**6.1. Immunological markers of renal inflammatory state**

**Macrophage inflammatory protein 3alpha (MIP-3alpha), chemokine C-C ligand 20 (CCL20)** is a major chemokine expressed by epithelial cells that attracts immature dendritic cells (DC). Graft-infiltrating dendritic cells (DC) and alloreactive T lymphocytes play a critical role in renal allograft rejection. Renal proximal tubular epithelial cells (TEC) are considered as an active players in the attraction of leukocytes during renal inflammatory responses. A significant increase in the excretion of major intrinsic protein MIP-3α/CCL20 to urine was observed in renal graft recipients with symptoms of graft rejection (Woltman et al., 2005; Peng et al., 2008).

**Growth-related oncogene-alpha (Gro-alpha)** is an analog of the keratinocyte-derived chemokine(KC). An increase in serum and urinary analog of Gro-alpha in the experimental renal damage appears the earliest and persists the longest among the 18 chosen cytokines and chemokines (Molls et al., 2006). Serum and urinary Gro-alpha were the highest 3 hours after ischemia, while histological changes were evident after one hour, whereas serum creatinine increased 24 hours after ischemia. Urinary concentration of Gro-α increased significantly in renal graft recipients who required dialysis in comparison to people with normal renal graft function. Urinary Gro-α is considered as an early marker of diagnosis and prognosis of acute kidney injury (AKI) resulted from ischemia (Molls et al., 2006). It was reported that Gro-alfa, significantly increased in patients who received cadaver kidney with poor function and from living donors with minimal ischemia. Therefore determination of KC and Gro-α may be used as biomarkers in the diagnosis of ischemic acute renal failure (ARF) and in the early diagnosis and prognosis of renal ischemia-reperfusion injury (IRI). IRI is the most frequent cause of acute kidney injury (AKI) and acute renal failure in delayed function graft received from a cadaver (Molls et al., 2006).

**Interleukin (IL-18)** is a proinflammatory cytokine released to urine by epithelium of renal proximal tubules after stimuli of nephrotoxic factor. Urinary IL-18 concentration >100 pg/mg of urinary creatinine is a good diagnostic marker of the acute renal damage and mortality of patients in intensive care units (Parikh et al., 2005) as well as a predictor of the delayed graft function. It seems that urinary IL-18 helps for a detection of the very early stage of kidney damage caused by ischemia or tubular nephrotoxins and plays a role in detecting prerenal nitrogenemia, chronic renal insufficiency and urinary tract infection (Parikh et al., 2005). Furthermore urinary Il-18 is an early predictive biomarker of the acute kidney injury after cardiac surgery (Parikh et al., 2005). IL-18- proinflammatory cytokine caspase-1 dependent (both derived from ischemic renal proximal tubular cells) is a proinflammatory cytokine activated in damaged renal tubules by caspase -1 and released to urine in a case of the acute kidney injury (AKI). A significant increase in urinary concentration of IL –18 and NGAL after transplantation, however before delay in the renal graft function, was found. IL-18 is also a predictor of the AKI severity preceding the increase in serum creatinine (Dinarello, 1999).

**Granzymes (***granule-associated enzymes***)** are serine proteases (27-32kDa) of the chymotrypsin family. Granzyme B (GzmB) and Fas-ligand (FAS-L) are cytotoxic molecules involved in the acute renal graft rejection (AR) by the induction of DNA fragmentation of damaged cells (Yannaraki et al., 2006). Granzyme A (GzmA) is a specific noninvasive immunological biomarker for monitoring renal graft condition which facilitate diagnosis and treatment after transplant complications. Granzyme A (GzmA) besides involvement in apoptosis may act as mitogen of B lymphocytes. GzmA is a noninvasive biomarker differentiating patients with subclinical and acute renal graft rejection from patients with renal tubular necrosis or persons with stabile renal graft (van Ham et al., 2010 ).

