent Advances in Clinical Diagnosis and Pharmacotherapy Options of Membranous… DOI: http://dx.doi.org/10.5772/intechopen.109186*

on the level of proteinuria. Hundred and fifty-eight patients with idiopathic MN were treated in a subsequent prospective randomized study for 6-month course of alternate prednisolone, and the results showed that corticosteroid therapy alone had no discernible benefits on either renal function preservation or remission induction after a mean of 45 months. Corticosteroid monotherapy has therefore been demonstrated to be ineffective in bringing about remission in MN patients [43].

#### **9.7 Cyclosporine as an immunosuppressive drug**

A high percentage of recurrence was found in early uncontrolled investigations using cyclosporine A (CSA), despite initial benefits being reported.

Thus, these drugs have been used for a long time for the maintenance in patients with CR or PR, particularly those at a high risk of recurrence. This must be balanced against the possibility of kidney damage from prolonged CSA exposure [29].

#### **9.8 Treatment of patients at high risk**

This patient category is characterized by persistently high proteinuria (28 g/day), highly elevated anti-PLA2R antibodies, or progressive kidney failure.

#### **10. Corticosteroids**

In the UK, a prospective double-blind randomized controlled experiment revealed no difference that was statistically significant between the control and treatment groups in the mean values of proteinuria and other kidney function markers like urea, creatinine, and uric acid.

Primary MN was treated with cytotoxic medications in conjunction with intravenous or oral corticosteroids [44]. Numerous research findings have shown how effective chlorambucil (leukeran) is at treating MN. Another trial randomly assigned participants to either supportive therapy only or a combination of prednisolone and leukeran. When compared to either the cyclosporine A (CSA) or supportive treatment groups, the prednisolone and chlorambucil group accrued the major end point, a further 20% fall in creatinine clearance, less frequently [29].

#### **11. Cyclosporine**

Renal functions and proteinuria significantly improved [45].

#### **11.1 Mycophenolate mofetil (MMF)**

Report by Miller and others found that treating 16 medium- or high-risk MN patients with MMF had no discernible effect on levels of creatinine or over the course of the experiment. An analysis of MN patients who had MMF treatment revealed similar results. Combined with steroids, three out of six patients' proteinuria improved by 61%.studies have shown that the majority of patients with primary glomerulopathies handled empirical MMF therapy well and were able to withdraw from steroids, ameliorate their nephrotic syndrome, and stabilize their renal function.

#### **11.2 Rituximab**

In different studies, the use of rituximab for patients with idiopathic MN showed more effective in both proteinuria and kidney function improvement [6, 46, 47].

#### **11.3 Eculizumab**

Eculizumab is a humanized anti-C5-monoclonal antibody designed to prevent the cleavage of C5 into its pro-inflammatory byproducts. More extensive research must be conducted in order to its involvement in the therapy of MN [48].

#### **11.4 Adrenocortical tropic hormone**

The use of synthetic adrenocorticotrophic hormone (ACTH) in the form of i.m injection in one study compared with combined corticosteroid and cyclophosphamide or chlorambucil showed no significant changes in the rate of remission (proteinuria/kidney function) [29].

#### **Acknowledgements**

The authors would like to thank all the doctors and researchers for providing the information on Iraqi patients with MN.

#### **Conflicts of interest**

The authors declare no conflict of interest.

#### **Information about data availability**

The authors will make the raw data used to support this article's conclusion available without undue reservation.

#### **Nomenclature and abbreviations**


*Recent Advances in Clinical Diagnosis and Pharmacotherapy Options of Membranous… DOI: http://dx.doi.org/10.5772/intechopen.109186*


#### **Author details**

Ali Lateif Al Geboury<sup>1</sup> \*, Maha Hameed Al-Bahrani<sup>2</sup> and Nawar Mohameed Alsayhood<sup>3</sup>

1 Baghdad private Hospital, Baghdad, Iraq

2 Molecular and Medical Biotechnology Department, College of Biotechnology, Al-Nahrain University, Baghdad, Iraq

3 Al-Yarmouk Teaching Hospital, Baghdad, Iraq

\*Address all correspondence to: alialgeboury@yahoo.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 5**

## Complement-Mediated Kidney Disease

*Ivana Vuković Brinar and Matija Matošević*

#### **Abstract**

From very discovery of the complement cascade, it had an intriguing role in pathophysiology of kidney disease. The hallmark of complement cascade involvement in kidney diseases comprises of immune-complexes deposits in the glomeruli, acting as activation for the classical pathway. However, additional mechanisms of complement activation, namely alternative and lectin pathways are extremely important and prominent in complement-mediated kidney disease. Disease prototype of activation of complement is an atypical hemolytic uremic syndrome with solid activation of complement and C3 glomerulopathy is a hallmark of fluid phase activation of alternative complement pathway. Further research has shown that alternative pathway also plays a role in pathogenesis and progression of other kidney diseases including anti-neutrophil cytoplasmic antibody-associated vasculitis and immune complexmediated glomerulonephritis as well as IgA nephropathy. A better understanding of complement system's role in kidney disease has also brought forth novel therapeutic approaches in form of complement cascade inhibitors, revolutionizing the treatment of patients that were faced with unfavorable outcomes. Through this chapter, we bring to you an overview of most prevalent complement-mediated kidney diseases with emphasis on the role of complement in their pathogenesis and the potential for treatment targeting the complement cascade.

**Keywords:** complement, kidney, glomerulonephritis, aHUS, ANCA, vasculitis, nephropathy, complement inhibition

#### **1. Introduction**

Ever since the complement cascade was first described in the 19th century, researchers have been interested in understanding the role of complement system in pathogenesis of kidney diseases [1]. Plethora of studies has shown that complement deposits paired with decreased levels of complement components in serum play a significant role in complement-mediated kidney diseases, with serum C3 and C4 component levels and C3 and C1q staining of kidney biopsies samples being a mainstay in diagnostic approach and clinical outcome monitoring. As a progression of complement-mediated kidney disease, acute kidney injury (AKI) also stems from complement activation, which acts as an important factor in pathogenesis, affecting all the major structures within the kidney [2].

