**2.1. Biomarkers of renal glomeruli**

The oldest biomarkers of renal glomeruli injure are serum urea and creatinine as well as clearance of endogenic creatinine, which similarly to inulin (gold standard in GFR determi‐ nation) is excreted to urine and not absorbed in renal tubules. Clearance of endogenic creati‐ nine is 10-20% higher than clearance of inuline, which is a result trace excretion of creatinine by renal tubules (Finney et al., 2000).

**Cystatin C** (**CYC**) is a cysteine protease inhibitor that is stably secreted from all nucleated cells, freely filtered through the glomerulus, and completely reabsorbed by the proximal tubules. During efficient function of proximal renal tubules there are traces of urinary CYC, independ‐ ent of age and body mass. Given that cystatin C is not normally found in urine in significant amounts, the elevated level of urinary cystatin C may display dysfunction of tubular cells and tubulointerstitial disease. Concentration of CYC in normal urine accounts 0.03-0.3 mg/L (Filler et al., 2005). Increase in serum CYC is proportional to decrease in GFR (Campo et al., 2004 ). It was reported that serum CYC correlated better with GFR than creatinine (Filler et al., 2005; Schuck et al., 2004). After renal transplantation CYC concentration increased simultaneously to AKI development, because of decreased reabsorption from damaged tubules. Therefore

urinary concentration of CYC may be treated as a good marker of the proximal tubules and effective biomarker of delayed renal graft function due to lack of diurnal changes, and high stability in routine conditions of urinary storage, urinary CYC may be determined in single urinary samples. Urinary CYC / creatinine ratio is a good indicator of renal tubules dysfunction as in disorders of renal tubules, urinary CYC concentration may increase even 200 fold (Uchida & Gotoh, 2002; Lisowska-Myjak, 2010). Two fully automated and quick immunological methods for CYC determination: turbidimetric PETIA (particle enhanced immunoturbidimet‐ ric assay) and nephelometric – PENIA (particle enhanced nephelometric immunoassay) were developed in 1994-1997. Presently measurements of urinary CYC are utilized mainly with a

Utility of Urinary Biomarkers in Kidney Transplant Function Assessment

http://dx.doi.org/10.5772/54746

63

**Figure 1.** Nephron structure

**Figure 1.** Nephron structure

**2. Markers of nephrons damage**

62 Current Issues and Future Direction in Kidney Transplantation

Daclizumab, blocks receptors for IL-2.

**2.1. Biomarkers of renal glomeruli**

by renal tubules (Finney et al., 2000).

After kidney transplantation it is particularly important to monitor the biomarkers which allow to detect progress in disease process and determine which functional parts of kidney are going to be damaged, to enable application of a quick appropriate treatment (Lisowska-Myjak, 2010; Alachkar et al., 2010; Metzger et al., 2010). Administration of immunosuppressants for preventing renal graft rejection may lead to progressive damage to the renal tissue (interstitial fibrosis, tubular micro calcifications, atrophy of renal tubules) caused by high toxicity of suppressing drugs. Cyclosporine A(CsA), tacrolimus, mycophenolate mofetil, basiliximab, prednizon and sirolimus (rapamycin) are commonly used in immunosuppressive therapy following kidney transplantation. Cyclosporine A and tacrolimus generate immunosuppres‐ sive action by binding to cyclofiline and inhibiting the action of calcineurin 2, which stimulates proliferation and differentiation of lymphocytes T. Cyclosporine A inhibits synthesis of lymphokines by lymphocytes T. Lymphokines synthesized by lymphocytes T stimulate immunological system and have the ability to "kill" inflammatory and neoplastic cells. Mycophenolate mofetil selectively inhibits inosine monophosphate dehydrogenase, a basic enzyme in guanosine synthesis. Mycophenolate mofetil inhibits proliferation of lymphocytes T and B after stimulation with antigenes, cytokines and mitogens. Basiliximab similarly to

Majority of renal pathological changes concern glomerules, proximal and distal tubules as well as vascular endothelium. At first renal proximal tubular cells (Fig.1.) demonstrating highest metabolic activity, possessing high amounts of mitochondries, lysosomes and peroxysomes are damaged. Remaining sections of nephron such as: Henle's loop, distal tubules and collecting tubules are usually damaged later. There are numerous biomarkers that identify injury the area of the renal nephron, such as the glomerulus, the proximal, and the distal tubule.

