Novel Biomarkers of Renal Function Introduction and Overview

Updated: Sep 20, 2017
  • Author: Nikhil A Shah, MBBS, DNB(Neph); Chief Editor: Vecihi Batuman, MD, FASN  more...
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Introduction and Overview

Introduction and Overview

A biomarker (biologic marker) is a measurable indicator of a specific biological state, particularly one relevant to the risk for, the presence of, or the stage of a disease. Although historically the term often referred to a physical trait or physiological metric, it now typically refers to products of genomic, metobolomic, and proteomic research. [1] Biomarkers can be used clinically to screen for, diagnose, or monitor the activity of diseases and to guide molecularly targeted therapy or assess therapeutic response. [2]

The National Institutes of Health (NIH) Biomarkers Definitions Working Group has defined a biomarker as, “A characteristic that is ob­jectively measured and evaluated as an indicator of normal biologic pro­cesses, pathogenic processes, or pharmacologic responses to a therapeutic intervention." [3]

Biomarker discovery is complex and involves many phases. The biomarker discovery process has been described as having the following six stages, each of which has its own challenges [1] :

  1. Discovery
  2. Qualification
  3. Verification
  4. Research assay optimization
  5. Clinical validation
  6. Commercialization

Ideal biomarkers have the following general characteristics [4] :

  • Noninvasive, easily measured, inexpensive, and produce rapid results
  • Have readily available sources, such as blood or urine
  •  Have high sensitivity, allowing early detection, and no overlap in values between diseased patients and healthy controls
  • Have high specificity, being greatly upregulated (or downregulated) specifically in the diseased samples, and unaffected by comorbid conditions
  • Levels vary rapidly to reflect disease severity and in response to treatment
  • Levels aid in risk stratification and possess prognostic value in terms of hard clinical outcomes
  • Biologically plausible and provide insight into the underlying disease mechanism

At present, serum creatinine, which is used to calculate the glomerular filtration rate (GFR), is the most commonly used marker of renal function. However, it is far from an ideal biomarker, for the following reasons:

  • The creatinine level is influenced by multiple non-renal factors (eg, age, gender, muscle mass, muscle metabolism, diet, medications, amputations, hydration status).
  • In acute kidney injury (AKI), the serum creatinine level can take several hours or days to reach a new steady state and does not reflect the actual decrease in GFR. An increase in the serum creatinine level represents a delayed indication of a functional change in GFR that lags behind structural changes that occur in the kidney during the early stage of AKI.
  • Because of renal reserve, the serum creatinine level may not rise until substantial kidney function has been lost.
  • Serum creatinine measurement does not allow differentiation between hemodynamically mediated changes in renal function, such as pre-renal azotemia from intrinsic renal failure or obstructive uropathy.

Given the limitations of serum creatinine as a marker of renal function, different urinary and serum proteins; molecules; and, most recently, microRNAs have been rigorously investigated over the past decade as possible biomarkers for kidney diseases.

Although a comprehensive discussion of individual biomarkers is beyond the scope of this review, we will briefly describe selected biomarkers that are being investigated for use in various renal conditions, including the following:

  • AKI
  • Chronic kidney disease (CKD)
  • Various forms of glomerulonephritis (GN)
  • Autosomal dominant polycystic kidney disease (ADPKD)
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Characteristics of an Ideal Marker for Kidney Disease

An ideal biomarker for acute kidney injury (AKI) or chronic kidney disease (CKD) should have the following characteristics [5] :