#### **2. Overview of the complement Cascade system**

The complement cascade consists of soluble and cell-bound components, acting in many different roles, from regulatory and executive elements to receptors. White blood cells, platelets, and different tissues interact with the complement system, combining in an inflammatory response aimed to protect against pathogens both infectious and noninfectious [3–6]. Three distinct pathways play a role in complement cascade activation. The classical pathway is triggered by the formation of immune complexes comprised of immunoglobulin M (IgM) or G (IgG) paired with pathogens or other foreign antigens, which in turn activate C1 complex of the complement system. C1 complex, comprised of three types of subunits, C1q, C1r, and C1s, binds to the Fc portion of either IgG or IgM, inducing the cleaving process that yields C4 and C2 components, which are further cleaved to form a classical pathway C3 convertase, C4bC2a [3–6]. Lectin pathway is mediated by either mannose-binding lectin (MBL) or ficolin-carbohydrate complex on the pathogen surface. Both MBL and ficolin form complexes with MBL-associated proteins (MASPs), main of which is MASP2. Conformational alterations in MASP2, induced by binding to pathogens, lead to cleavage of C4, with C4b component attaching itself to the surface of the pathogen, bringing about the binding of C2 component and its subsequent cleavage by MASP2, thus producing C2a component and forming a lectin pathway C3 convertase C4bC2a [3–6]. An alternative pathway is set off by carbohydrates present on foreign surfaces, which causes C3 components to constantly be hydrolyzed in a "tick over" mechanism, forming C3b components. This brings about the recruitment of factor B, which binds to C3b, inducing factor D to cleave factor B and form an alternative pathway C3 convertase C3bBb, which is stabilized with plasma properdin [3–6].

Both C4bC2a formed through classical and lectin pathways and C3bBb of alternative pathway cleave C3 component, resulting in the release of C3a and C3b, with C3b further amplifying complement activation and phagocytosis. C3b also forms a complex with C3 convertase, forming C5 convertase (C3bBbC3b


#### **Table 1.**

*Overview of complement system regulators.*

and C4bC2aC3b), which cleaves C5 component into C5a and C5b, latter of which combines with C6, C7, C8, and multiple C9 components, forming the membrane attack complex (MAC, C5b-9), a terminal complement component responsible for formation of pores by inserting itself into cell membrane, effectively leading to cell lysis, while C3a and C5a components act as anaphylatoxins [3–6].

Considering the complexity of complement system and its multitude of interactions with various components of immune system, mechanisms of regulation are needed to limit the activity of complement system to proper levels, thus preventing damage to the host cells in both fluid and solid phases [3, 4, 6]. A list of regulatory components and their function is outlined in **Table 1**. Aside from complement's role in recognition and eradication of pathogens, it is also included in B and T cell function, acting as mediators and signal modulators in B-cells and playing a role in maintaining T-cell growth, proliferation, and differentiation [4, 5].

#### **3. Role of complement Cascade in kidney diseases**

As our knowledge of complement system and its intricate interactions with our immune system continues to grow, our understanding of its role in pathogenesis of kidney disease also changed. Long has been known that deposition of immune complexes (IC) in the glomeruli is the cause of classical pathway activation, thus involving the complement system directly in pathogenesis of immune complex-mediated glomerulonephritis [1, 5]. Due to fenestration of glomerular endothelium and negative charge, glomerular basement membrane (GBM) is susceptible to IC and antigen binding, resulting in immune response. Furthermore, antibodies can bind to antigens expressed by the glomerulus itself, either already present there or formed through conformational changes. All this elicits the activation of classical complement pathway [1, 5]. Genetic or acquired dysregulation of alternative pathways is also a major cause of kidney disease, particularly thrombotic microangiopathy, characterized by thrombocytopenia, hemolytic anemia, and acute kidney injury (AKI), is usually present in nonimmune-complex glomerulonephritis [1, 7]. Complement activation plays a major factor in AKI, although it is still to be determined whether complement activation represents a unique occurrence in each type of AKI or is it a general response to injury [2]. Genetic variations of complement regulatory proteins also play a role in pathogenesis of kidney diseases, mainly affecting AP regulators such as factor H and I and membrane cofactor protein (MCP) [5]. Effective application of complement inhibitory drugs, such as complement C5 inhibitor Eculizumab, also supports a key role of complement system in kidney disease, as well as sets up a novel target for potential treatments [5, 8].

#### **3.1 IgA glomerulonephritis**

IgA glomerulonephritis (IgAN), also known as IgA nephropathy, is the most common type of glomerulonephritis, with an estimated annual incidence between 0.2 and 5.7 per 100,000, and a leading cause of chronic kidney disease worldwide [1, 6, 9–11]. IgAN is an autoimmune disease of unanswered etiology with "multihit theory" usually used to describe the pathogenesis of this multifactor disease [6, 9–11]. Due to poor glycosylation, aberrant IgA1 antibodies (Gd-IgA1) are formed, characterized by presence of galactose-deficient *O*-glycans. This glycosylation defect appears to be hereditary, although this alone is not enough for the

