New Insights Into the Use of Biomarkers of Diabetic Nephropathy Jay C. Jha, Karin A. M. Jandeleit-Dahm, and Mark E. Cooper Diabetic nephropathy (DN) is a major microvascular complication of diabetes characterized by increasing albuminuria and progressive loss of kidney function. Increased excretion of albumin into the urine is a key feature of DN, and its assessment is considered to be an early marker predicting the onset and progression of DN. However, albuminuria has certain limitations; therefore, the quest for more reliable renal biomarkers with higher sensitivity and specificity are needed for early prediction of the onset and monitoring of the progression of DN. Furthermore, such biomarkers may also provide a better insight into identifying the complex pathophysiological processes responsible for DN. This article aims to provide a comprehensive and critical review of the current literature on relevant biomarkers of kidney injury, including markers of renal fibrosis, inflammation, and oxidative stress, as well as addressing contemporary proteomic approaches. Q 2014 by the National Kidney Foundation, Inc. All rights reserved. Key Words: Diabetes, Diabetic nephropathy, Biomarkers, Proteomics, Metabolomics
Introduction Diabetes mellitus represents a global threat for premature morbidity and mortality, and it is likely to be the 5th leading cause of death worldwide.1 According to the recently updated International Diabetes Federation’s Diabetes Atlas Report, more than 371 million people have diabetes, and this figure is increasing globally at an alarming rate. Diabetic nephropathy (DN) is a major chronic microvascular complication of diabetes, and it is the leading cause of ESRD worldwide, often requiring dialysis and/or transplantation.2 DN is characterized by a progressive increase in albuminuria and a decline in glomerular filtration rate (GFR), which often occur in association with an increase in blood pressure, ultimately leading to end-stage kidney failure.3 Proteinuria is considered a hallmark of DN, and it has generally been considered to primarily reflect glomerular injury and increased glomerular permeability to macromolecules.4 In addition, renal functional changes are associated with structural abnormalities, including glomerular basement membrane (GBM) thickening and mesangial expansion as a result of the accumulation of extracellular matrix (ECM), which leads to glomerulosclerosis and tubulointerstitial fibrosis. Mechanisms underlying nephropathy in diabetes include a range of hemodynamic and metabolic factors, such as hyperglycemia, dyslipidemia, systemic and intraglomerular hypertension, activation of the renin-
From JDRF Danielle Alberti Memorial Centre for Diabetic Complications, Diabetic Complications Division, Baker IDI Heart & Diabetes Institute, Department of Medicine, Monash University, Melbourne, Australia. Financial Disclosure: The authors declare that they have no relevant financial interests. Address correspondence to Mark E. Cooper, MBBS, PhD, Diabetic Complications, Baker IDI Heart & Diabetes Research Institute, PO Box 6492 St. Kilda Road, Melbourne, Victoria 8008, Australia. E-mail: Mark.Cooper@bakeridi. edu.au Ó 2014 by the National Kidney Foundation, Inc. All rights reserved. 1548-5595/$36.00 http://dx.doi.org/10.1053/j.ackd.2014.03.008
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angiotensin system, impaired insulin signaling, infiltration by inflammatory cells, increased growth factors and proinflammatory mediators, and activation of key intracellular signaling pathways and transcription factors leading to the development and progression of diabetesassociated kidney disease.5,6 Several mechanisms have been proposed to explain the detrimental effects of hyperglycemia-induced tissue damage, including flux through the polyol pathway, advanced glycation end product (AGE) formation, hexosamine pathway flux and activation of protein kinase C, angiotensin II, and nicotinamide adenine dinucleotide phosphate oxidase.3,6-8 A growing number of studies support that the generation of reactive oxygen species as a common downstream pathway of most of these mechanisms ultimately leads to inflammation and fibrosis7-9 (Fig 1). However, the exact pathogenesis of DN is complex and poorly understood. The early assessment of the nature, severity, and rate of progression of DN could assist in successful therapeutic intervention and in the development of more effective treatment strategies for diabetic patients. Therefore, sensitive and specific biomarkers need to be carefully considered and their usefulness validated. Furthermore, such biomarkers will help to elucidate the pathophysiology of DN and direct investigators toward the use of new therapeutic agents.
Biomarkers of DN In general, a biomarker is a biological substance that may reflect the pathophysiological processes or pharmacological responses to a therapeutic intervention. The ideal renal biomarker should be easy to measure and noninvasive; it should be accurate and highly reproducible; and it should demonstrate high sensitivity, and high specificity, cost-effectiveness in its ability to predict the presence of disease, prognosis of the disorder, and progression of the condition. Advanced technologies have resulted in the identification of several potential biomarkers in the serum, urine, and
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kidney tissues of patients with diabetic kidney disease, and they are often initially explored in animal models. However, many of these biomarkers require further validation. These biomarkers have been categorized based on their ability to detect glomerular and tubular injuries as well as inflammation or oxidative stress (Fig 1). Furthermore, recent proteomic approaches have provided further information about a range of potential renal biomarkers.
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Transferrin
Transferrin is a glycoprotein with a molecular weight of 76.5 kDa, which is slightly greater than that of albumin. However, it is more readily filtered through the glomerular barrier than albumin because it is less anionic.10 Therefore, urinary transferrin has been considered to be a more sensitive early marker for glomerular damage, with transferrinuria occurring before the onset of microalbuminuria in diabetic patients. Indeed, microtransferrinuria has been shown in some type 2 diabetic patients Biomarkers of Kidney Injury in DN with diffuse glomerular lesions in the context of normoalbuminuria.17 Furthermore, many of the early studies in Albuminuria DN involved the use of transferrin rather than albumin in assessing the progression of DN.18,19 A direct Proteinuria is a hallmark of DN, and its presence is a strong correlation between the urinary excretion of transferrin prognostic indicator of the likelihood of kidney disease and other kidney biomarkers, such as albumin, a1progression. It primarily reflects glomerular injury and microglobulin, or N-acetyl-b-D-glucosaminidase (NAG), increased glomerular permeability to macromolecules, has been observed in type 2 diabetic patients.15,17 including in particular a low-molecular-weight protein, alTransferrinuria also correlated with the degree of bumin (65 kDa).4 The glomerular portion of the nephron interstitial fibrosis, tubular atrophy, and interstitial participates in filtration of albumin followed by tubular inflammatory cell infilreabsorption. Alteration in tration in patients with structure and function of DN.17 These findings indithese 2 compartments results CLINICAL SUMMARY cate that urinary transferrin in excretion of excessive almay be useful in detecting bumin into the urine and is Albuminuria is a key feature of DN and is considered an DN at an early stage even early marker of predicting the onset and progression of DN. considered to represent an before the onset of microalearly clinical manifestation A range of potential biomarkers have been identified but to buminuria. However, uri10 of DN. The severity and date, albuminuria remains the gold-standard for diagnosing nary transferrin excretion and categorizing DN. progression of DN is categois also elevated in primary rized into at least 3 stages Proteomics-based approaches may lead to identification of glomerulonephritis and by the rate of urinary albunew biomarkers for prognosis and prediction of treatment other kidney diseases, and response in patients with DN. min excretion: microalbumiit is not specific for DN.20 nuria (30-300 mg/day), The identification of albumacroalbuminuria (300 mg min as another protein to 3 g/day), and nephroticmarker in urine that could be used to monitor kidney range albuminuria (.3 g/day). Another approach is to disease progression led most researchers to choose classify the degree of albuminuria by calculating the urine albumin rather than transferrin as a preferred marker of albumin-to-creatinine ratio (ACR). This ratio is unaffected proteinuria. by variations in urine concentration and avoids the 11 requirement for timed specimens. Microalbuminuria has been considered to be a predictor of the progression Markers of Glomerular Injury of kidney disease in type 1 and type 2 diabetic patients.12,13 Furthermore, the measurement of urinary albumin levels Adiponectin in diabetic patients provides an important prognostic 14 indicator of cardiovascular outcomes. However, not all Adiponectin is a 30-kDa adipocyte-derived vasoactive patients with proteinuria will undergo progressive kidney peptide hormone. High concentrations of adiponectin dysfunction, and not all diabetic patients with progressive have been reported in the urine and serum samples of pakidney impairment will develop proteinuria. Therefore, it tients with diabetic kidney disease.21,22 It has been has been argued that the assessment of microalbuminuria/ suggested that adiponectin levels in urine are as proteinuria has limitations with respect to its specificity for reliable as the albumin excretion rate in predicting the DN and prognostic value for kidney outcomes.15 Indeed, it progression from macroalbuminuria to ESRD in type 1 diabetic patients.21 A recent study in type 2 diabetic pais likely that some normoalbuminuric subjects will have a significant decline in GFR, and as yet we do not have tients with microalbuminuria has suggested that quantiappropriate biomarkers in the urine of such subjects to fication of urinary adiponectin excretion appeared to be predict subsequent decline in GFR.16 an independent indicator of vascular damage potentially
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Figure 1. Biomarkers of diabetic nephropathy during the development and progression of kidney diseases. AGEs3,5-6, ANG II3,5-6, PKC3,5-6; NADPH oxidase6-8; ROS7-9; MCP-160-61; IL-665-67; IL-1865-67; TNF-a63,64; TNFR163,64; CD40-ligand68,69; collagen type IV53-56; BMP-757,58; 8-OHdG72,73; 8(F2a)TGF-b57,58; isoprostane74; 4-hydroxy-2nonenal74; 3-nitrotyrosine peptide75, pentosidine74,76; albuminuria4,10-14; adiponectin21-23, transferrin10,17-20; ceruloplasmin24-26; laminin27,28; podocyte markers29-33; NGAL34-36; NAG37,38; KIM-139,40; L-FABP46-49; H-FABP50; RBP451,52; a1- and b2-microglobulin42-45. Abbreviations: AGEs, advanced glycation end products; ANG II, angiotensin II; BMP-7, bone morphogenetic protein-7; 8-OHdG, 8 hydroxy-20 -deoxyguanosine; H-FABP, heart-type fatty acid binding protein; IL-6, interleukin-6; IL-18, interleukin-18; KIM-1, kidney injury molecule-1; L-FABP, liver-type fatty acid binding protein; MCP-1, monocyte chemoattractant protein-1; NADPH, nicotinamide adenine dinucleotide phosphate; NAG, N-acetyl-b-Dglucosaminidase; NGAL, neutrophil gelatinaseassociated lipocalin; PKC, protein kinase C; RBP4, retinol binding protein-4; ROS, reactive oxygen species; TGF-b, transforming growth factor-b; TNF-a, tumor necrosis factor-a; TNFR1, tumor necrosis factor receptor-1.
