**7. Tubular enzymes**

people with normal function of renal graft. Therefore monitoring of urinary VEGF was proposed as a marker of detection acute renal graft rejection and the evaluation of the

**Hepatocyte growth factor (HGF)** induces angiogenesis by stimulation proliferation, migration and adhesion of endothelial cells. Urinary HGF concentration was highest at the first day after transplantation, decreased quickly within next week and later remained on the same level. Determination of the urinary HGF immediately after kidney transplantation may be a quick,

**Endothelin-1 (ET-1)** is the strongest vasoconstrictory factor produced by endothelium of blood vessels, glomerular mesangium, renal tubular cells, fibroblasts and macrophages. ET-1 regulates fibrosis by joining interstitial fibroblasts, initiation its proliferation and synthesis of extracellular matrix as well as chemotactic action on macrophages. ET-1 is degraded mostly in lungs and kidneys. Urinary ET-1 excretion reflects its renal production. Increase in ET-1 gene expression and urinary excretion correlates positively with proteinuria and negatively with creatinine clearance (Grenda et al., 2007; Saurina et al., 2007). Plasma and urinary ET-1 concentrations are increased in patients treated with Cyclosporine A and FKJ506. Cyclosporine A and FK506 are calcineurine inhibitors broadly applied for immunosuppression in kidney transplant patients. Cyclosporine A and FK506 significantly improve graft survival. However graft recipients may die because of cardiovascular complications as 80% of renal graft recipi‐ ents reveal vascular hypertension. Increased ET-1 concentration may reflect activation of the

noninvasive marker of long lasting renal graft function (Kwiatkowska et al., 2010).

ET-1 system in chronic insufficiency of transplanted kidney (Slowinski et al., 2002).

et al., 2010).

of acute renal graft rejection (Peng et al., 2008).

**Monocyte chemotactic peptide-1 (MCP-1/CCL2)** mediates recruitment of inflammatory cells: monocytes/macrophages and lymphocytes T, to renal tubules damaged by high concentrations of albumin in tubules. A strict relationship between albuminuria, urinary MCP-1/CCL2 and macrophage infiltration in damaged loci, was demonstrated (Urbschat et al., 2011; Viedt & Orth, 2002). In patients with acute renal graft rejection urinary concentration of MPC-1, determined by ELISA, was ten times higher than in patients with stable graft function (Dubiński et al., 2008). Since chronic damage to renal graft as a result of gradual fibrosis and tubular damage (IF/TA) is the most frequent cause of graft loss, urinary CCL2 may be treated as an independent prognostic marker of development of IF/TA during the next 24 months (Ho

**Fractalkine** (**CX3CL1**) is a chemokine from the CX3C group of complement system, stimulated by CX3CR1 receptor connected to G protein. In experimental renal disease induced by albumin overload and proceeding with proteinuria, increased expression of fractalkine gene correlates with applied albumin dose and time of albumin interaction with the renal tubular cells (Donadelli et al., 2003). Fractalkine urinary concentration is a noninvasive method for detection

**Angiotensin II (Ang II)** is an important intrarenal factor favoring processes of inflammation and fibrosis by an increase in the expression of the proinflammatory genes (IL-6, TNFα, MCP-1, RANTES). According to latest opinions the urinary concentration of angiotensinogen, reflects amounts of produced Ang II inside the kidney better, than immediate evaluation of Ang II in

effectiveness of immunotherapy (Peng et al., 2008; Alachkar, 2012).

70 Current Issues and Future Direction in Kidney Transplantation

Currently, in clinical diagnostic practice for renal parenchymal tubular impairment, assess‐ ment of urinary enzymes is used. Particular advantage of urinary enzymes determination is its localization in appropriate renal cells (glomeruli, tubules) and their organelles (cytoplasm, lysosomes, membranes), which may deliver detailed information concerning nature and dimension of the renal cells damage and an evaluation of their dysfunction or necrosis (Westhuyzen et al., 2003; Trof et al., 2006). Routine, simple, cheap and broadly available spectrophotometric methods are applied for measurement of urinary enzymes activity. An increase in urinary excretion of enzymes reflects damage of particular renal section (D'Amico & Bazzi, 2003; Jung et al., 1986). Determination of urinary FBP-1,6, NAG, glutathione-Stransferase and pyruvate kinase has recently been recommended for the diagnosis of kidney disease and early detection of transplant rejection (Kotanko et al., 1997; Kotanko et al., 1986).

#### **7.1. Enzymes of brush border membranes**

**Gamma-glutamyltransferase** (**GGT**) – is connected with cellular membranes of liver, kidney, pancreas and prostatic gland (Kuźniar et al., 2006). Serial determination of urinary enzymes is a reliable proof for nephrotoxicity resulted from long term cyclosporine A treatment. Lack of enzymuria indicates a recovery of renal tubules to normal function (Tataranni et al., 1992).

**Alkaline phosphatase (AP)** – is present in cellular membranes of many tissues, mainly bonds, liver and intestine where it participates in metabolism of organic phosphates. Frequent cause of deterioration to the renal graft function is nephrotoxicity of immunosuppressive drugs (e.g. cyclosporin A) reflected by increase in activity of urinary enzymes : ALP, LDH, GGT, betaglucuronidase (Refaie et al., 2000; Takahashi et al., 1989; Simić-Ogrizović et al., 1994).

**8. Markers of renal ischemia/reperfusion injury**

(Zynek-Litwin et al., 2010).

(Bhoola et al., 1992).

tubules (Gautier et al., 2010).