development of disease levels of Gd-IgA1 correlate with the severity and outcome [11, 12]. Gd-IgA1 is targeted by antiglycan autoantibodies, due to exposed terminal *N*-acetylgalactosamine residues acting as neoepitopes for IgG antiglycan autoantibodies [11, 12]. After the formation of Gd-IgA1-antiglycan IgG immune complexes (Gd-IgA1-IC), they are deposited in the mesangium of kidney, where they initiate inflammatory and proliferative signaling cascades, promoting local inflammation, mesangial matrix production, and mesangial cells proliferation, ending in glomerular and interstitial fibrosis leading to renal injury [10–12]. Aforementioned Gd-IgA1-IC also causes activation of complement cascade, specifically alternative and lectin pathway, causing the accumulation of C3 components and its subsequent deposition, along with mesangial C4. *FHR-*gene cluster is also involved in development of IgAN, with elevated plasma FHR1 contributing to inflammation and stronger C3 fragment deposition due to inadequate inhibition through factor H (6,10–13. Furthermore, on one hand, genetic variants of FHR5 contribute to disease susceptibility, due to its role as a complement activator, but on the other hand, FHR1 and FHR3 deficiency have demonstrated a protective role [6]. As for the lectin pathway, mannose-binding lectin and L-ficolin, both acting as pattern recognition molecules in charge of activating lectin pathway, have been detected in the glomeruli of IgAN patients, with genetic variants in genes for mannose-binding lectine-2 (MBL-2) representing a risk factor in progression of IgAN [6, 10–13]. In addition to primary IgAN, there is also so-called "secondary IgAN," usually associated with other systemic conditions and sharing pathophysiological processes, histological and clinical manifestation with primary IgAN [11].

Considering a complex and multiple interactions needed for development of disease, clinical manifestations of IgAN can come in a plethora of clinical syndromes [11]. The most common clinical manifestation of IgAN in adults includes a combination of asymptomatic hematuria with fluctuating levels of proteinuria with or without chronic kidney disease [10–12]. Hematuria in IgAN can be either macroscopic gross hematuria (MGH) or microscopic hematuria, with MGH being more common in pediatric population and early stages of adult IgAN [12]. MGH is usually accompanied by mucosal infections (either respiratory or gastrointestinal), pointing at dysregulation of mucosal immune system and mucosa-kidney axis as potential contributing factors to pathogenesis of hematuria [12]. Proteinuria paired with hypertension and reduction in glomerular filtration is key factor in chronic kidney disease, with amount of proteinuria being especially relevant, considering that more than 0.5 g/day of protein is associated with poor renal outcome. On rare occasions, nephrotic syndrome can occur and should not be confused with more common "nephrotic-range" proteinuria, which usually is not paired with hypoalbuminemia. Although renal function impairment in IgAN usually progresses chronically, acute kidney injury (AKI) can develop in two specific cases: First being in rapidly progressive glomerulonephritis and second due to acute tubular injury from red blood cells or heme toxicity. Aside from all of the above, systemic forms of IgAN can also occur, more frequently in pediatric population. These include Henoch-Schönlein purpura and IgA vasculitis. Also, pathology finding of thrombotic microangiopathy (TMA) is present, in spite of insufficient laboratory evidence. Clinical outcomes are also varied, ranging from spontaneous remission in 5–15% to rapidly progressive glomerulonephritis and end-stage renal disease [10, 12, 11]. Considering that light microscopy findings can be varied, diagnosis of IgAN relies heavily on immunofluorescent microscopy, which is used not only to visualize IgA deposits in the mesangium but also to show C3 deposits, pointing toward complement activation in glomeruli [10, 11].

#### *Complement-Mediated Kidney Disease DOI: http://dx.doi.org/10.5772/intechopen.108555*

For now, there is no curative treatment available for IgAN. Treatment is usually focused on comprehensive supportive care, including angiotensin-converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers for regulation of hypertension, statins, low sodium diet, and avoiding nephrotoxic drugs. Control of proteinuria is essential for renal function improvement. If renal function continues to deteriorate, a 6-month course of corticosteroids can be recommended [10, 11]. With new knowledge coming to light regarding the role of complement in IgAN, complement inhibition presents a novel treatment modality for IgAN [11, 12, 14]. Eculizumab, anti C5 complement monoclonal antibody, was used off-label for IgAN in 2 patients [15, 16], with results being lackluster, with only temporary clinical improvement, due to the fact that IgAN is characterized by primarily C3 deposits, while eculizumab blocks C5 component of complement, thus not preventing accumulation of C3 [1]. This implies that other complement inhibitors, targeting higher instances of complement cascade, such as factor B inhibitor LNP023, MASP-2 inhibitor OMS721, and anti-C3 compstatin and APL-2, could potentially be more useful in preventing the role of complement in IgAN [11, 12, 17].

#### **3.2 Membranous nephropathy**

Membranous nephropathy (MN), also known as membranous glomerulonephritis, is an autoimmune disease caused by an accumulation of immune complexes in the subepithelial space of the glomerular capillary wall, and thus it is the most common cause of nephrotic syndrome in adults. Rarely affecting children, usual age of onset is between 50 and 60 years of age [1, 6, 10, 18, 19]. Main cause of MN is accumulation of circulating autoantibodies that target specific antigens on the surface of podocytes. Being of IgG type, these antibodies form immune complexes that accumulate in the subepithelial space, thus distorting physiological function of basement membrane [10, 18]. Commonly targeted surface antigen is the podocyte protein called M-type phospholipase A2 receptor (PLA2R), which is affected in 70 to 80% of cases, while in around 3 to 5% of cases anti-thrombospondin type-1 domain-containing protein 7A (THSD7A) is the main antigen [10, 20]. MN is classified as either primary, caused by autoantibodies targeting aforementioned proteins, or secondary, caused by other autoimmune diseases, malignant tumors, infection, or drugs [10, 18, 19]. The role of complement system is not fully elucidated in pathophysiology of MN. Complement components, especially C3 and C5b-9, can be found alongside immune-complex deposits in primary MN. Although both anti-PLA2R and anti-THSD7A antibodies are mostly of IgG4 subtype, which are unable to interact with C1q complement component making it subsequently unable to activate the classical pathway of complement activation, there is still evidence of classical pathway activation, along with lectin pathway and alternative pathway activation. Classical pathway can be activated through other subtypes of antibodies, namely IgG1,2, and 3, which could explain the presence of C1q components in renal biopsies. On the other hand, lectin pathway activates in patients exhibiting mannose-binding lectin (MBL) in the glomeruli. Due to "tick-over" phenomenon, alternative pathway is constitutively active. Taking into account all of the aforementioned complement pathways, it is highly likely that different levels of activity are present in different patients [6, 10, 18, 21]. Pathohistological approach to diagnosing MN includes light microscopy, which shows thickened glomerular capillary wall, due to deposition of immune complexes and complement components, with visible spikes, immunofluorescent microscopy, which displays IgG staining abetted by C3 complement components, and anti-PLA2R antibodies. Furthermore, subclass