identifying subjects at increased risk for vascular events.21,22 However, further study is needed to validate adiponectin as an early or prognostic marker in DN because the former study by Eynatten and colleagues21 showed no additional benefit of urinary adiponectin over urinary albumin in predicting diabetic kidney disease. Furthermore, the role of adiponectin as a factor that improves kidney function warrants further examination.23
Ceruloplasmin Ceruloplasmin is the major copper-carrying protein (151 kDa) in the blood, and it is more negatively charged than albumin. High levels of urinary ceruloplasmin were found in normoalbuminuric type 2 diabetic patients when compared with controls,24 which was associated with the development of microalbuminuria in normoalbuminuric diabetic patients.25 Urinary ceruloplasmin/ creatinine ratios were increased in patients with DN. However, improved glycemic control and low-dose losartan treatment were found to be associated with decreased urinary ceruloplasmin excretion in normoalbuminuric diabetic patients.15,26 Its role remains to be fully clarified given no clear-cut evidence that it is superior to urinary albumin.
Laminin Laminin, a high-molecular-weight (900 kDa) glycoprotein, is a major component of the kidney GBM and mesangium. It is believed that serum laminin is not
filtered by the normal glomerulus, and urinary laminin is secreted from the kidney.27 Urinary laminin excretion was found to be higher in diabetic patients when compared with healthy controls before the onset of microalbuminuria. In addition, there was a positive correlation between the urinary excretion of laminin and type IV collagen, the main constituent of the GBM.28 A significantly higher urinary laminin-to-albumin ratio was seen in type 2 diabetic patients with evidence of nephropathy compared with subjects with nephropathy of nondiabetic origin,28 which suggested that this marker may be more specific for DN than for other kidney diseases.
Podocyte Number and Excretion of PodocyteSpecific Proteins In recent years, the urinary excretion of podocytes or their associated proteins has drawn great attention as potential markers of early glomerulopathy. Urinary excretion of podocytes has been demonstrated in diabetic patients with micro- and macroalbuminuria.29 Moreover, the urinary excretion of podocyte-specific proteins, such as nephrin, synaptopodin, podocalyxin, and podocin, are increased in diabetic patients when compared with controls. However, only urinary nephrin and synaptopodin excretion were shown to have a positive correlation with proteinuria and declining kidney function.30 A recent study has demonstrated higher urinary podocalyxin levels in patients with diabetes and found positive correlations between urinary podocalyxin levels and urinary b2-microglobulin, a1-microglobulin, and urinary NAG but not with serum creatinine, estimated GFR, or
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proteinuria.31 However, podocalyxin is expressed not only in podocytes but is also present in many other cell types (epithelial cells, endothelial cells, platelets, megakaryocytes, and a subset of neurons) and cannot be considered a podocyte-specific biomarker.32 Indeed, podocyturia and excretion of podocyte-specific proteins such as nephrin, synaptopodin, and podocalyxin occur in other forms of nondiabetic CKD.32,33
Markers of Tubular Injury Although the previously described biomarkers are generally considered to reflect glomerular injury, many biomarkers notably reflect tubular injuries. These include neutrophil gelatinase-associated lipocalin (NGAL), NAG, kidney injury molecule-1 (KIM-1), a1- and b2microglobulin, liver-type fatty acid binding protein (L-FABP), and retinal binding protein 4 (RBP4). The specificity of tubular markers remains to be fully determined, but the excretion of these tubular proteins into the urine may not reflect structural damage but could occur in association with polyuria and glycosuria in the absence of nephrological injuries.
NGAL NGAL, also known as lipocalin-2, is an iron-transporting, low-molecular-weight protein (25 kDa) covalently bound to neutrophil-derived gelatinase. It is almost completely reabsorbed by tubules in the normal kidney. Urinary NGAL levels can be easily measured by enzyme-linked immunoabsorbent assay (ELISA). NGAL was recently considered one of the most sensitive and specific tubular biomarkers for detecting acute tubular injury. High levels of NGAL in the urine after acute nephrotoxic and ischemic insults indicate dysfunction of proximal tubular reabsorption.34 It has been demonstrated that urinary NGAL levels are elevated in patients with type 1 diabetes with or without albuminuria, indicating tubular damage at an early stage of DN before the onset of microalbuminuria.35 Furthermore, urinary NGAL could be a more promising early marker than ACR for detecting and predicting kidney injury in type 2 diabetic patients.36
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dependency potentially limits the utility of NAG as a biomarker of DN.
KIM-1 KIM-1, a transmembrane protein secreted by the proximal tubule, is undetectable in the urine under normal conditions. However, high levels of KIM-1 appear in the urine in its stable form after ischemic or nephrotoxic acute kidney injury (AKI).10 Recent studies have demonstrated a close association between KIM-1 and progression of DN toward ESRD in patients with type 1 diabetes as well as in hospitalized patients with AKI.39 Increased urine levels of KIM-1 can be detected by ELISA, a microbead assay, or by an immunochromatographic dipstick in patients with tubulointerstitial damage, and they appear to correlate with tubular expression of this molecule.40 Despite some limitations, KIM-1 is considered a sensitive and specific biomarker for the early diagnosis of AKI and as a prognostic marker for its clinical outcomes. However, the role of KIM-1 in more chronic conditions such as DN remains to be fully defined. A recent study identified a role for KIM-1 in predicting nephropathy in type 1 diabetic subjects.41 Ongoing as yet unpublished studies are in progress to explore the potential added benefits of urinary KIM-1 to albumin in the FinnDiane cohort.
a1- and b2-Microglobulin a1- and b2-microglobulin are low-molecular-weight glycoproteins and are thus completely filtered by the glomerulus and reabsorbed by proximal tubular cells.10,42 Injury to proximal tubules results in increased urinary excretion of a1- and b2-microglobulin, which are potentially useful markers for the early detection of DN. High levels of urinary a1- and b2-microglobulin were found in patients with type 2 diabetes and were directly correlated with albuminuria and with hemoglobin A1c levels. However, glycemic control attenuates levels of a1- and b2-microglobulin.43,44 Furthermore, conflicting results have been reported regarding the utility of these proteins in detecting early stages of albuminuria or kidney damage.10,45
NAG NAG is a relatively high-molecular-weight (.130 kDa) lysosomal enzyme that is secreted during kidney injury by proximal tubules. NAG is a well-studied, sensitive, and robust urinary marker for tubular injury. Increased urinary excretion of NAG was observed in diabetic patients with normoalbuminuria.37 It has recently been demonstrated that changes in urinary levels of NAG were also correlated with regression of microalbuminuria in patients with type 1 diabetes.38 However, urinary NAG concentrations directly increase with hyperglycemia and decrease with improved glycemia; this glucose-
Fatty Acid Binding Protein L-FABP is a low-molecular-weight (14 kDa) protein that is expressed abundantly in the proximal tubule.46 Urinary excretion of L-FABP is a sensitive indicator of acute and chronic tubulointerstitial injury.40 Elevated levels of urinary L-FABP have been detected in diabetic patients before the onset of glomerular damage, microalbuminuria, or overt nephropathy.47 Notably, a positive correlation exists between urinary L-FABP levels and proteinuria and serum creatinine levels in CKD attributed to diabetes or hypertension.40,48 A recent study in
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the very well-phenotyped FinnDiane cohort of type 1 diabetic subjects demonstrated that urinary L-FABP was not superior to albumin in predicting DN, but when combined with albuminuria levels, it modestly improved prediction of the progression from normoalbuminuria to microalbuminuria.49 Furthermore, heart-type fatty acid binding protein (H-FABP) is expressed in the distal tubule of the kidney. In a cross-sectional study involving type 1 and type 2 diabetic subjects, a positive association between urinary H-FABP and estimated GFR independent of albuminuria suggested that HFABP could be a potential marker to assess diabetic kidney disease,50 although this is not a universal view.
RBP4 RBP4 is a low-molecular-weight protein (21 kDa) with diverse functions. RBP4 can be easily filtered by the glomerulus and is almost completely reabsorbed by the proximal tubule. An elevated level of urinary RBP4 can be observed even with minor alterations in renal tubular function.51 Indeed, high levels of urinary RBP4 were demonstrated in patients with type 2 diabetes in the presence of microvascular and macrovascular complications when compared with patients without complications.52 Therefore, urinary RBP4 is viewed to be a sensitive biomarker of tubular function and may be useful in the early detection of renal dysfunction, especially in conjunction with increased albuminuria.
Biomarkers of Kidney Fibrosis and Inflammation in DN Fibrotic Markers Collagen Type IV Collagen type IV, a high-molecular-weight protein (540 kDa), is the main constituent of glomerular and tubular basement membranes as well as the mesangial matrix. It has been found to be increased during the progression of renal fibrosis.40,53 Collagen type IV is too large to be filtered by glomeruli. Its urinary excretion, which is not significantly affected by its serum levels, appears to reflect the rate of matrix synthesis/degradation in damaged kidneys.10,54 An elevated urinary excretion of collagen type IV was observed in patients with immunoglobulin A nephropathy as well as DN and correlates with albuminuria and declining kidney function.40,55 Furthermore, a positive correlation of the urinary excretion of collagen type IV was seen with the urinary excretion of laminin, NAG, and a1microglobulin.56 Hence, the measurement of urinary levels of collagen type IV could be considered a useful potential biomarker for the early assessment of progression of kidney disease in diabetic patients.