The oldest biomarkers of renal glomeruli injure are serum urea and creatinine as well as clearance of endogenic creatinine, which similarly to inulin (gold standard in GFR determi‐ nation) is excreted to urine and not absorbed in renal tubules. Clearance of endogenic creati‐ nine is 10-20% higher than clearance of inuline, which is a result trace excretion of creatinine

**Cystatin C** (**CYC**) is a cysteine protease inhibitor that is stably secreted from all nucleated cells, freely filtered through the glomerulus, and completely reabsorbed by the proximal tubules. During efficient function of proximal renal tubules there are traces of urinary CYC, independ‐ ent of age and body mass. Given that cystatin C is not normally found in urine in significant amounts, the elevated level of urinary cystatin C may display dysfunction of tubular cells and tubulointerstitial disease. Concentration of CYC in normal urine accounts 0.03-0.3 mg/L (Filler et al., 2005). Increase in serum CYC is proportional to decrease in GFR (Campo et al., 2004 ). It was reported that serum CYC correlated better with GFR than creatinine (Filler et al., 2005; Schuck et al., 2004). After renal transplantation CYC concentration increased simultaneously to AKI development, because of decreased reabsorption from damaged tubules. Therefore

urinary concentration of CYC may be treated as a good marker of the proximal tubules and effective biomarker of delayed renal graft function due to lack of diurnal changes, and high stability in routine conditions of urinary storage, urinary CYC may be determined in single urinary samples. Urinary CYC / creatinine ratio is a good indicator of renal tubules dysfunction as in disorders of renal tubules, urinary CYC concentration may increase even 200 fold (Uchida & Gotoh, 2002; Lisowska-Myjak, 2010). Two fully automated and quick immunological methods for CYC determination: turbidimetric PETIA (particle enhanced immunoturbidimet‐ ric assay) and nephelometric – PENIA (particle enhanced nephelometric immunoassay) were developed in 1994-1997. Presently measurements of urinary CYC are utilized mainly with a PENIA method designed primary for CYC determination in serum (Herget-Rosenthal et al., 2004). PENIA method (particle enhanced nephelometric immunoassay)allows for a CYC detection at a concentration of 0.05-10.47 mg/L.

**2.2. Adhesion molecules connected podocytes with basement membrane**

laminins, and fibronectins (Srivastava et al., 2011).

**3. Biomarkers of proximal tubules**

increase in urinary RBP concentration is observed.

2007).

(Teppo et al., 2004).

**Integrin α3 and integrin β3** are particularly recommended biomarkers for monitoring the function of transplanted kidney both at early and remote period after transplantation. The integrin family of cell adhesion proteins promotes the attachment and migration of cells on the surrounding extra cellular matrix (ECM). The signals initiated by integrin binding to ECM proteins are necessary to maintain cell survival, adhesion, migration and invasion. Integrins are transmembrane glycoproteins consisting of two units: α and β. Beta1 family of integrins represents the major class of cell substrate receptors with specificities primarily for collagens,

Utility of Urinary Biomarkers in Kidney Transplant Function Assessment

http://dx.doi.org/10.5772/54746

65

**Vascular cell adhesion molecule-1 (VCAM-1), sVCAM-1 (CD106) (soluble vascular cell adhesion molecule 1) and anti-intercellular adhesion molecule-1 (ICAM-1)** The ICAM and VCAM – members of the immunoglobulin (Ig) superfamily, are the chief endothelial cell proteins that are recognized by the white cell integrins. Elevated urinary sVCAM-1, IL6, sIL6R and TNFR1 concentrations indicate an acute kidney transplant rejection in the first 2 weeks after transplantation (Reinhold et al., 2012). It was reported that increased urinary concentra‐ tions of sICAM-1, determined by ELISA, occured in patients with acute renal graft rejection (Teppo et al., 2001), and in people with proteinuria, high concentrations of sVCAM and sICAM were observed (van Ree et al., 2008). Recently a non-invasive monitoring of the acute renal graft rejection by determination of cell adhesion molecules has been recommended (Gwinner,