staining for IgG may help in classification. Electron microscopy can be used, where available, to show subepithelial deposits. Findings of IgG4 antibodies and deposits located only in subepithelial space point toward primary MN, while additional findings of deposits on intramembranous and mesangial levels with IgG1, 2, and 3 subclasses of antibodies imply a secondary MN [10]. MN usually presents as nephrotic syndrome, although non-nephrotic proteinuria is present in 20% of patients [10]. Treatment of MN is largely only supportive and aimed at managing hypertension, reducing edema, and reducing protein excretion. To that effect, ACEI or ARB is prescribed, preferable over other antihypertensive drugs due to their renoprotective properties. Patients with hyperlipidemia and coagulation dysregulation should be introduced to statins and anticoagulants, respectively. If the patients are unresponsive to conservative treatment, alkylating agents (such as cyclophosphamide or chlorambucil) paired with corticosteroids can be attempted. Besides that, calcineurin inhibitors as monotherapy and mycophenolate mofetil can be used. Considering that complement system plays a role in pathogenesis of MN, targeting specific key steps in complement cascade is an enticing therapeutic target. Ongoing studies regarding APL-2, which binds C3 and C3b, act as their inhibitor, while eculizumab, a C5 complement component blocker, has already seen use in MN, although to poor effect regarding proteinuria [19, 10].

#### **3.3 C3 Glomerulopathy**

C3 glomerulopathy (C3G) is a clinical and pathological condition primarily caused by dysregulation of the alternative pathway of complement system, specifically the fluid phase [6, 10, 22]. C3G is relatively rare, with incidence ranging from 1 to 3 cases per million per year, and affects children and adolescents more often [22]. There are two main subtypes of C3G, one of them being dense deposit disease (DDD) characterized by electron-dense deposits within GBM and other being C3 glomerulonephritis (C3GN), in which deposits are found along subendothelial side of GBM but also in the mesangium, differentiating two subtypes [1, 6, 10, 22]. Classical definition of C3G lies upon positive immunofluorescence staining for C3, which should be either isolated or at minimum two orders of magnitude greater than other immune reactants. But this criterion is not infallible, as immunofluorescence results can be borderline and inconclusive. Furthermore, complement abnormalities can also be found in membranoproliferative glomerulonephritis (MPGN) and lastly, immunofluorescence stain used in clinical settings marks primarily C3c, but in C3G, the predominant accumulated compound is C3dg. Taking all of this into an account, a novel pathogenic classification was proposed, identifying four different clusters with main differences being in levels of C3 and prevalence of complement genetic abnormalities [22, 23].

The focal point of pathogenesis of C3G is a dysregulation of alternative complement pathway brought forth by genetic abnormalities, autoantibodies against complement components, or nephritic factors (NeFs) responsible for C3 and C5 convertase stabilization [6, 10, 22]. Genetic abnormalities are present in around 25% of patients, with C3 gene, complement factor B, H, and I genes (CHB, CFH, and CHI) and complement factor H-related protein gene (CFHR) being the most affected. Pathogenic variants in C3 disrupt cleavage of the molecule due to erroneous recognition sites for CFH and CHI binding, while on the other hand, CFB pathological variants bestow gain-of-function properties. Variants in CFI genes diminish its cofactor activity and CFH variants have a profound effect on N-terminal regions, causing disruption in fluid phase regulation [6, 10, 22]. NeFs are a heterogenous group of antibodies that target neoepitopes in C3 or C5 convertase. This results in stabilization

#### *Complement-Mediated Kidney Disease DOI: http://dx.doi.org/10.5772/intechopen.108555*

of both convertases, leading to a prolonged half-life. Two main types of NeFs have been described: C5NeFs, properdin-dependent NeFs-targeting epitopes of C5 convertase, mainly found in C3GN, and C3NeFs, properdin-independent NeFs targeting C3 epitopes in DDD [6, 10, 22]. In addition to these main antibodies, C4neFs, targeting classic and lectin pathway C3 convertase, was also described, along with autoantibodies against CFH, CFB, and C3b but their role remains uncertain [22].

Although C3G usually presents in a wide array of clinical manifestations, ranging from incidental asymptomatic microscopic hematuria to chronic kidney disease, most frequent manifestation is proteinuria with preserved kidney function [1, 6, 10, 22, 24]. Considering an infection can precede C3G, differential diagnosis must include postinfectious glomerulonephritis, with specific markers like anti-streptolysin O and deoxyribonuclease B antibodies. There is a number of possible external, non-renal manifestations, such as retinal drusen and atrophy and acquired partial lipodystrophy. In majority of cases, C3G is progressive, with almost half the patients progress to end-stage kidney disease in 5-year period [1, 6, 10, 22, 24].

Diagnosis of C3G depends on histopathologic findings of kidney biopsies. Under light microscopy the most prevailing finding is that of membranoproliferative glomerulonephritis, found in more than 50% of cases, but findings of mesangial proliferative, diffuse endocapillary proliferative and diffuse sclerosing glomerulonephritis can also be present. Crescents, either cellular or fibrocellular can also be found. Immunofluorescence staining is used to highlight C3 in the mesangium and capillary walls, with no considerable staining for immunoglobulins, C1q, and light chains. Electron microscopy is used to distinguish between C3GN and DDD. In addition to histopathologic procedures, genetic and molecular analysis pertaining to complement systems can be helpful to acquire valuable information regarding pathophysiological mechanisms [22, 24].