Transforming Growth Factor-b Axis The development and progression of renal fibrosis is mediated by overproduction of profibrotic growth factors such as transforming growth factor-b1 (TGF-b1) and connective tissue growth factor, with their urinary excretion measurable by ELISA. Progression of CKD was associated with higher urine levels of TGF-b1 and connective tissue growth factor in patients with diabetes.57 Another secretory cytokine belonging to the TGF-b superfamily is bone morphogenetic protein-7 (BMP-7). A randomized clinical study has demonstrated higher levels of circulating total TGF-b1, lower BMP-7, and a higher total TGF-b1-to-BMP-7 ratio at baseline in type 2 diabetic patients with ESRD, suggesting that this ratio could be useful as a better predictor of kidney disease progression than the conventional markers such as albuminuria in patients with DN.58
Proinflammatory Molecules DN is considered at least in part to be an inflammatory disease. Accumulating evidence now indicates that abnormal production of chemokines and cytokines plays a significant role in the immunopathological mechanisms leading to the development and progression of DN.10,59 Furthermore, recent studies have identified the involvement of chemokines, such as monocyte chemoattractant protein-1 (MCP-1), and cytokines, such as tumor necrosis factor-a (TNF-a), tumor necrosis factor receptor-1 (TNFR1), interleukin-6 (IL-6), interleukin-18 (IL-18), and soluble CD40 ligand, in kidney inflammation. The serum or urine levels of these inflammatory markers can be detected by ELISA; thus, it has been suggested that measurement of these proteins could serve as potential markers of progressive DN. Leukocytes are recruited into the kidney by chemokines, leading to a kidney inflammatory response. MCP-1 has been the most extensively studied proinflammatory chemokine in the context of DN and is considered to be the most potent chemokine for recruiting monocytes/macrophages. Kidney MCP-1 is mostly secreted by glomerular and tubular epithelial cells through activation of nuclear factor kappa B.60 High levels of urinary MCP-1 have been demonstrated in patients with DN and advanced tubulointerstitial lesions.60,61 Furthermore, a direct correlation of urinary MCP-1 was observed with albuminuria, macrophage infiltration into the tubulointerstitium, and urinary excretion of NAG.10 These results are ultimately consistent with the potential utility of urinary MCP-1 as a marker of progression of DN. However its diagnostic role in DN remains to be validated. Other potent proinflammatory cytokines include TNFa and its receptor, TNFR1, which are expressed on infiltrating macrophages and by some glomerular and tubular epithelial cells during renal inflammation.62 A gradual increase in serum and urine levels of TNF-a
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and soluble TNFR1 have been observed in diabetic patients with progressive nephropathy40,63,64; therefore, they have the potential to be considered as early markers of kidney injury. Other proinflammatory cytokines involved in the pathogenesis of DN include IL-6 and IL-18, which are produced by leukocytes, blood vessels, and kidney tubules.10,40 High serum and urine levels of IL-6 and IL-18 were reported in patients with DN.40,65,66 In addition, urinary IL-6 levels correlate directly with its own kidney gene expression as well as with urinary albumin excretion and thickening of the GBM.67 The interaction between a receptor CD40 and its immunomodulating ligand (CD40 L) appears to play an important role in the pathogenesis of the renal inflammatory response involving immune cells and renal epithelial cells.10,68 A significantly elevated urinary level of soluble CD40 L (sCD40 L) was seen in type 1 and type 2 diabetes patients with nephropathy when compared with normoalbuminuric patients, and this molecule positively correlated with albuminuria.69 Moreover, it was reported that increased levels of sCD40 L may help to identify type 1 diabetic adolescents and young adults at risk for developing persistent microalbuminuria.69a In contrast, another study demonstrated that a high plasma level of sCD40 L in type 1 diabetic patients with nephropathy was not a predictor of all-cause mortality or deterioration in kidney function.69b Therefore, further studies are needed to determine the applicability of this particular protein as a renal biomarker.
Biomarkers of Oxidative Stress in DN Oxidative stress has been identified as a key contributor to the pathogenesis and progression of diabetes-related complications including DN.5,40,70 Recent studies have strongly supported the presence of increased plasma and urinary reactive oxygen species in patients with moderate to severe CKD including DN.71 Oxidative stress has been shown to have systemic effects leading to lipid peroxidation as well as damage to proteins and DNA leading to cellular dysfunction. Several serum and urine biomarkers of oxidative stress have been identified in patients as well as in animal models of diabetes-related complications, including 8-hydroxy20 -deoxyguanosine (8-OHdG), 8(F2a)-isoprostane, 4hydroxy-2-nonenal, and 3-nitrotyrosine peptides as well as AGEs including carboxymethyl lysine and pentosidine, which are generated as a result of glycation and oxidation, also known as ‘‘glycoxidation.’’ During increased oxidative stress, one of the nucleobases of DNA, guanine, undergoes metabolic oxidation releasing a stable product, 8-OHdG, which appears in the urine. An elevated level of 8-OHdG was found in the urine of diabetic patients with nephropathy and was positively
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correlated with hemoglobin A1c, suggesting that 8OHdG could represent a potential sensitive biomarker of oxidative DNA damage in diabetes.72,73 Lipid peroxidation is another event in the oxidative stress process resulting in the production of 8(F2a)isoprostane and 4-hydroxy-2-nonenal.74 Urinary and serum levels of 8-isoprostane and 4-hydroxy-2-nonenal were found to be elevated in patients with CKD including DN74 and can be measured by ELISA or high-performance liquid chromatography.40 Furthermore, renal peroxinitrite is the downstream product of superoxide that is involved in the nitration of tyrosine residues to form stable 3-nitrotyrosine peptides. Increased expression of 3-nitrotyrosine peptides was found in the kidneys of patients with DN.75 Indeed, measurement of these peptides in serum or urine by liquid chromatography and ELISA may prove to be useful for assessing nitrosative stress in kidney disease, particularly in the settings of diabetes. Several studies have supported that pentosidine, an AGE, accumulates in the kidney in diabetes and causes cellular dysfunction.10,76 Elevated levels of serum and urinary pentosidine were reported in type 2 diabetic patients with progressive nephropathy when compared with control subjects.74
Proteomics Approaches in DN Proteomic approaches have recently received increased attention as potential tools for the identification of certain proteomic signatures as diagnostic and prognostic biomarkers of kidney diseases.77 Indeed, characterization and validation of these proteomic signatures may represent an important step forward in the noninvasive diagnosis of kidney diseases. Urinary and plasma proteome analyses have identified several biomarkers that are significantly associated with DN. The most commonly used proteomic techniques applied to DN involve mass spectrometry and 2-dimensional gel electrophoresis.78 A recent proteomics-based study has reported that at least 34 proteins were upregulated and a similar number of proteins were downregulated in DN.77 In addition, a recent analysis performed in urine samples obtained from Pima Indians with type 2 diabetes identified a 12peak proteomic mass spectrometer signature that can predict cases of DN in 74% of type 2 diabetic patients before the onset of microalbuminuria.79 Furthermore, other proteomic studies have identified various potential biomarkers. These include specific collagen fragments, b2-microglobulin, ubiquitin, proinflammatory cytokines, RBP4, transthyretin, apolipoprotein A1, apolipoprotein C1, and cystatin C.10,80 A urinary peptidome study has identified that type 1 diabetic patients with a decline in kidney function had decreased levels of peptide fragments of a1(IV) and a1(V) collagens and tenascin-X and increased peptide fragments of inositol pentakisphosphate 2-kinase, zona occludens 3, and FAT tumor
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suppressor 2 in the urine.81 Altogether, these findings suggest that proteomics-based approaches hold great promise not only as a new diagnostic strategy for DN, but they may also help to better understand the pathogenesis of diabetic kidney disease. In a recent prospective study of type 1 and type 2 diabetic subjects,82 a previous urine proteomic signature of at least 34 peptides, primarily collagen fragments, could be identified in urine 3 to 5 years before subjects developed microalbuminuria and increased the ability to detect progression to overt nephropathy when compared with urinary albumin alone. In another prospective study of type 2 diabetic subjects,83 a proteomic approach classifying subjects on the basis of the urinary excretion of 273 peptides was associated with a modest improvement in the prediction of kidney disease progression. Major differences in urinary excretion of collagen and a2-HS-glycoprotein fragments were observed among subjects who progressed from normoalbuminuria to microalbuminuria or microalbuminuria to macroalbuminuria when compared with nonprogressing control subjects. After an initial proteomic analysis of urine samples from type 2 diabetic subjects from the Veteran Affairs Diabetes Trial, urinary haptoglobin was identified as a potentially useful biomarker.84 Indeed, addition of the urinary haptoglobin-to-creatinine ratio to ACR improved the prediction of identifying those subjects with early decline in estimated GFR.
Metabolic Profiling in DN Although this review focuses on the role of various proteins and peptide fragments as biomarkers of DN, it needs to be appreciated that other approaches include metabolic profiling85 of serum and urine samples. Indeed, with fewer molecules to examine when compared with proteomics, it may be possible to identify a novel signature in urine of a much smaller number of metabolites. Indeed, a novel signature of at least 12 metabolites has been identified in urine samples that appears to be linked to diabetic kidney disease.86 These metabolites include members of the tricarboxylic acid cycle, which further underscores the potential role of mitochondrial dysfunction in the pathogenesis of DN. Ongoing studies are in progress to further define the role of metabolomics in DN.
Conclusion Recent advances in molecular and biochemical analyses have identified a wide range of potential serum and urine biomarkers for monitoring kidney function and injury as well as predicting the development and progression of DN. However, to date, the measurement of urinary albumin has remained the gold standard marker for diag-
nosing and categorizing DN. Ongoing evaluation of proteomics-based approaches may lead to novel strategies improving the understanding of DN and assessing the prognosis and treatment response in patients with DN.