#### **6.2. Immunological markers of renal fibrosis**

CXCL10 concentration above 1,97 ng /mmol of creatinine is a threshold for consideration of

**Macrophage inflammatory protein 3alpha (MIP-3alpha), chemokine C-C ligand 20 (CCL20)** is a major chemokine expressed by epithelial cells that attracts immature dendritic cells (DC). Graft-infiltrating dendritic cells (DC) and alloreactive T lymphocytes play a critical role in renal allograft rejection. Renal proximal tubular epithelial cells (TEC) are considered as an active players in the attraction of leukocytes during renal inflammatory responses. A significant increase in the excretion of major intrinsic protein MIP-3α/CCL20 to urine was observed in renal graft recipients with symptoms of graft rejection (Woltman et al., 2005; Peng

**Growth-related oncogene-alpha (Gro-alpha)** is an analog of the keratinocyte-derived chemokine(KC). An increase in serum and urinary analog of Gro-alpha in the experimental renal damage appears the earliest and persists the longest among the 18 chosen cytokines and chemokines (Molls et al., 2006). Serum and urinary Gro-alpha were the highest 3 hours after ischemia, while histological changes were evident after one hour, whereas serum creatinine increased 24 hours after ischemia. Urinary concentration of Gro-α increased significantly in renal graft recipients who required dialysis in comparison to people with normal renal graft function. Urinary Gro-α is considered as an early marker of diagnosis and prognosis of acute kidney injury (AKI) resulted from ischemia (Molls et al., 2006). It was reported that Gro-alfa, significantly increased in patients who received cadaver kidney with poor function and from living donors with minimal ischemia. Therefore determination of KC and Gro-α may be used as biomarkers in the diagnosis of ischemic acute renal failure (ARF) and in the early diagnosis and prognosis of renal ischemia-reperfusion injury (IRI). IRI is the most frequent cause of acute kidney injury (AKI) and acute renal failure in delayed function graft received from a cadaver

**Interleukin (IL-18)** is a proinflammatory cytokine released to urine by epithelium of renal proximal tubules after stimuli of nephrotoxic factor. Urinary IL-18 concentration >100 pg/mg of urinary creatinine is a good diagnostic marker of the acute renal damage and mortality of patients in intensive care units (Parikh et al., 2005) as well as a predictor of the delayed graft function. It seems that urinary IL-18 helps for a detection of the very early stage of kidney damage caused by ischemia or tubular nephrotoxins and plays a role in detecting prerenal nitrogenemia, chronic renal insufficiency and urinary tract infection (Parikh et al., 2005). Furthermore urinary Il-18 is an early predictive biomarker of the acute kidney injury after cardiac surgery (Parikh et al., 2005). IL-18- proinflammatory cytokine caspase-1 dependent (both derived from ischemic renal proximal tubular cells) is a proinflammatory cytokine activated in damaged renal tubules by caspase -1 and released to urine in a case of the acute kidney injury (AKI). A significant increase in urinary concentration of IL –18 and NGAL after transplantation, however before delay in the renal graft function, was found. IL-18 is also a predictor of the AKI severity preceding the increase in serum creatinine (Dinarello, 1999).

renal biopsy (Ho et al., 2011).

68 Current Issues and Future Direction in Kidney Transplantation

et al., 2008).

(Molls et al., 2006).

**6.1. Immunological markers of renal inflammatory state**

**Chemokine regulated upon activation in normal T cells expressed and secreted (RANTES/ CCL5)** is a chemokine of the beta subfamily secreted by macrophages and T lymphocytes. RANTES can signal through CCR1, CCR3, CCR5 and US28(cytomegalovirus receptor) receptors. It is chemoattractant towards monocytes, memory T cells(CD4+/CD45RO+), basophils, and neutrophils. RANTES occurs in increased amounts in diseased kidneys and indicate on interstitial inflammatory changes of the tubular cells at the early stages of acute kidney injury, skin or heart graft rejection (Koga et al., 2000; Gwinner, 2007). RANTES expressed in renal different cells (mesanglial cells, endothelium of renal tubules, fibroblasts, lymphocytes) plays an active role in acute and chronic kidney inflammation and development of tubule-interstitial damage. (Baer et al., 2005) reported absence of significant differences in plasma and urinary RANTES in patients with acute renal graft rejection and recipients with normal graft function. Therefore RANTES is not suitable for detection of early kidney graft rejection. However an significant increase in the serum and urinary RANTES was observed immediately after renal transplantation which may reflect an activation of the immunological systems.