There is no single ideal treatment for C3G, as treatment modalities are etiologically dependent. Renin-angiotensin system (RAAS) inhibitors are used as a supportive therapy, targeting hypertension and proteinuria. Furthermore, recent studies have shown that renin possesses an intrinsic ability to cleave C3, thus acting as an accelerator of alternative pathway activation, which further justifies the use of RAAS inhibitors [10, 22]. Immunosuppression, usually consisting of either mycophenolate mofetil or rituximab, has also been proven to produce better outcomes, especially if paired with corticosteroids [22, 24]. The use of complement inhibitors is justified, considering involvement of complement activity in pathophysiological mechanism of C3G, but evidence remains limited, with varying results [6, 10, 22, 24].

#### **3.4 Postinfectious glomerulonephritis**

Postinfectious glomerulonephritis (PIGN) is a type of glomerulonephritis that develops after infections, most common of which include streptococcal pharyngitis, other upper respiratory tract infections, gastroenteritis, skin infections, and pneumonia, although there have been reports of no recent infections preceding PIGN [1, 10, 25]. PIGN shares its clinical and pathological traits with C3G, with both entities being preceded by infections and sharing dysregulations in alternative complement pathways, suggested by low C3 and normal C4 plasma levels. PIGN is usually a rare occurrence as a result of adequate and effective treatment of preceding infection, but when it does occur, it is self-limiting. However, "atypical postinfectious glomerulonephritis" is a phrase used to describe a type of PIGN characterized by alternative pathway activation that causes it to persist, with possibility of progressing, with

persistent proteinuria and hypocomplementemia, into end-stage kidney disease due to decline in renal function [1, 10, 25]. This complement involvement is in support of findings pointing toward similarities between PIGN and C3G that could potentially cause a diagnostic challenge [1, 10, 25, 26].

Clinical presentation of PIGN is varied, typically consisting of hematuria, nephrotic or less than nephrotic range proteinuria, azotemia, hypertension, peripheral edema, and anemia [1, 10, 25].

Pathological findings pointing toward diagnosis of PIGN consist of proliferative glomerulonephritis with subepithelial "humps" visible on electron microscopy, paired with bright C3 staining with or without IgG deposits on immunofluorescence microscopy [1, 10, 25].

Due to PIGN being a primarily self-limiting disease, supportive therapy is modality of choice, consisting mainly of fluid and electrolyte control paired with antihypertensives, correction of potential acidosis and, if required, dialysis [1, 10, 25].

#### **3.5 Atypical hemolytic uremic syndrome**

Atypical hemolytic uremic syndrome (aHUS) is a disease primarily caused by dysregulation in an alternative pathway of complement. Majority of aHUS patients are adults, but around 5 to 10% are pediatric patients, making it a relatively rare but complicated disease to battle [27–29]. Although mutations of complement regulatory proteins are present in majority of cases, in some instances, they are not phenotypically active, thus requiring a trigger, usually in form of bacterial or viral infections, parasites, other autoimmune diseases, and drugs, including chemotherapy and vaccines. Due to active COVID-19 pandemic, special mention needs to be put on SARS-CoV-2 as a potential novel trigger of aHUS [27, 29].

The main pathophysiological mechanism of aHUS is the loss-of-inhibition of regulatory proteins of the alternative pathway of complement system, namely CFH, CFI, and membrane-cofactor protein (MCP). This occurs either through genetic mutations targeting specific genes coding for said regulatory proteins or through activity of anti-CFH antibodies. These changes, paired with a trigger that induces complement activation through an alternative pathway, cause the loss of protection of endothelial cells, making them susceptible to complement-induced damage. Loss-of-inhibition of regulatory proteins is not the only possible mechanism. Gain-of-function mutations in genes coding for C3 complement component and CFB, main components of alternative pathway C3 convertase are also connected to overactivation of alternative complement pathway, thus resulting in pro-inflammatory and procoagulant activity of endothelial cells. This pathophysiological mechanism results in development of thrombotic microangiopathy (TMA), a characteristic pathology finding in aHUS and source of its clinical presentation [1, 27–29].

Clinical presentation mainly consists of hemolytic anemia, thrombocytopenia, and end-organ damage, most commonly in form of renal failure, which is characterized by oliguria and hypertension [27–29]. Other symptoms can also occur, most often cardiovascular and neurological but also pulmonary, gastrointestinal, and dermatological, making aHUS a complicated clinical entity to recognize [27–29].

Diagnostic approach to aHUS is one of exclusion, considering there are no direct tests for it and available biomarkers are not completely dependable. There must be no associated disease present, no criteria met for *Shiga* toxin-producing *E. coli* HUS (STEC HUS), which is the most common cause of aHUS, and no criteria met for thrombotic thrombocytopenic purpura (TTP), namely serum ADAMTS 13 activity

*Complement-Mediated Kidney Disease DOI: http://dx.doi.org/10.5772/intechopen.108555*

should be under 10%. Complement system activity investigation is strongly advised including measuring of C3, C4, CFH, and CFI plasma concentrations, measuring of MCP expression, anti-CFH antibody levels, and sC5b-9 levels [27–29].

Eculizumab and ravulizumab, monoclonal antibodies that act as complement component C5 blockers, are treatment of choice in aHUS. Other than that, plasma exchange therapy and plasma infusions can be used if complement blockers are unavailable or until an aHUS diagnosis can be confirmed with exclusion. Complement C5 blockers prevent cleavage of C5 into C5a and C5b, which has a twofold effect, negating pro-inflammatory activity of C5a and also disrupting the formation of C5b-9 (MAC) complexes, altogether resulting in end of acute hemolysis and stabilization in both platelet count and renal function [10, 27–29].