References 1. Roglic G, Unwin N, Bennett PH, et al. The burden of mortality attributable to diabetes: realistic estimates for the year 2000. Diabetes Care. 2005;28(9):2130-2135. 2. Molitch ME, DeFronzo RA, Franz MJ, et al. Nephropathy in diabetes. Diabetes Care. 2004;27(Suppl 1):S79-S83. 3. Cooper ME. Pathogenesis, prevention, and treatment of diabetic nephropathy. Lancet. 1998;352(9123):213-219. 4. Brenner BM, Hostetter TH, Humes HD. Molecular basis of proteinuria of glomerular origin. N Engl J Med. 1978;298(15): 826-833. 5. Forbes JM, Coughlan MT, Cooper ME. Oxidative stress as a major culprit in kidney disease in diabetes. Diabetes. 2008;57(6):14461454. 6. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature. 2001;414(6865):813-820. 7. Kaneto H, Katakami N, Kawamori D, et al. Involvement of oxidative stress in the pathogenesis of diabetes. Antioxid Redox Signal. 2007;9(3):355-366. 8. Gill PS, Wilcox CS. NADPH oxidases in the kidney. Antioxid Redox Signal. 2006;8(9-10):1597-1607. 9. Palicz A, Foubert TR, Jesaitis AJ, Marodi L, McPhail LC. Phosphatidic acid and diacylglycerol directly activate NADPH oxidase by interacting with enzyme components. J Biol Chem. 2001;276(5): 3090-3097. 10. Moresco RN, Sangoi MB, De Carvalho JA, Tatsch E, Bochi GV. Diabetic nephropathy: traditional to proteomic markers. Clin Chim Acta. 2013;421:17-30. 11. Keane WF, Eknoyan G. Proteinuria, albuminuria, risk, assessment, detection, elimination (PARADE): a position paper of the National Kidney Foundation. Am J Kidney Dis. 1999;33(5):1004-1010. 12. Perkins BA, Ficociello LH, Roshan B, Warram JH, Krolewski AS. In patients with type 1 diabetes and new-onset microalbuminuria the development of advanced chronic kidney disease may not require progression to proteinuria. Kidney Int. 2010;77(1):57-64. 13. Adler AI, Stevens RJ, Manley SE, Bilous RW, Cull CA, Holman RR. Development and progression of nephropathy in type 2 diabetes: the United Kingdom Prospective Diabetes Study (UKPDS 64). Kidney Int. 2003;63(1):225-232. 14. Yokoyama H, Araki S, Haneda M, et al. Chronic kidney disease categories and renal-cardiovascular outcomes in type 2 diabetes without prevalent cardiovascular disease: a prospective cohort study (JDDM25). Diabetologia. 2012;55(7):1911-1918. 15. Cohen-Bucay A, Viswanathan G. Urinary markers of glomerular injury in diabetic nephropathy. Int J Nephrol. 2012;2012:146987. 16. Macisaac RJ, Tsalamandris C, Thomas MC, et al. Estimating glomerular filtration rate in diabetes: a comparison of cystatinC- and creatinine-based methods. Diabetologia. 2006;49(7):16861689. 17. Kanauchi M, Akai Y, Hashimoto T. Transferrinuria in type 2 diabetic patients with early nephropathy and tubulointerstitial injury. Eur J Intern Med. 2002;13(3):190-193. 18. Jerums G, Cooper ME, Seeman E, Murray RM, McNeil JJ. Spectrum of proteinuria in type I and type II diabetes. Diabetes Care. 1987;10(4):419-427. 19. Jerums G, Allen TJ, Cooper ME. Triphasic changes in selectivity with increasing proteinuria in type 1 and type 2 diabetes. Diabet Med. 1989;6(9):772-779.
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20. Yaqoob M, McClelland P, Patrick AW, Stevenson A, Mason H, Bell GM. Tubular damage in microalbuminuric patients with primary glomerulonephritis and diabetic nephropathy. Ren Fail. 1995;17(1):43-49. 21. von Eynatten M, Liu D, Hock C, et al. Urinary adiponectin excretion: a novel marker for vascular damage in type 2 diabetes. Diabetes. 2009;58(9):2093-2099. 22. Saraheimo M, Forsblom C, Thorn L, et al. Serum adiponectin and progression of diabetic nephropathy in patients with type 1 diabetes. Diabetes Care. 2008;31(6):1165-1169. 23. Sharma K, Ramachandrarao S, Qiu G, et al. Adiponectin regulates albuminuria and podocyte function in mice. J Clin Invest. 2008;118(5):1645-1656. 24. Narita T, Sasaki H, Hosoba M, et al. Parallel increase in urinary excretion rates of immunoglobulin G, ceruloplasmin, transferrin, and orosomucoid in normoalbuminuric type 2 diabetic patients. Diabetes Care. 2004;27(5):1176-1181. 25. Narita T, Hosoba M, Kakei M, Ito S. Increased urinary excretions of immunoglobulin G, ceruloplasmin, and transferrin predict development of microalbuminuria in patients with type 2 diabetes. Diabetes Care. 2006;29(1):142-144. 26. Qin LX, Zeng X, Huang G. Changes in serum and urine ceruloplasmin concentrations in type 2 diabetes. Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2004;29(2):208-211. 27. Hong CY, Chia KS. Markers of diabetic nephropathy. J Diabetes Complications. 1998;12(1):43-60. 28. Inoue M, Oishi C, Shimajiri Y, Furuta M, Ueyama M, Sanke T. Clinical usefulness of measurement of urine type IV collagen for detection of early phase of nephropathy in type 2 diabetic patients. Rinsho Byori. 2008;56(7):564-569. 29. Nakamura T, Ushiyama C, Suzuki S, et al. Urinary excretion of podocytes in patients with diabetic nephropathy. Nephrol Dial Transplant. 2000;15(9):1379-1383. 30. Wang G, Lai FM, Lai KB, Chow KM, Li KT, Szeto CC. Messenger RNA expression of podocyte-associated molecules in the urinary sediment of patients with diabetic nephropathy. Nephron Clin Pract. 2007;106(4):c169-c179. 31. Hara M, Yamagata K, Tomino Y, et al. Urinary podocalyxin is an early marker for podocyte injury in patients with diabetes: establishment of a highly sensitive ELISA to detect urinary podocalyxin. Diabetologia. 2012;55(11):2913-2919. 32. Nielsen JS, McNagny KM. The role of podocalyxin in health and disease. J Am Soc Nephrol. 2009;20(8):1669-1676. 33. Koop K, Eikmans M, Baelde HJ, et al. Expression of podocyteassociated molecules in acquired human kidney diseases. J Am Soc Nephrol. 2003;14(8):2063-2071. 34. Mishra J, Ma Q, Prada A, et al. Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol. 2003;14(10):2534-2543. 35. Nielsen SE, Schjoedt KJ, Astrup AS, et al. Neutrophil gelatinaseassociated lipocalin (NGAL) and kidney injury molecule 1 (KIM1) in patients with diabetic nephropathy: a cross-sectional study and the effects of lisinopril. Diabetic Med. 2010;27(10):1144-1150. 36. Nielsen SE, Reinhard H, Zdunek D, et al. Tubular markers are associated with decline in kidney function in proteinuric type 2 diabetic patients. Diabetes Res Clin Pract. 2012;97(1):71-76. 37. Piwowar A, Knapik-Kordecka M, Fus I, Warwas M. Urinary activities of cathepsin B, N-acetyl-beta-D-glucosaminidase, and albuminuria in patients with type 2 diabetes mellitus. Med Sci Monit. 2006;12(5):CR210-CR214. 38. Vaidya VS, Niewczas MA, Ficociello LH, et al. Regression of microalbuminuria in type 1 diabetes is associated with lower levels of urinary tubular injury biomarkers, kidney injury molecule-1, and N-acetyl-beta-D-glucosaminidase. Kidney Int. 2011;79(4):464-470. 39. Vaidya VS, Ferguson MA, Bonventre JV. Biomarkers of acute kidney injury. Annu Rev Pharmacol Toxicol. 2008;48:463-493.