**Transforming growth factor beta (TGF-β)** is responsible for exacerbation of fibrosis, controls growth and differentiation of cells and production of extracellular matrix as well as regulates cellular migration. A participation of TGF-ß1 in lung and kidney fibrosis during chronic allograft rejection was reported by (Awad et al.,1998; Bartnard et al., 1990). Increased excretion of urinary TGF-β was proposed as a marker of the intrarenal production and activity of TGFß1 in kidney. An increase in the urinary TGF-ß1 was reported in different nephropathies particularly significant in patients with heavy proteinuria (Schnaper et al., 2003; Böttinger & Bitzer, 2002). The 6-12 month immunosuppression with cyclosporine in renal-transplant recipients caused development of a chronic interstitial nephropathy with decreased GFR. Cyclosporine A (CsA) facilitate the expression of TGF-β in renal tubular cells and cells of renal juxtaglomerular apparatus. Furthermore, CsA stimulates T lymphocytes and endothelial cell to a TGF-β 1 production. Expression of TGF-β 1 is CsA dose dependent. High doses of CsA are risk factors of chronic graft dysfunction, among kidney recipients (Boratyńska et al., 2003).

**Vascular endothelial growth factor (VEGF)** a dimeric protein containing subunits constituted of 121, 165, 189 or 206 amino acids is a proangiogenic growth factor. In patients with symptoms of acute renal graft rejection high urinary VEGF concentration was found in comparison to 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 effectiveness of immunotherapy (Peng et al., 2008; Alachkar, 2012).

the urine. Improvement of the ELISA method for determination of human urinary angioten‐ sinogen, may allow to disclose influence of Ang II on intrarenal destructive processes

Utility of Urinary Biomarkers in Kidney Transplant Function Assessment

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

71

**Complement** is a major mediator system in pathogenesis of various kidney diseases. The presence and localization of complement components in glomerulus and/or the tubuleinterstitial area provides diagnostic tools for several human renal diseases (Zoja et al., 2003; Lisowska-Myjak, 2010). Increase in urinary excretion of complement components in patients with proteinuria significantly correlate with urinary excretion of total proteins and decrease in renal function. Therefore increase in urinary C5b-9 in patients with proteinuria may by prognostic marker for the development of kidney insufficiency (Eddy, 2002). Accelerated C3 activation at renal proximal tubules in diseases proceeding with proteinuria are result of increased intratubular protein catabolism, with accompanying increase in ammonia (activator of alternative pathway of complement activation) (Morita et al., 2000; Abbate et al., 2008; Sheerin et al., 2008; Lederer et al., 2003). Everyday urinary determination of C5A and TCC may be a sensitive and reliable marker of the acute insufficiency of the transplanted kidney and

**Galectin 3 (Gal-3)** is a beta-galactoside-binding lectin in diverse fibrotic tissues. Gal 3 plays an important role in fibrosis of transplanted kidney and may be a potential marker of chronic

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).

**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

(Yamamoto et al., 2007; Katsurada et al., 2007).

predictor of graft rejection (Müller et al., 1997).

allograft impairment (CAI) (Dang et al., 2012).

**7.1. Enzymes of brush border membranes**

**7. Tubular enzymes**

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

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

**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 et al., 2010).

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

**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 the urine. Improvement of the ELISA method for determination of human urinary angioten‐ sinogen, may allow to disclose influence of Ang II on intrarenal destructive processes (Yamamoto et al., 2007; Katsurada et al., 2007).

**Complement** is a major mediator system in pathogenesis of various kidney diseases. The presence and localization of complement components in glomerulus and/or the tubuleinterstitial area provides diagnostic tools for several human renal diseases (Zoja et al., 2003; Lisowska-Myjak, 2010). Increase in urinary excretion of complement components in patients with proteinuria significantly correlate with urinary excretion of total proteins and decrease in renal function. Therefore increase in urinary C5b-9 in patients with proteinuria may by prognostic marker for the development of kidney insufficiency (Eddy, 2002). Accelerated C3 activation at renal proximal tubules in diseases proceeding with proteinuria are result of increased intratubular protein catabolism, with accompanying increase in ammonia (activator of alternative pathway of complement activation) (Morita et al., 2000; Abbate et al., 2008; Sheerin et al., 2008; Lederer et al., 2003). Everyday urinary determination of C5A and TCC may be a sensitive and reliable marker of the acute insufficiency of the transplanted kidney and predictor of graft rejection (Müller et al., 1997).

**Galectin 3 (Gal-3)** is a beta-galactoside-binding lectin in diverse fibrotic tissues. Gal 3 plays an important role in fibrosis of transplanted kidney and may be a potential marker of chronic allograft impairment (CAI) (Dang et al., 2012).