#### **3.6 ANCA-glomerulonephritis**

Vasculitis is an inflammation of the blood vessels, specifically their wall, which brings about structural and functional damage. Vasculitis is classified using the 2012 International Chapel Hill Consensus Conference (CHCC), which defines vasculitis according to vessel size [1, 30, 31]. Antineutrophil cytoplasmic antibody-associated (ANCA) vasculitis (AAV) is subdivided into three clinical entities: granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA). AAV is relatively uncommon, with incidence ranging up to twenty per million population per year. It is also worth noting that incidence increases with age, reaching its peak around 60 to 70 years of age [1, 30, 31]. Familial hereditary forms of AAV have been described but are rare [30]. The main role in AAV is given to ANCA autoantibodies, which target cytoplasmic antigens of primary neutrophil granules and monocytic lysosomes. Most commonly targeted antigens are proteinase 3 (PR3) and myeloperoxidase (MPO). PR3-ANCA is commonly related to GPA while MPO-ANCA is found in MPA or renal-limited vasculitis [1, 30, 31]. Other than that, there exists ANCA-negative patients, which exhibit clinical features and pathology usually found in AAV. These patients usually have a renal-limited disease, with fewer systemic features [30].

Neutrophils are the main arbiters of vessel injury found in AAV, stimulated by inflammatory cytokines (namely tumor necrosis factor α and interleukin 1), lipopolysaccharide, or complement component C5a, due to either infection or other types of inflammation. This causes a shift of MPO and PR3 to neutrophil surface, priming the neutrophils, making it easy for ANCAs to bind to these antigens, evocating a potent cellular activation [1, 30, 31]. All of this results in degranulation of neutrophils and release of reactive oxygen species, causing vessel damage. Due to extrusion of neutrophil extracellular traps (NETs), neutrophil undergo NETosis, a special form of cell death. These NETs have a multitude of functions, including directly injuring endothelium, transferring MPO or PR3 to endothelium and dendritic cells for antigen presentation, and activating the alternative pathway of complement [1, 30, 31]. Tissue deposition of PR3 and MPO, paired with chemokines, brings about the recruitment of autoreactive T cells and monocytes, further propagating tissue injury [1, 30, 31]. Originally thought that complement involvement was limited due to scarce complement depositions in kidney biopsies and lack of hypocomplementemia, novel studies demonstrate a role of alternative pathway of complement in pathogenesis of AAV, with C5a and C5a receptor (CD88) being the center of this process [30–32]. Activated neutrophils release properdin, an alternative pathway promotor, which activity propagates generation of C5a through an alternative pathway, which in turn binds to C5a receptor, further priming and activating neutrophils, thus creating an amplification loop [30–33].

ANCA-GN is characterized by necrotizing and/or crescentic GN without significant immune-complex depositions, either on immunofluorescent or electron microscopy. In some cases, small amounts of IgG and C3 deposits can be found, which is associated with more severe disease [30].

Clinical presentation of GPA consists of constitutional symptoms, chronic respiratory illness, arthralgia and leukocytoclastic skin rash, lung nodules and, most importantly, acute kidney injury (AKI), which has been proven by pathological findings, including necrotizing and crescentic pauci-immune GN and PR3-ANCA positive results. On the other hand, MPA is distinguished from GPA by absence of granulomatous manifestations and positive MPO-ANCA finding [1, 30, 31]. AKI is very common in AAV and the most important factor in predicting mortality, with patients who present with glomerular filtration rate lesser than 50 mL/min having a 50% risk of death of kidney failure in 5-year time. Typical presentation includes rapidly progressive GN paired with sub-nephrotic range proteinuria, microscopic hematuria, and hypertension. To minimize the progression of kidney presentation, timely initiation of therapy is crucial [1, 30, 31]. Aside from kidney involvement and constitutional symptoms (fatigue, myalgia, and fever), systemic features include lung involvement, namely pulmonary necrotizing granulomatous lesions, upper respiratory tract disease, including rhinitis, sinusitis, otitis media, and granulomatous inflammation, purpuric rash on extremities (secondary to leukocytoclastic vasculitis), cutaneous nodular lesions, peripheral neuropathy, mesenteric vasculitis (with abdominal pain and blood in stool), and cardiac involvement can be present. In rare instances, vasculitis of the liver and pancreas can mimic hepatitis or pancreatitis, making differential diagnosis difficult [1, 30, 31].

Approach to treatment includes several phases, the first being induction phase, which lasts for three to six months and is used to stop the inflammation process and lessen tissue damage. Next is the maintenance phase, which lasts from 24 to 48 months with the aim of preventing relapse. Glucocorticoids combined with either cyclophosphamide or rituximab represent a standard of care for induction phase, while in refractory disease, it is recommended to switch the initial induction agent. Aside from cyclophosphamide and rituximab, mycophenolate mofetil can also be used, but with a greater risk of relapse. Plasma exchange can also be used due to its ability to remove ANCAs and other inflammatory mediators. With newly identified role of alternative complement pathway, complement inhibition is a novel approach to treatment. Avacopan, oral C5a receptor (CD88) inhibitor, and IFX-1, a monoclonal antibody targeting C5a, are in clinical trials, with avacopan already being a feasible therapeutic option instead of corticosteroids [1, 30–32].

#### **4. Conclusion**

Over the years, a large body of evidence has been accumulated pointing toward complement system as a major pathogenesis factor in many kidney diseases considering that kidneys are uniquely susceptible to complement-mediated inflammation. It is clear that involvement of complement in kidney disease represents a fruitful field of research, hoping to better understand pathogenesis of many kidney diseases and also provides a novel target for potential treatment.