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40. Tesch GH. Review: serum and urine biomarkers of kidney disease: a pathophysiological perspective. Nephrology (Carlton). 2010;15(6): 609-616. 41. Nielsen SE, Andersen S, Zdunek D, Hess G, Parving HH, Rossing P. Tubular markers do not predict the decline in glomerular filtration rate in type 1 diabetic patients with overt nephropathy. Kidney Int. 2011;79(10):1113-1118. 42. Ekstrom B, Peterson PA, Berggard I. A urinary and plasma alpha1glycoprotein of low molecular weight: isolation and some properties. Biochem Biophys Res Commun. 1975;65(4):1427-1433. 43. Hong CY, Hughes K, Chia KS, Ng V, Ling SL. Urinary alpha1microglobulin as a marker of nephropathy in type 2 diabetic Asian subjects in Singapore. Diabetes Care. 2003;26(2):338-342. 44. Yoshikawa R, Wada J, Seiki K, et al. Urinary PGDS levels are associated with vascular injury in type 2 diabetes patients. Diabetes Res Clin Pract. 2007;76(3):358-367. 45. Helaly MA, Sheashaa HA, Hatata el SZ, Youssef AB, Hegazi A, Abdel-Aal IA. Endothelial dysfunction in geriatric diabetic patients: the role of microalbuminuria in elderly type 2 diabetic patients? A randomized controlled study. Int Urol Nephrol. 2007;39(1):333-338. 46. Portilla D, Dent C, Sugaya T, et al. Liver fatty acid-binding protein as a biomarker of acute kidney injury after cardiac surgery. Kidney Int. 2008;73(4):465-472. 47. Nielsen SE, Sugaya T, Tarnow L, et al. Tubular and glomerular injury in diabetes and the impact of ACE inhibition. Diabetes Care. 2009;32(9):1684-1688. 48. Ishimitsu T, Ohta S, Saito M, et al. Urinary excretion of liver fatty acid-binding protein in health-check participants. Clin Exp Nephrol. 2005;9(1):34-39. 49. Panduru NM, Forsblom C, Saraheimo M, et al. Urinary liver-type fatty acid-binding protein and progression of diabetic nephropathy in type 1 diabetes. Diabetes Care. 2013;36(7):2077-2083. 50. Nauta FL, Boertien WE, Bakker SJ, et al. Glomerular and tubular damage markers are elevated in patients with diabetes. Diabetes Care. 2011;34(4):975-981. 51. Lisowska-Myjak B. Serum and urinary biomarkers of acute kidney injury. Blood Purif. 2010;29(4):357-365. 52. Hong CY, Chia KS, Ling SL. Urinary protein excretion in Type 2 diabetes with complications. J Diabetes Complications. 2000;14(5): 259-265. 53. Kado S, Aoki A, Wada S, et al. Urinary type IV collagen as a marker for early diabetic nephropathy. Diabetes Res Clin Pract. 1996;31(1-3): 103-108. 54. Iijima T, Suzuki S, Sekizuka K, et al. Follow-up study on urinary type IV collagen in patients with early stage diabetic nephropathy. J Clin Lab Anal. 1998;12(6):378-382. 55. Takizawa H, Satoh T, Kurusu A, et al. Increase of urinary type IV collagen in normoalbuminuric patients with impaired glucose tolerance. Nephron. 1998;79(4):474-475. 56. Cohen MP, Lautenslager GT, Shearman CW. Increased collagen IV excretion in diabetes. A marker of compromised filtration function. Diabetes Care. 2001;24(5):914-918. 57. Tsakas S, Goumenos DS. Accurate measurement and clinical significance of urinary transforming growth factor-beta1. Am J Nephrol. 2006;26(2):186-193. 58. Wong MG, Perkovic V, Woodward M, et al. Circulating bone morphogenetic protein-7 and transforming growth factor-beta1 are better predictors of renal end points in patients with type 2 diabetes mellitus. Kidney Int. 2013;83(2):278-284. 59. Wong CK, Ho AW, Tong PC, et al. Aberrant activation profile of cytokines and mitogen-activated protein kinases in type 2 diabetic patients with nephropathy. Clin Exp Immunol. 2007;149(1): 123-131. 60. Wada T, Furuichi K, Sakai N, et al. Up-regulation of monocyte chemoattractant protein-1 in tubulointerstitial lesions of human diabetic nephropathy. Kidney Int. 2000;58(4):1492-1499.
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61. Tashiro K, Koyanagi I, Saitoh A, et al. Urinary levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8), and renal injuries in patients with type 2 diabetic nephropathy. J Clin Lab Anal. 2002;16(1):1-4. 62. Ortiz A, Egido J. Is there a role for specific anti-TNF strategies in glomerular diseases? Nephrol Dial Transplant. 1995;10(3):309-311. 63. Niewczas MA, Ficociello LH, Johnson AC, et al. Serum concentrations of markers of TNFalpha and Fas-mediated pathways and renal function in nonproteinuric patients with type 1 diabetes. Clin J Am Soc Nephrol. 2009;4(1):62-70. 64. Navarro JF, Mora C, Gomez M, Muros M, Lopez-Aguilar C, Garcia J. Influence of renal involvement on peripheral blood mononuclear cell expression behaviour of tumour necrosis factor-alpha and interleukin-6 in type 2 diabetic patients. Nephrol Dial Transplant. 2008;23(3):919-926. 65. Nakamura A, Shikata K, Hiramatsu M, et al. Serum interleukin-18 levels are associated with nephropathy and atherosclerosis in Japanese patients with type 2 diabetes. Diabetes Care. 2005;28(12):2890-2895. 66. Pickup JC, Chusney GD, Thomas SM, Burt D. Plasma interleukin6, tumour necrosis factor alpha and blood cytokine production in type 2 diabetes. Life Sci. 2000;67(3):291-300. 67. Navarro JF, Mora C, Muros M, Garcia J. Urinary tumour necrosis factor-alpha excretion independently correlates with clinical markers of glomerular and tubulointerstitial injury in type 2 diabetic patients. Nephrol Dial Transplant. 2006;21(12):3428-3434. 68. Dechanet J, Grosset C, Taupin JL, et al. CD40 ligand stimulates proinflammatory cytokine production by human endothelial cells. J Immunol. 1997;159(11):5640-5647. 69. El-Asrar MA, Adly AA, Ismail EA. Soluble CD40L in children and adolescents with type 1 diabetes: relation to microvascular complications and glycemic control. Pediatr Diabetes. 2012;13(8):616-624. 69a. Chiarelli F, Giannini C, Verrotii A, Mezzett A, Mohn A. Increased concentrations of soluble CD40 ligand may help to identify type 1 diabetic adolescents and young adults at risk for developing persistent microalbuminuria. Diabetes Metab Res Rev. 2008;24(7):570-576. 69b. Lajer M, Tarnow I, Michelson AD, et al. Soluble CD40 ligand is elevated in type 1 diabetic nephropathy but not predictive of mortality, cardiovascular events or kidney function. Platelets. 2010;21(7): 525-532. 70. Giacco F, Brownlee M. Oxidative stress and diabetic complications. Circ Res. 2010;107(9):1058-1070. 71. Oberg BP, McMenamin E, Lucas FL, et al. Increased prevalence of oxidant stress and inflammation in patients with moderate to severe chronic kidney disease. Kidney Int. 2004;65(3):1009-1016.
72. Wu LL, Chiou CC, Chang PY, Wu JT. Urinary 8-OHdG: a marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics. Clin Chim Acta. 2004;339(1-2):1-9. 73. Kanauchi M, Nishioka H, Hashimoto T. Oxidative DNA damage and tubulointerstitial injury in diabetic nephropathy. Nephron. 2002;91(2):327-329. 74. Calabrese V, Mancuso C, Sapienza M, et al. Oxidative stress and cellular stress response in diabetic nephropathy. Cell Stress Chaperones. 2007;12(4):299-306. 75. Thuraisingham RC, Nott CA, Dodd SM, Yaqoob MM. Increased nitrotyrosine staining in kidneys from patients with diabetic nephropathy. Kidney Int. 2000;57(5):1968-1972. 76. Sugiyama S, Miyata T, Ueda Y, et al. Plasma levels of pentosidine in diabetic patients: an advanced glycation end product. J Am Soc Nephrol. 1998;9(9):1681-1688. 77. Ben Ameur R, Molina L, Bolvin C, et al. Proteomic approaches for discovering biomarkers of diabetic nephropathy. Nephrol Dial Transplant. 2010;25(9):2866-2875. 78. Thongboonkerd V. Study of diabetic nephropathy in the proteomic era. Contrib Nephrol. 2011;170:172-183. 79. Otu HH, Can H, Spentzos D, et al. Prediction of diabetic nephropathy using urine proteomic profiling 10 years prior to development of nephropathy. Diabetes Care. 2007;30(3):638-643. 80. Starkey JM, Tilton RG. Proteomics and systems biology for understanding diabetic nephropathy. J Cardiovasc Transl Res. 2012;5(4): 479-490. 81. Merchant ML, Perkins BA, Boratyn GM, et al. Urinary peptidome may predict renal function decline in type 1 diabetes and microalbuminuria. J Am Soc Nephrol. 2009;20(9):2065-2074. 82. Zurbig P, Jerums G, Hovind P, et al. Urinary proteomics for early diagnosis in diabetic nephropathy. Diabetes. 2012;61(12): 3304-3313. 83. Roscioni SS, de Zeeuw D, Hellemons ME, et al. A urinary peptide biomarker set predicts worsening of albuminuria in type 2 diabetes mellitus. Diabetologia. 2013;56(2):259-267. 84. Bhensdadia NM, Hunt KJ, Lopes-Virella MF, et al. Urine haptoglobin levels predict early renal functional decline in patients with type 2 diabetes. Kidney Int. 2013;83(6):1136-1143. 85. Lewis GD, Asnani A, Gerszten RE. Application of metabolomics to cardiovascular biomarker and pathway discovery. J Am Coll Cardiol. 2008;52(2):117-123. 86. Sharma K, Karl B, Mathew AV, et al. Metabolomics reveals signature of mitochondrial dysfunction in diabetic kidney disease. J Am Soc Nephrol. 2013;24(11):1901-1912.
Introduction Diabetes mellitus represents a global threat for premature morbidity and mortality, and it is likely to be the 5th leading cause of death worldwide.1 According to the recently updated International Diabetes Federation’s Diabetes Atlas Report, more than 371 million people have diabetes, and this figure is increasing globally at an alarming rate. Diabetic nephropathy (DN) is a major chronic microvascular complication of diabetes, and it is the leading cause of ESRD worldwide, often requiring dialysis and/or transplantation.2 DN is characterized by a progressive increase in albuminuria and a decline in glomerular filtration rate (GFR), which often occur in association with an increase in blood pressure, ultimately leading to end-stage kidney failure.3 Proteinuria is considered a hallmark of DN, and it has generally been considered to primarily reflect glomerular injury and increased glomerular permeability to macromolecules.4 In addition, renal functional changes are associated with structural abnormalities, including glomerular basement membrane (GBM) thickening and mesangial expansion as a result of the accumulation of extracellular matrix (ECM), which leads to glomerulosclerosis and tubulointerstitial fibrosis. Mechanisms underlying nephropathy in diabetes include a range of hemodynamic and metabolic factors, such as hyperglycemia, dyslipidemia, systemic and intraglomerular hypertension, activation of the renin-
From JDRF Danielle Alberti Memorial Centre for Diabetic Complications, Diabetic Complications Division, Baker IDI Heart & Diabetes Institute, Department of Medicine, Monash University, Melbourne, Australia. Financial Disclosure: The authors declare that they have no relevant financial interests. Address correspondence to Mark E. Cooper, MBBS, PhD, Diabetic Complications, Baker IDI Heart & Diabetes Research Institute, PO Box 6492 St. Kilda Road, Melbourne, Victoria 8008, Australia. E-mail: Mark.Cooper@bakeridi. edu.au Ó 2014 by the National Kidney Foundation, Inc. All rights reserved. 1548-5595/$36.00 http://dx.doi.org/10.1053/j.ackd.2014.03.008
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angiotensin system, impaired insulin signaling, infiltration by inflammatory cells, increased growth factors and proinflammatory mediators, and activation of key intracellular signaling pathways and transcription factors leading to the development and progression of diabetesassociated kidney disease.5,6 Several mechanisms have been proposed to explain the detrimental effects of hyperglycemia-induced tissue damage, including flux through the polyol pathway, advanced glycation end product (AGE) formation, hexosamine pathway flux and activation of protein kinase C, angiotensin II, and nicotinamide adenine dinucleotide phosphate oxidase.3,6-8 A growing number of studies support that the generation of reactive oxygen species as a common downstream pathway of most of these mechanisms ultimately leads to inflammation and fibrosis7-9 (Fig 1). However, the exact pathogenesis of DN is complex and poorly understood. The early assessment of the nature, severity, and rate of progression of DN could assist in successful therapeutic intervention and in the development of more effective treatment strategies for diabetic patients. Therefore, sensitive and specific biomarkers need to be carefully considered and their usefulness validated. Furthermore, such biomarkers will help to elucidate the pathophysiology of DN and direct investigators toward the use of new therapeutic agents.