**9. Biomarkers of dystal renal tubules**

**Leukocyte elastase** (LE, neutrophil elastase), is a 30-kDa glycoprotein serine protease released from neutrophils as a mediator of ischemia/reperfusion injury after renal transplantation. Urinary LE is a simple noninvasive marker of the neutrophil activation after renal transplantat

Utility of Urinary Biomarkers in Kidney Transplant Function Assessment

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

73

In the assessment of distal renal tubule dysfunction it is advised to examine urine osmo‐ larity and/or determination Tamm-Horsfall glycoprotein as well as urinary kallikrein

**Renal kallikrein** is a serine protease which releases vasodilatatory peptides: bradykinine and calidine, from kininogen. Renal kallikrein is present in renal collecting tubules and is released to tubular fluid by terminal section of dystal segment of nephron (Manucha & Vallés, 1999; Thongboonkerd & Malasit, 2005). An increase in activity of urinary kallikrein was observed

**AnnexinA11** (**ANXA11**). Annexins are calcium-binding proteins which binds to acidic phospholipid and F-actin. Depending on calcium concentration Annexin A 11 participate in signal transduction, cell proliferation, regulation of vesicular transport and interaction with the cell membranes. Annexin occurs in high quantities in renal distal tubular cells and epithelium of renal glomeruli. Annexin physiologic role seems to be related to cell apoptosis (Rodrigues-Garcia et al., 1996). Significant correlation between urinary Annexin V and other proteins and lack of correlation with urinary urea and creatinine concentration suggests that Annexin V is not an indicator of kidney function, but rather reflects local kidney damage (Matsuda et al., 2000). Annexin A11 may act as an atypical calcium channel and useful marker

**Renal papillary antigen-1 (RPA-1)** a renal papillary antigen-1, sensitive and specific antigen of renal papillary cells is a sensitive and specific urinary marker of damage renal collecting

**Prominin-2 (PROM-2**) analog of **CD133 (prominin-1)** is an membrane glycoprotein (112kD) with the highest expression in epithelial cells of matured kidney. Prominin-2 is a cholesterolbinding protein associated with apical and basolateral plasmalemmal protrusions in polarized epithelial cells and released into urine (Florek et al., 2007) and a novel marker of distal tubules

**μ-glutathione-S-transferase (μ-GST)** is a conjugating glutathione with electrophilic com‐ pounds that occurs in epithelial cells of ascending part of Henle's loop (Gautier et al., 2010; Holmquist *&* Torffvit, 2008). After nephrotoxic drugs treatment (e.g. cisplatin) μ-GST quickly appear in urine. μ-GST is a more specific marker of nephrotoxicity (AUC 1.000) than α-GST

in insufficiency and loss of the renal graft function (Krimkevich, 1990).

of acute and chronic renal graft rejection (Srivastava et al., 2011)

and collecting ducts of the human and murine kidney (Jászai et al., 2010).

**Alanylaminopeptidase (AAP)** – proteolytic enzyme degrading oligopeptides. Increases in urinary concentration of hexosaminidase and AAP accompany acute renal tubular necrosis, renal graft rejection or nephrotoxic action of immunosuppresive drugs (e.g. cyclosporin A) administered to patients after kidney transplantat (Kuźniar et al., 2006; Lisowska-Myjak, 2010; Santos et al., 2010). Increases in urinary excretion of tubular enzymes testifies tubular brush border membrane damage with a loss of microvillus structure (Westhuyzen et al., 2003).

#### **7.2. Cytosolic enzymes**

**Glutathione S-transferase (alpha-GST, pi-GST)** is a specific cytoplasmic enzyme of tubular epithelial cells consisting of two isoenzymes: α-GST with alkaline and πi-GST with acidic pH optimum. GST-α appears in epithelium of proximal tubular cells and GST-π in distal tubules (Branten et al., 2000). Determination in urine α-GST and, π-GST is applied to diagnosis acute renal graft rejection with acute tubular necrosis (Kuźniar et al., 2006; Polak, 1999). Differenti‐ ated increase in urinary GST- alpha and GST- pi excretion may point to localization of an nephron damage (Westhuyzen et al., 2003; Trof et al., 2006; Herget-Rosenthal et al., 2004; Branten et al., 2000; Gautier et al., 2010).

**Fructose-1,6-bisphosphatase (FBP-1,6)** is localized mostly in contorted and to less extend in straight part of proximal renal tubules, similarly to hexosaminidase and GST, points to accurate localization of damaged nephron (Trof et al., 2006; Kotanko et al., 1986). Increase in urinary FBP-1,6 was observed in patients after kidney transplantat. Urinary FBP-1,6 excretion was significantly lower in patients with median of cold ischemia below 22 hours, than above 22 hours. Even in lack of graft dysfunction, in situation where it is a long time of cold ischemia, urinary excretion of FBP-ase correlates with a degree of damage to the renal tubules (Kotanko et al., 1997). It was reported that a panel of urinary enzymes activities: FBP-ase, glutathione Stransferase, N-acetyl-beta-D-glucosaminidase and pyruvate kinase is a good marker of the cyclosporin A nephrotoxicity (Kotanko et al., 1986).

#### **7.3. Renal lysosomal enzymes**

**N-acetyl-β-D-hexosaminidase (HEX)** is one of the most frequently determined urinary markers of renal tubules damage, because its activity increased at early steps of the renal tubules damage, before occurrence of disturbances in renal excretory function. Hexosamini‐ dase localized mainly in renal proximal tubular cells, is a specific marker for proximal tubular cells because its high molecular weight (> 130 kDa) excludes its glomerular filtration. In the course of active kidney disease HEX activity is constantly increased. An increase in urinary activity HEX and its isoenzyme B indicate on damage in the renal tubular cells. Therefore urinary HEX and particularly HEX B activity may be treated as a specific marker of damage in the renal proximal tubules of the transplanted kidney (Liangos et al., 2007; Holdt−Lehmann et al., 2000;).