*Complement-Mediated Kidney Disease DOI: http://dx.doi.org/10.5772/intechopen.108555*

#### **Author details**

Ivana Vuković Brinar1,2\* and Matija Matošević2

1 University Hospital Center Zagreb, Zagreb, Croatia

2 School of Medicine, University of Zagreb, Croatia

\*Address all correspondence to: ivemedex@yahoo.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*Complement-Mediated Kidney Disease DOI: http://dx.doi.org/10.5772/intechopen.108555*

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#### **Chapter 6**

## Investigation of Sickle Cell Nephropathy

*Rumeysa Duyuran and Hülya Çiçek*

#### **Abstract**

Sickle cell nephropathy is a complication of sickle cell anemia (SCD), a genetically inherited blood disease. It is a genetic disorder characterized by the presence of HbS modified due to amino acid mutation. The mutation causes hemoglobin to assume a sickle shape under certain conditions, leading to various complications such as decreased elasticity, increased hemolysis, and vascular occlusion. Polymerization of HbS in an oxygen-free environment causes organ dysfunction by contributing to vascular occlusion and tissue hypoxia. These sickle-shaped cells can cause blockages in the circulatory system and tissue hypoxia, leading to damage to various organs. Nephropathy is known as one of the common complications of sickle cell disease. Sickle cell nephropathy is generally characterized by impaired renal function, proteinuria, hematuria, hyposthenuria, and sometimes renal failure. Treatment of sickle cell nephropathy focuses on symptomatic supportive treatments, and in advanced cases such as renal failure, kidney transplantation may be required. In summary, sickle cell nephropathy is a condition that causes damage to the kidneys as a result of sickle cell anemia and can lead to serious complications. For these reasons, it becomes important to conduct further research to support the literature.

**Keywords:** sickle cell anemia, nephropathy, erythrocyte, hemoglobinopathy, HbS

#### **1. Introduction**

In 1910, Herrick JB first described sickle cell nephropathy in a black student. He described anemia with elongated sickle-shaped red blood cells, increased urine volume, and decreased urine density [1]. Sickle cell disease (SCD) is a genetically inherited disease that affects many systems and is a clinical condition that occurs when red blood cells contain modified HbS. The main features of the disease are recurrent painful attacks, chronic hemolytic anemia, and acute and chronic organ dysfunction [2]. HbS is formed by the replacement of glutamine at position 6 at the amino (-NH2) end of the beta-globin chain with the amino acid valine. It is formed when GTG (Guanine-Tymine-Guanine) replaces GAG (Guanine-Adenine-Guanine) at the base level. As a result of this mutation, the oxygen-free HbS polymerizes, and the solid precipitates into crystals. Erythrocytes take from a biconcave disc shape to a crescent-like sickle shape (**Figure 1**) [3]. The deformed cells are destroyed early in the spleen, and it also reduces blood flow and causes congestion, especially in small vessels.

**Figure 2.**

Hemoglobin distributes oxygen to the tissues and its high density inside the erythrocytes provides the ability of the erythrocyte to maintain and change shape. Normal human hemoglobin has 4 polypeptide chains and 4 heme groups. Polypeptide chains consist of 2 alpha and 2 beta chains. The main hemoglobin found in adults is HbA and the amount of HbA2 is very small. The HbF level is high throughout fetal life and its proportion in erythrocytes decreases after birth. Hemoglobinopathies result from changes that result in abnormal hemoglobin synthesis [4].

*Investigation of Sickle Cell Nephropathy DOI: http://dx.doi.org/10.5772/intechopen.113757*

Hemoglobinopathies are examined in 5 basic groups and the most common ones are sickle cell syndromes (structural hemoglobinopathies) and thalassemias (globin chain synthesis disorder). Structural hemoglobinopathies, including sickle cell anemia, occur due to a mutation that changes the amino acid sequence of a globin chain, which changes the physiological properties of hemoglobin and creates the typical clinical symptoms of the disease (**Figure 2**) [5].

With a single point mutation, the replacement of glutamic acid in the 6th position of the β globin chain with valine, that is, the replacement of adenine in deoxyribonucleic acid (DNA) with thymine (GAG-GTG) results in the HbS molecule. A small genetic change significantly affects the molecular balance [6].

#### **2. Overview of sickle cell nephropathy**

The most common hemoglobinopathy in the world is SCD. Clinical findings in SCD are quite variable. While some patients are asymptomatic and detected in community screenings, some patients may apply to a doctor with a severe crisis. Most of the patients live completely asymptomatic except during these crisis periods [7]. Atherosclerosis seen in patients with sickle cell anemia is an important cause of morbidity and mortality due to organ damage and disease [8]. One of the patients' most frequently affected organs is the kidneys. Atherosclerosis develops as a result of the sickling of erythrocytes and the adhesion of sickle cells to the endothelium. This causes structural and functional disorders in the kidneys. Changes are seen throughout the nephron from the glomeruli to the papillary end. Side effects such as hyposthenuria, renal acidification potassium (K) secretion disorders, hypertension, hematuria, proteinuria, nephrotic syndrome, renal papillary necrosis (RPN), renal medullary carcinoma, acute, and chronic renal failure may occur [9, 10].

After the erythrocytes take the shape of a sickle, they slow down the blood flow by reducing the fluidity in the circulation, and this causes congestion and an oxygen-free environment in the small vessels. Generally, sickle erythrocytes regain their former shape with re-oxygenation, while some of them cannot return to their normal shape due to permanent damage to the cell membranes. These cells cause vascular occlusion, creating hypoxia in the tissues, cause the painful crises, organ necrosis, and ultimately acute and chronic tissue destruction [11]. The mechanisms of irreversible sickling of cells are not fully understood. The number of irreversibly sickled cells for each patient is usually constant and is mainly related to the degree of anemia. This cell number is not associated with the patient's crises, nor is it a determinant of conditions such as pain attacks. Recent studies on the pathogenesis of SCD have focused on the pathological conditions that occur during avascular occlusion by polymerization of HbS in an oxygen-free environment [12]. Structural and functional disorders are found in the kidneys in sickle cell anemia patients. Sickling of erythrocytes as a result of HbS polymerization, resulting in vascular occlusion and hypoxia, causes dysfunction in the renal medulla. Inhibition of sickling is the most important mechanism to prevent renal involvement of SCD. Symptoms progressing to chronic kidney disease and end-stage renal disease can be seen in SCD [13, 14]. In tubular dysfunctions, the most common renal side effect in sickle cell patients is hyposthenuria, which is defined as the inability to achieve the maximum concentration of urine. This situation manifests itself with increased frequency of urination and enuresis in early childhood in homozygous SCD patients, and often with nocturia in later periods [15].