Biomarkers of DN In general, a biomarker is a biological substance that may reflect the pathophysiological processes or pharmacological responses to a therapeutic intervention. The ideal renal biomarker should be easy to measure and noninvasive; it should be accurate and highly reproducible; and it should demonstrate high sensitivity, and high specificity, cost-effectiveness in its ability to predict the presence of disease, prognosis of the disorder, and progression of the condition. Advanced technologies have resulted in the identification of several potential biomarkers in the serum, urine, and
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kidney tissues of patients with diabetic kidney disease, and they are often initially explored in animal models. However, many of these biomarkers require further validation. These biomarkers have been categorized based on their ability to detect glomerular and tubular injuries as well as inflammation or oxidative stress (Fig 1). Furthermore, recent proteomic approaches have provided further information about a range of potential renal biomarkers.
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Transferrin
Transferrin is a glycoprotein with a molecular weight of 76.5 kDa, which is slightly greater than that of albumin. However, it is more readily filtered through the glomerular barrier than albumin because it is less anionic.10 Therefore, urinary transferrin has been considered to be a more sensitive early marker for glomerular damage, with transferrinuria occurring before the onset of microalbuminuria in diabetic patients. Indeed, microtransferrinuria has been shown in some type 2 diabetic patients Biomarkers of Kidney Injury in DN with diffuse glomerular lesions in the context of normoalbuminuria.17 Furthermore, many of the early studies in Albuminuria DN involved the use of transferrin rather than albumin in assessing the progression of DN.18,19 A direct Proteinuria is a hallmark of DN, and its presence is a strong correlation between the urinary excretion of transferrin prognostic indicator of the likelihood of kidney disease and other kidney biomarkers, such as albumin, a1progression. It primarily reflects glomerular injury and microglobulin, or N-acetyl-b-D-glucosaminidase (NAG), increased glomerular permeability to macromolecules, has been observed in type 2 diabetic patients.15,17 including in particular a low-molecular-weight protein, alTransferrinuria also correlated with the degree of bumin (65 kDa).4 The glomerular portion of the nephron interstitial fibrosis, tubular atrophy, and interstitial participates in filtration of albumin followed by tubular inflammatory cell infilreabsorption. Alteration in tration in patients with structure and function of DN.17 These findings indithese 2 compartments results CLINICAL SUMMARY cate that urinary transferrin in excretion of excessive almay be useful in detecting bumin into the urine and is Albuminuria is a key feature of DN and is considered an DN at an early stage even early marker of predicting the onset and progression of DN. considered to represent an before the onset of microalearly clinical manifestation A range of potential biomarkers have been identified but to buminuria. However, uri10 of DN. The severity and date, albuminuria remains the gold-standard for diagnosing nary transferrin excretion and categorizing DN. progression of DN is categois also elevated in primary rized into at least 3 stages Proteomics-based approaches may lead to identification of glomerulonephritis and by the rate of urinary albunew biomarkers for prognosis and prediction of treatment other kidney diseases, and response in patients with DN. min excretion: microalbumiit is not specific for DN.20 nuria (30-300 mg/day), The identification of albumacroalbuminuria (300 mg min as another protein to 3 g/day), and nephroticmarker in urine that could be used to monitor kidney range albuminuria (.3 g/day). Another approach is to disease progression led most researchers to choose classify the degree of albuminuria by calculating the urine albumin rather than transferrin as a preferred marker of albumin-to-creatinine ratio (ACR). This ratio is unaffected proteinuria. by variations in urine concentration and avoids the 11 requirement for timed specimens. Microalbuminuria has been considered to be a predictor of the progression Markers of Glomerular Injury of kidney disease in type 1 and type 2 diabetic patients.12,13 Furthermore, the measurement of urinary albumin levels Adiponectin in diabetic patients provides an important prognostic 14 indicator of cardiovascular outcomes. However, not all Adiponectin is a 30-kDa adipocyte-derived vasoactive patients with proteinuria will undergo progressive kidney peptide hormone. High concentrations of adiponectin dysfunction, and not all diabetic patients with progressive have been reported in the urine and serum samples of pakidney impairment will develop proteinuria. Therefore, it tients with diabetic kidney disease.21,22 It has been has been argued that the assessment of microalbuminuria/ suggested that adiponectin levels in urine are as proteinuria has limitations with respect to its specificity for reliable as the albumin excretion rate in predicting the DN and prognostic value for kidney outcomes.15 Indeed, it progression from macroalbuminuria to ESRD in type 1 diabetic patients.21 A recent study in type 2 diabetic pais likely that some normoalbuminuric subjects will have a significant decline in GFR, and as yet we do not have tients with microalbuminuria has suggested that quantiappropriate biomarkers in the urine of such subjects to fication of urinary adiponectin excretion appeared to be predict subsequent decline in GFR.16 an independent indicator of vascular damage potentially
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Figure 1. Biomarkers of diabetic nephropathy during the development and progression of kidney diseases. AGEs3,5-6, ANG II3,5-6, PKC3,5-6; NADPH oxidase6-8; ROS7-9; MCP-160-61; IL-665-67; IL-1865-67; TNF-a63,64; TNFR163,64; CD40-ligand68,69; collagen type IV53-56; BMP-757,58; 8-OHdG72,73; 8(F2a)TGF-b57,58; isoprostane74; 4-hydroxy-2nonenal74; 3-nitrotyrosine peptide75, pentosidine74,76; albuminuria4,10-14; adiponectin21-23, transferrin10,17-20; ceruloplasmin24-26; laminin27,28; podocyte markers29-33; NGAL34-36; NAG37,38; KIM-139,40; L-FABP46-49; H-FABP50; RBP451,52; a1- and b2-microglobulin42-45. Abbreviations: AGEs, advanced glycation end products; ANG II, angiotensin II; BMP-7, bone morphogenetic protein-7; 8-OHdG, 8 hydroxy-20 -deoxyguanosine; H-FABP, heart-type fatty acid binding protein; IL-6, interleukin-6; IL-18, interleukin-18; KIM-1, kidney injury molecule-1; L-FABP, liver-type fatty acid binding protein; MCP-1, monocyte chemoattractant protein-1; NADPH, nicotinamide adenine dinucleotide phosphate; NAG, N-acetyl-b-Dglucosaminidase; NGAL, neutrophil gelatinaseassociated lipocalin; PKC, protein kinase C; RBP4, retinol binding protein-4; ROS, reactive oxygen species; TGF-b, transforming growth factor-b; TNF-a, tumor necrosis factor-a; TNFR1, tumor necrosis factor receptor-1.
identifying subjects at increased risk for vascular events.21,22 However, further study is needed to validate adiponectin as an early or prognostic marker in DN because the former study by Eynatten and colleagues21 showed no additional benefit of urinary adiponectin over urinary albumin in predicting diabetic kidney disease. Furthermore, the role of adiponectin as a factor that improves kidney function warrants further examination.23
Ceruloplasmin Ceruloplasmin is the major copper-carrying protein (151 kDa) in the blood, and it is more negatively charged than albumin. High levels of urinary ceruloplasmin were found in normoalbuminuric type 2 diabetic patients when compared with controls,24 which was associated with the development of microalbuminuria in normoalbuminuric diabetic patients.25 Urinary ceruloplasmin/ creatinine ratios were increased in patients with DN. However, improved glycemic control and low-dose losartan treatment were found to be associated with decreased urinary ceruloplasmin excretion in normoalbuminuric diabetic patients.15,26 Its role remains to be fully clarified given no clear-cut evidence that it is superior to urinary albumin.
Laminin Laminin, a high-molecular-weight (900 kDa) glycoprotein, is a major component of the kidney GBM and mesangium. It is believed that serum laminin is not
filtered by the normal glomerulus, and urinary laminin is secreted from the kidney.27 Urinary laminin excretion was found to be higher in diabetic patients when compared with healthy controls before the onset of microalbuminuria. In addition, there was a positive correlation between the urinary excretion of laminin and type IV collagen, the main constituent of the GBM.28 A significantly higher urinary laminin-to-albumin ratio was seen in type 2 diabetic patients with evidence of nephropathy compared with subjects with nephropathy of nondiabetic origin,28 which suggested that this marker may be more specific for DN than for other kidney diseases.