**α1-microglobulin (α1M)**is **a** 27 kDa glycoprotein related to retinol binding protein synthesized by liver cells, engaged in immunoregulation (binds lymphocytes T and B) and heme catabo‐ lism. Determination of α1M (stable in acid urine) is a sensitive indicator of renal proximal tubules damage (Guder, 2008; Lisowska-Myjak, 2010; Câmara et al., 2009). (Teppo et al., 2004) reported that six month after transplantation, 32% of patients presented microalbumi‐ nuria. Evaluation of a damage to renal proximal tubules, on the basis of an increase in urinary α1M concentration may be a consequence to a deterioration of glomerular filtration. Increase in α1M /creatinine ratio is an early and sensitive indicator of a poor function of the transplanted kidney, and indicates a poor prognosis of long term survival of renal transplanted patients

**Retinol binding protein (RBP)**, protein of the lipocalin family, synthesized mainly in a liver, supplies retinol to peripheral tissues. RBP removed from plasma by glomerular filtration is subsequently absorbed and catabolized in renal proximal tubules. Increased urinary RBP is caused by a disorder in glomerular filtration and reabsorption in renal proximal tubules (Guder, 2008; Kuźniar et al., 2006; Uchida & Gotoh, 2002; Câmara et al., 2009). It seems that urinary RBP is a better biomarker of proximal tubules damage than β2M, as RBP has greater stability in acid urine than β2M and renal insufficiency is only a clinical situation where an

**Proteinuria** reflects increased filtration plasma proteins to tubular fluid and disturbed protein reabsorption by renal proximal tubular cells (Haraldsson & Sörensson, 2004; Halbesma et al., 2006; Giorgio et al., 2004). Proteinuria over 0.5g/24 hours is a marker the severity of the tubular damage, independent risk factor of progressive tubular-interstitial fibrosis and strong predictor of the end-stage renal insufficiency. Evident proteinuria is symptomatic for estab‐ lished renal damage significantly connected with decreased GFR (Abbate et al., 2006; Eddy, 2004; Tryggvason & Pettersson, 2003; Zoja et al., 2003; Ofstad & Iversen, 2005). Even minimal proteinuria lasting one year after renal transplantation is an indicator of a poor renal graft function and may be a risk factor for renal graft failure (Kang et al., 2009). Urinary protein is a non-invasive and easy to perform parameter. It was reported that proteinuria (<0.5 g/24 h) occurred in half of patients within 3 month after renal transplantation (Sancho Calabuig et al., 2009). Higher than standard doses of everolimus (EVL) resulted in an increase of proteinuria. Therefore the standard doses of EVL are recommended which seems to be suitable for protecting against an acute graft rejection with better prognosis of renal function in longer perspective (Loriga et al., 2010). As chronic allograft nephropathy (CAN) is the most frequent reason for late loss of graft, immunosuppression with mycophenolate mofetil significantly improves graft function and in such circumstances evaluation of proteinuria seems to have prognostic value (Grebe et al., 2004).

**Albuminuria** as a marker of glomerular filtration is more sensitive than proteinuria. Urinary albumin border value of 200 μg/min differentiates patients with albuminuria and proteinuria. Increase in urinary albumin excretion above 200 μg/min (macroalbuminuria) indicates damage to glomerular filtration membrane, a start of evident proteinuria, progression of kidney disease and cardiovascular changes (Ruggenenti & Remuzzi,2006). After exceeding maximal reab‐ sorption capacity of proximal tubular cells, protein of primary urine appeared in final urine (Luke, 1999; Remuzzi et al., 2006; Zoja et al., 2003). Excessive accumulation, in proximal tubular cells, plasma proteins excreted to primary urine, induced increase in local expression of cytokines and chemokines, which presence in final urine is a specific indicator of development and extent of renal damage (Lisowska-Myjak, 2010; Alachkar et al., 2010). Renal tubular cells exposed to increased amounts of filtered plasma proteins resulting cell injury. Microalbumi‐ nuria predicts a loss of renal graft. Determination of urinary albumin and UACR-urine albumin-to-creatinine ratios (UACR) are particularly recommended indicators for detection of changes in transplanted kidney (Erman et al., 2011). Microalbuminuria is considered to be a better indicator of kidney transplant condition than proteinuria (Bandukwala et al., 2009). Albuminuria, the marker of renal glomeruli damage and chronic damage of transplanted kidney, which may also reflect interstitial inflammatory process, is considered a predictor of long-term allograft outcomes in a kidney graft recipient (Nauta et al., 2011).