Dehydration occurs more quickly in hot weather, due to the inability to achieve the maximum concentration ability of urine. Since vasopressin production is normal, the concentration defect in SCD does not respond to vasopressin. The degree of hyposthenuria is different in sickle cell anemia carriers. Impairment in the ability to concentrate urine may be temporarily corrected by blood transfusions in the early stages of the disease. However, medullary fibrosis and permanent damage to the collecting ducts render the concentration defect irreversible [16]. It is recommended that hypostenuric patients take more fluids orally or intravenously to compensate for fluid loss. Urine dilution is dependent on solute reabsorption in the ascending limb of the loop of Henle in the cortical nephron. Since there is no involvement in these regions in SCD, patients can dilute the urine normally. Proximal tubular functions are higher than normal in sickle cell patients. As a result of increased sodium (Na) reabsorption from the proximal tubule in patients, less Na is transferred to the distal region, resulting in a poorer response to diuretics [17]. It has been suggested that these changes are a balancing mechanism against medullary damage for Na and water retention [18]. Phosphorus (P) reabsorption and uric acid secretion are also increased in patients with SCD. Therefore, patients may develop hyperphosphatemia and hyperuricemia as a result of hemolysis. In sickle cell patients, serum potassium and uric acid levels were high, and tubular phosphate reabsorption and potassium excretion were low. Although the renin-aldosterone axis functions normally in sickle cell patients, there may be an increase in plasma renin and aldosterone levels due to medullary fibrosis [8]. The tubular secretion of creatinine is increased in patients. Therefore, creatinine clearance (Cr/Kl) is found to be higher than the value estimated as glomerular filtration rate (GFR). Serum creatinine level is low in SCD. Decreased CrKl in SCD suggests decreased functional ability of renal tubules [19]. The pathogenesis of hematuria is explained by sickled erythrocytes causing vascular occlusion in the medulla and then extravasation of blood cells. Painless macroscopic hematuria is the most dramatic clinical picture in SCD [20]. Bleeding is unilateral in 80–90% of cases and mostly originates from the left kidney. This is due to the higher venous pressure in the left kidney. Occlusion in the renal vessels causes damage to the vasa recta and small infarctions, resulting in the development of renal papillary necrosis (RPN). The most important finding of patients with RPN is hematuria. RPN occurs in more than 40% of homozygous patients. In RPN due to SCD, unlike papillary necrosis due to the use of analgesics, the vasa recta are empty and the peritubular capillaries are primarily involved [21, 22].

Acute renal failure is one of the common side effects in sickle cell patients, and it is especially seen in hospitalized patients. Acute renal failure is most commonly seen due to infections and rhabdomyolysis. Fluid loss is the most important trigger for acute renal failure. The use of anti-inflammatory drugs is also accepted as a factor that increases this deficiency [23].

Another sign of kidney involvement in sickle cell anemia is proteinuria, and it is among the most common findings in kidney disease related to SCD. Proteinuria is detected in 17–33% of patients with SCD with the dipstick method. Its incidence is age-related, and it is less common in children and more common in older ages. Proteinuria is seen at a higher rate in homozygous SCD than in heterozygous sickle cell carriers [24]. Proteinuria was associated with the severity of the disease. Microalbuminuria and the presence of immunoglobulin G (IgG) in the urine are accepted as early predictors of glomerular damage [25]. The association of significant proteinuria with hematuria in sickle cell anemia is well known. Sickle cell glomerulopathy is defined as proteinuria at the nephrotic border. SCD glomerulopathy has a

#### *Investigation of Sickle Cell Nephropathy DOI: http://dx.doi.org/10.5772/intechopen.113757*

faster course than other causes of nephrotic syndrome. Glomerulopathy, proteinuria, and nephrotic syndrome may progress in sickle cell patients [26]. While hypertension alone is not observed in patients with SCD, hypertension can be detected if nephrotic syndrome is present with SCD. Sickled cells block glomerular capillaries, causing direct endothelial damage, endothelial hyperplasia, and glomerular fibrosis. The development of focal segmental glomerulosclerosis in SCD is believed to be due to hyperfiltration. In SCD nephropathy, enlargement of the glomeruli, perihilar focal segmental glomerulosclerosis, and hemosiderosis are seen in the early period, while membranoproliferative glomerulonephritis develops in advanced cases [13, 27].

### **Author details**

Rumeysa Duyuran and Hülya Çiçek\* Faculty of Medicine, Department of Medical Biochemistry, Gaziantep University, Gaziantep, Turkey

\*Address all correspondence to: drhulyacicek@hotmail.com

© 2024 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### *Edited by Hülya Çiçek*

This book discusses the diagnosis and treatment of several kidney diseases. For example, nephrotic syndrome is a disease in which the protein permeability of the basement membranes in the renal glomerular capillaries increases. Nephrotic syndrome may occur due to secondary causes or may be idiopathic. Glomerular diseases in children may occur secondary to primary renal involvement or systemic diseases. The clinical picture is classified as nephritic syndrome, rapidly progressive glomerulonephritis, nephrotic syndrome, and isolated proteinuria or hematuria. Primary examinations that help define the clinical picture include a review of signs and symptoms, urine examination, amount of proteinuria, and renal function. A biopsy is performed on patients to determine the underlying disease, and the appropriate treatment is arranged according to this result. This book discusses all these issues and more.

Published in London, UK © 2024 IntechOpen © Dr\_Microbe / iStock

Novel Topics in the Diagnosis, Treatment, and Follow-Up of Nephritis, Nephrotic Syndrome, and Nephrosis

Novel Topics in the Diagnosis,

Treatment, and Follow-

Up of Nephritis, Nephrotic

Syndrome, and Nephrosis

*Edited by Hülya Çiçek*