Podocyte Number and Excretion of PodocyteSpecific Proteins In recent years, the urinary excretion of podocytes or their associated proteins has drawn great attention as potential markers of early glomerulopathy. Urinary excretion of podocytes has been demonstrated in diabetic patients with micro- and macroalbuminuria.29 Moreover, the urinary excretion of podocyte-specific proteins, such as nephrin, synaptopodin, podocalyxin, and podocin, are increased in diabetic patients when compared with controls. However, only urinary nephrin and synaptopodin excretion were shown to have a positive correlation with proteinuria and declining kidney function.30 A recent study has demonstrated higher urinary podocalyxin levels in patients with diabetes and found positive correlations between urinary podocalyxin levels and urinary b2-microglobulin, a1-microglobulin, and urinary NAG but not with serum creatinine, estimated GFR, or
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proteinuria.31 However, podocalyxin is expressed not only in podocytes but is also present in many other cell types (epithelial cells, endothelial cells, platelets, megakaryocytes, and a subset of neurons) and cannot be considered a podocyte-specific biomarker.32 Indeed, podocyturia and excretion of podocyte-specific proteins such as nephrin, synaptopodin, and podocalyxin occur in other forms of nondiabetic CKD.32,33
Markers of Tubular Injury Although the previously described biomarkers are generally considered to reflect glomerular injury, many biomarkers notably reflect tubular injuries. These include neutrophil gelatinase-associated lipocalin (NGAL), NAG, kidney injury molecule-1 (KIM-1), a1- and b2microglobulin, liver-type fatty acid binding protein (L-FABP), and retinal binding protein 4 (RBP4). The specificity of tubular markers remains to be fully determined, but the excretion of these tubular proteins into the urine may not reflect structural damage but could occur in association with polyuria and glycosuria in the absence of nephrological injuries.
NGAL NGAL, also known as lipocalin-2, is an iron-transporting, low-molecular-weight protein (25 kDa) covalently bound to neutrophil-derived gelatinase. It is almost completely reabsorbed by tubules in the normal kidney. Urinary NGAL levels can be easily measured by enzyme-linked immunoabsorbent assay (ELISA). NGAL was recently considered one of the most sensitive and specific tubular biomarkers for detecting acute tubular injury. High levels of NGAL in the urine after acute nephrotoxic and ischemic insults indicate dysfunction of proximal tubular reabsorption.34 It has been demonstrated that urinary NGAL levels are elevated in patients with type 1 diabetes with or without albuminuria, indicating tubular damage at an early stage of DN before the onset of microalbuminuria.35 Furthermore, urinary NGAL could be a more promising early marker than ACR for detecting and predicting kidney injury in type 2 diabetic patients.36
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dependency potentially limits the utility of NAG as a biomarker of DN.
KIM-1 KIM-1, a transmembrane protein secreted by the proximal tubule, is undetectable in the urine under normal conditions. However, high levels of KIM-1 appear in the urine in its stable form after ischemic or nephrotoxic acute kidney injury (AKI).10 Recent studies have demonstrated a close association between KIM-1 and progression of DN toward ESRD in patients with type 1 diabetes as well as in hospitalized patients with AKI.39 Increased urine levels of KIM-1 can be detected by ELISA, a microbead assay, or by an immunochromatographic dipstick in patients with tubulointerstitial damage, and they appear to correlate with tubular expression of this molecule.40 Despite some limitations, KIM-1 is considered a sensitive and specific biomarker for the early diagnosis of AKI and as a prognostic marker for its clinical outcomes. However, the role of KIM-1 in more chronic conditions such as DN remains to be fully defined. A recent study identified a role for KIM-1 in predicting nephropathy in type 1 diabetic subjects.41 Ongoing as yet unpublished studies are in progress to explore the potential added benefits of urinary KIM-1 to albumin in the FinnDiane cohort.
a1- and b2-Microglobulin a1- and b2-microglobulin are low-molecular-weight glycoproteins and are thus completely filtered by the glomerulus and reabsorbed by proximal tubular cells.10,42 Injury to proximal tubules results in increased urinary excretion of a1- and b2-microglobulin, which are potentially useful markers for the early detection of DN. High levels of urinary a1- and b2-microglobulin were found in patients with type 2 diabetes and were directly correlated with albuminuria and with hemoglobin A1c levels. However, glycemic control attenuates levels of a1- and b2-microglobulin.43,44 Furthermore, conflicting results have been reported regarding the utility of these proteins in detecting early stages of albuminuria or kidney damage.10,45
NAG NAG is a relatively high-molecular-weight (.130 kDa) lysosomal enzyme that is secreted during kidney injury by proximal tubules. NAG is a well-studied, sensitive, and robust urinary marker for tubular injury. Increased urinary excretion of NAG was observed in diabetic patients with normoalbuminuria.37 It has recently been demonstrated that changes in urinary levels of NAG were also correlated with regression of microalbuminuria in patients with type 1 diabetes.38 However, urinary NAG concentrations directly increase with hyperglycemia and decrease with improved glycemia; this glucose-
Fatty Acid Binding Protein L-FABP is a low-molecular-weight (14 kDa) protein that is expressed abundantly in the proximal tubule.46 Urinary excretion of L-FABP is a sensitive indicator of acute and chronic tubulointerstitial injury.40 Elevated levels of urinary L-FABP have been detected in diabetic patients before the onset of glomerular damage, microalbuminuria, or overt nephropathy.47 Notably, a positive correlation exists between urinary L-FABP levels and proteinuria and serum creatinine levels in CKD attributed to diabetes or hypertension.40,48 A recent study in
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the very well-phenotyped FinnDiane cohort of type 1 diabetic subjects demonstrated that urinary L-FABP was not superior to albumin in predicting DN, but when combined with albuminuria levels, it modestly improved prediction of the progression from normoalbuminuria to microalbuminuria.49 Furthermore, heart-type fatty acid binding protein (H-FABP) is expressed in the distal tubule of the kidney. In a cross-sectional study involving type 1 and type 2 diabetic subjects, a positive association between urinary H-FABP and estimated GFR independent of albuminuria suggested that HFABP could be a potential marker to assess diabetic kidney disease,50 although this is not a universal view.
RBP4 RBP4 is a low-molecular-weight protein (21 kDa) with diverse functions. RBP4 can be easily filtered by the glomerulus and is almost completely reabsorbed by the proximal tubule. An elevated level of urinary RBP4 can be observed even with minor alterations in renal tubular function.51 Indeed, high levels of urinary RBP4 were demonstrated in patients with type 2 diabetes in the presence of microvascular and macrovascular complications when compared with patients without complications.52 Therefore, urinary RBP4 is viewed to be a sensitive biomarker of tubular function and may be useful in the early detection of renal dysfunction, especially in conjunction with increased albuminuria.
Biomarkers of Kidney Fibrosis and Inflammation in DN Fibrotic Markers Collagen Type IV Collagen type IV, a high-molecular-weight protein (540 kDa), is the main constituent of glomerular and tubular basement membranes as well as the mesangial matrix. It has been found to be increased during the progression of renal fibrosis.40,53 Collagen type IV is too large to be filtered by glomeruli. Its urinary excretion, which is not significantly affected by its serum levels, appears to reflect the rate of matrix synthesis/degradation in damaged kidneys.10,54 An elevated urinary excretion of collagen type IV was observed in patients with immunoglobulin A nephropathy as well as DN and correlates with albuminuria and declining kidney function.40,55 Furthermore, a positive correlation of the urinary excretion of collagen type IV was seen with the urinary excretion of laminin, NAG, and a1microglobulin.56 Hence, the measurement of urinary levels of collagen type IV could be considered a useful potential biomarker for the early assessment of progression of kidney disease in diabetic patients.
Transforming Growth Factor-b Axis The development and progression of renal fibrosis is mediated by overproduction of profibrotic growth factors such as transforming growth factor-b1 (TGF-b1) and connective tissue growth factor, with their urinary excretion measurable by ELISA. Progression of CKD was associated with higher urine levels of TGF-b1 and connective tissue growth factor in patients with diabetes.57 Another secretory cytokine belonging to the TGF-b superfamily is bone morphogenetic protein-7 (BMP-7). A randomized clinical study has demonstrated higher levels of circulating total TGF-b1, lower BMP-7, and a higher total TGF-b1-to-BMP-7 ratio at baseline in type 2 diabetic patients with ESRD, suggesting that this ratio could be useful as a better predictor of kidney disease progression than the conventional markers such as albuminuria in patients with DN.58
Proinflammatory Molecules DN is considered at least in part to be an inflammatory disease. Accumulating evidence now indicates that abnormal production of chemokines and cytokines plays a significant role in the immunopathological mechanisms leading to the development and progression of DN.10,59 Furthermore, recent studies have identified the involvement of chemokines, such as monocyte chemoattractant protein-1 (MCP-1), and cytokines, such as tumor necrosis factor-a (TNF-a), tumor necrosis factor receptor-1 (TNFR1), interleukin-6 (IL-6), interleukin-18 (IL-18), and soluble CD40 ligand, in kidney inflammation. The serum or urine levels of these inflammatory markers can be detected by ELISA; thus, it has been suggested that measurement of these proteins could serve as potential markers of progressive DN. Leukocytes are recruited into the kidney by chemokines, leading to a kidney inflammatory response. MCP-1 has been the most extensively studied proinflammatory chemokine in the context of DN and is considered to be the most potent chemokine for recruiting monocytes/macrophages. Kidney MCP-1 is mostly secreted by glomerular and tubular epithelial cells through activation of nuclear factor kappa B.60 High levels of urinary MCP-1 have been demonstrated in patients with DN and advanced tubulointerstitial lesions.60,61 Furthermore, a direct correlation of urinary MCP-1 was observed with albuminuria, macrophage infiltration into the tubulointerstitium, and urinary excretion of NAG.10 These results are ultimately consistent with the potential utility of urinary MCP-1 as a marker of progression of DN. However its diagnostic role in DN remains to be validated. Other potent proinflammatory cytokines include TNFa and its receptor, TNFR1, which are expressed on infiltrating macrophages and by some glomerular and tubular epithelial cells during renal inflammation.62 A gradual increase in serum and urine levels of TNF-a
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and soluble TNFR1 have been observed in diabetic patients with progressive nephropathy40,63,64; therefore, they have the potential to be considered as early markers of kidney injury. Other proinflammatory cytokines involved in the pathogenesis of DN include IL-6 and IL-18, which are produced by leukocytes, blood vessels, and kidney tubules.10,40 High serum and urine levels of IL-6 and IL-18 were reported in patients with DN.40,65,66 In addition, urinary IL-6 levels correlate directly with its own kidney gene expression as well as with urinary albumin excretion and thickening of the GBM.67 The interaction between a receptor CD40 and its immunomodulating ligand (CD40 L) appears to play an important role in the pathogenesis of the renal inflammatory response involving immune cells and renal epithelial cells.10,68 A significantly elevated urinary level of soluble CD40 L (sCD40 L) was seen in type 1 and type 2 diabetes patients with nephropathy when compared with normoalbuminuric patients, and this molecule positively correlated with albuminuria.69 Moreover, it was reported that increased levels of sCD40 L may help to identify type 1 diabetic adolescents and young adults at risk for developing persistent microalbuminuria.69a In contrast, another study demonstrated that a high plasma level of sCD40 L in type 1 diabetic patients with nephropathy was not a predictor of all-cause mortality or deterioration in kidney function.69b Therefore, further studies are needed to determine the applicability of this particular protein as a renal biomarker.
Biomarkers of Oxidative Stress in DN Oxidative stress has been identified as a key contributor to the pathogenesis and progression of diabetes-related complications including DN.5,40,70 Recent studies have strongly supported the presence of increased plasma and urinary reactive oxygen species in patients with moderate to severe CKD including DN.71 Oxidative stress has been shown to have systemic effects leading to lipid peroxidation as well as damage to proteins and DNA leading to cellular dysfunction. Several serum and urine biomarkers of oxidative stress have been identified in patients as well as in animal models of diabetes-related complications, including 8-hydroxy20 -deoxyguanosine (8-OHdG), 8(F2a)-isoprostane, 4hydroxy-2-nonenal, and 3-nitrotyrosine peptides as well as AGEs including carboxymethyl lysine and pentosidine, which are generated as a result of glycation and oxidation, also known as ‘‘glycoxidation.’’ During increased oxidative stress, one of the nucleobases of DNA, guanine, undergoes metabolic oxidation releasing a stable product, 8-OHdG, which appears in the urine. An elevated level of 8-OHdG was found in the urine of diabetic patients with nephropathy and was positively
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correlated with hemoglobin A1c, suggesting that 8OHdG could represent a potential sensitive biomarker of oxidative DNA damage in diabetes.72,73 Lipid peroxidation is another event in the oxidative stress process resulting in the production of 8(F2a)isoprostane and 4-hydroxy-2-nonenal.74 Urinary and serum levels of 8-isoprostane and 4-hydroxy-2-nonenal were found to be elevated in patients with CKD including DN74 and can be measured by ELISA or high-performance liquid chromatography.40 Furthermore, renal peroxinitrite is the downstream product of superoxide that is involved in the nitration of tyrosine residues to form stable 3-nitrotyrosine peptides. Increased expression of 3-nitrotyrosine peptides was found in the kidneys of patients with DN.75 Indeed, measurement of these peptides in serum or urine by liquid chromatography and ELISA may prove to be useful for assessing nitrosative stress in kidney disease, particularly in the settings of diabetes. Several studies have supported that pentosidine, an AGE, accumulates in the kidney in diabetes and causes cellular dysfunction.10,76 Elevated levels of serum and urinary pentosidine were reported in type 2 diabetic patients with progressive nephropathy when compared with control subjects.74
Proteomics Approaches in DN Proteomic approaches have recently received increased attention as potential tools for the identification of certain proteomic signatures as diagnostic and prognostic biomarkers of kidney diseases.77 Indeed, characterization and validation of these proteomic signatures may represent an important step forward in the noninvasive diagnosis of kidney diseases. Urinary and plasma proteome analyses have identified several biomarkers that are significantly associated with DN. The most commonly used proteomic techniques applied to DN involve mass spectrometry and 2-dimensional gel electrophoresis.78 A recent proteomics-based study has reported that at least 34 proteins were upregulated and a similar number of proteins were downregulated in DN.77 In addition, a recent analysis performed in urine samples obtained from Pima Indians with type 2 diabetes identified a 12peak proteomic mass spectrometer signature that can predict cases of DN in 74% of type 2 diabetic patients before the onset of microalbuminuria.79 Furthermore, other proteomic studies have identified various potential biomarkers. These include specific collagen fragments, b2-microglobulin, ubiquitin, proinflammatory cytokines, RBP4, transthyretin, apolipoprotein A1, apolipoprotein C1, and cystatin C.10,80 A urinary peptidome study has identified that type 1 diabetic patients with a decline in kidney function had decreased levels of peptide fragments of a1(IV) and a1(V) collagens and tenascin-X and increased peptide fragments of inositol pentakisphosphate 2-kinase, zona occludens 3, and FAT tumor
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suppressor 2 in the urine.81 Altogether, these findings suggest that proteomics-based approaches hold great promise not only as a new diagnostic strategy for DN, but they may also help to better understand the pathogenesis of diabetic kidney disease. In a recent prospective study of type 1 and type 2 diabetic subjects,82 a previous urine proteomic signature of at least 34 peptides, primarily collagen fragments, could be identified in urine 3 to 5 years before subjects developed microalbuminuria and increased the ability to detect progression to overt nephropathy when compared with urinary albumin alone. In another prospective study of type 2 diabetic subjects,83 a proteomic approach classifying subjects on the basis of the urinary excretion of 273 peptides was associated with a modest improvement in the prediction of kidney disease progression. Major differences in urinary excretion of collagen and a2-HS-glycoprotein fragments were observed among subjects who progressed from normoalbuminuria to microalbuminuria or microalbuminuria to macroalbuminuria when compared with nonprogressing control subjects. After an initial proteomic analysis of urine samples from type 2 diabetic subjects from the Veteran Affairs Diabetes Trial, urinary haptoglobin was identified as a potentially useful biomarker.84 Indeed, addition of the urinary haptoglobin-to-creatinine ratio to ACR improved the prediction of identifying those subjects with early decline in estimated GFR.
Metabolic Profiling in DN Although this review focuses on the role of various proteins and peptide fragments as biomarkers of DN, it needs to be appreciated that other approaches include metabolic profiling85 of serum and urine samples. Indeed, with fewer molecules to examine when compared with proteomics, it may be possible to identify a novel signature in urine of a much smaller number of metabolites. Indeed, a novel signature of at least 12 metabolites has been identified in urine samples that appears to be linked to diabetic kidney disease.86 These metabolites include members of the tricarboxylic acid cycle, which further underscores the potential role of mitochondrial dysfunction in the pathogenesis of DN. Ongoing studies are in progress to further define the role of metabolomics in DN.
Conclusion Recent advances in molecular and biochemical analyses have identified a wide range of potential serum and urine biomarkers for monitoring kidney function and injury as well as predicting the development and progression of DN. However, to date, the measurement of urinary albumin has remained the gold standard marker for diag-
nosing and categorizing DN. Ongoing evaluation of proteomics-based approaches may lead to novel strategies improving the understanding of DN and assessing the prognosis and treatment response in patients with DN.
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61. Tashiro K, Koyanagi I, Saitoh A, et al. Urinary levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8), and renal injuries in patients with type 2 diabetic nephropathy. J Clin Lab Anal. 2002;16(1):1-4. 62. Ortiz A, Egido J. Is there a role for specific anti-TNF strategies in glomerular diseases? Nephrol Dial Transplant. 1995;10(3):309-311. 63. Niewczas MA, Ficociello LH, Johnson AC, et al. Serum concentrations of markers of TNFalpha and Fas-mediated pathways and renal function in nonproteinuric patients with type 1 diabetes. Clin J Am Soc Nephrol. 2009;4(1):62-70. 64. Navarro JF, Mora C, Gomez M, Muros M, Lopez-Aguilar C, Garcia J. Influence of renal involvement on peripheral blood mononuclear cell expression behaviour of tumour necrosis factor-alpha and interleukin-6 in type 2 diabetic patients. Nephrol Dial Transplant. 2008;23(3):919-926. 65. Nakamura A, Shikata K, Hiramatsu M, et al. Serum interleukin-18 levels are associated with nephropathy and atherosclerosis in Japanese patients with type 2 diabetes. Diabetes Care. 2005;28(12):2890-2895. 66. Pickup JC, Chusney GD, Thomas SM, Burt D. Plasma interleukin6, tumour necrosis factor alpha and blood cytokine production in type 2 diabetes. Life Sci. 2000;67(3):291-300. 67. Navarro JF, Mora C, Muros M, Garcia J. Urinary tumour necrosis factor-alpha excretion independently correlates with clinical markers of glomerular and tubulointerstitial injury in type 2 diabetic patients. Nephrol Dial Transplant. 2006;21(12):3428-3434. 68. Dechanet J, Grosset C, Taupin JL, et al. CD40 ligand stimulates proinflammatory cytokine production by human endothelial cells. J Immunol. 1997;159(11):5640-5647. 69. El-Asrar MA, Adly AA, Ismail EA. Soluble CD40L in children and adolescents with type 1 diabetes: relation to microvascular complications and glycemic control. Pediatr Diabetes. 2012;13(8):616-624. 69a. Chiarelli F, Giannini C, Verrotii A, Mezzett A, Mohn A. Increased concentrations of soluble CD40 ligand may help to identify type 1 diabetic adolescents and young adults at risk for developing persistent microalbuminuria. Diabetes Metab Res Rev. 2008;24(7):570-576. 69b. Lajer M, Tarnow I, Michelson AD, et al. Soluble CD40 ligand is elevated in type 1 diabetic nephropathy but not predictive of mortality, cardiovascular events or kidney function. Platelets. 2010;21(7): 525-532. 70. Giacco F, Brownlee M. Oxidative stress and diabetic complications. Circ Res. 2010;107(9):1058-1070. 71. Oberg BP, McMenamin E, Lucas FL, et al. Increased prevalence of oxidant stress and inflammation in patients with moderate to severe chronic kidney disease. Kidney Int. 2004;65(3):1009-1016.
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