  • Shows rapid and reliable increase in response to kidney diseases
  • Is highly sensitive and specific for acute and/or chronic kidney disease
  • Shows good correlation with degree of renal injury
  • Provides risk stratification and prognostic information (severity of kidney disease, need for dialysis, length of hospital stay, and mortality)
  • Is site-specific to detect early injury (proximal, distal, interstitium, or vasculature) and identify pathologic changes in specific segments of renal tubules
  • Is applicable across different races and age groups
  • Allows recognition of the cause of kidney injury or disease (eg, ischemia, toxins, sepsis, cardiovascular disease, diabetic nephropathy, lupus, combined factors)
  • Is organ-specific and allows differentiation among intrarenal, prerenal,and extrarenal causes of kidney injury
  • Noninvasively identifies the duration of kidney failure (AKI versus CKD)
  • Is useful to monitor the response to therapeutic interventions
  • Provides information on the risk of complications from comorbid conditions (especially in CKD)
  • Is stable over time across different temperature and pH conditions, with clinically relevant storage conditions
  • Is rapidly and easily measurable
  • Is not subject to interference by drugs or endogenous substances

 

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Biomarkers of Acute Kidney Injury

Biomarkers of acute kidney injury (AKI) include the following [6] :

  • Neutrophil gelatinase-associated lipocalin (NGAL)
  • Interleukin-18 (IL-18)
  • Kidney injury molecule 1 (KIM-1)
  • Liver-type fatty acid–binding protein (L-FABP)
  • Insulinlike growth factor–binding protein 7 (IGFBP7) x tissue inhibitor of metalloproteinases–2 (TIMP-2)
  • Calprotectin
  • Urinary angiotensinogen
  • Urinary microRNA

Neutrophil Gelatinase-associated Lipocalin

NGAL is a 25-kD protein of the lipocalin family. Elevation of NGAL levels has been documented in the plasma and urine of animal models of ischemic and nephrotoxic AKI; hence, NGAL is considered to be a novel urinary biomarker for ischemic injury. [7] The thick ascending limb of the loop of Henle and the intercalated cells of the collecting duct are the primary sites of NGAL production in the kidney. [8, 9, 10]

In human studies, the expression of the NGAL messenger ribonucleic acid (mRNA) and protein has been shown to be significantly increased in the kidney tubules in the following settings:

  • Ischemic, septic, or post-transplantation AKI [11]
  • Within 2-6 hours after cardiopulmonary bypass surgery [12]
  • At frequent intervals for 24 hours post–cardiopulmonary bypass surgery in children [13]
  • Following contrast administration [14]

NGAL has been thought to reduce injury by inhibiting apoptosis and increasing the normal proliferation of kidney tubule cells. More specifically, NGAL has been reported to up-regulate heme oxygenase-1, which preserves proximal tubule N-cadherin, and subsequently inhibits cell death. [15]

NGAL elevation is detected in 3 h after injury, peaks in 6-12 h, and remains elevated for up to 5 days, depending on the severity of injury. [16]

NGAL has been tested in multiple studies of patients at risk for AKI. Using samples from the Translational Research Investigating Biomarker Endpoints in AKI study (TRIBE-AKI), researchers tried to determine whether biomarkers measured at the time of first clinical diagnosis of early AKI after cardiac surgery can potentially predict AKI severity. Biomarkers such as urinary IL-18, urinary albumin-to-creatinine ratio (ACR), and urinary and plasma NGAL were demonstrated to improve risk classification compared with the clinical model alone, with plasma NGAL performing the best (category-free net reclassification improvement of 0.69, P <0.0001). The authors concluded that biomarkers measured on the day of AKI diagnosis improve risk stratification and identify patients at higher risk for progression of AKI and worse outcomes. [17]

In a study of 157 adults treated in an emergency department with in 24 hours of poisoning, baseline NGAL levels allowed for better prediction of AKI than initial creatinine levels; higher plasma NGAL levels were found in patients with AKI than in those without AKI  (median, 310 vs 86 ng/mL; P <0.001). The researchers concluded that plasma NGAL may serve as a good predictor of AKI in cases of adult poisoning. [18]

Moreover, NGAL has shown some potential to aid in the diagnosis of early acute tubular necrosis (ATN) and to differentiate it from pre-renal disease. Paragas et al found that in a mouse strain with a gene for bioluminescence and fluorescence inserted into the NGAL gene, imaging after ischemia reperfusion demonstrated illumination of specific cells of the distal nephron, indicating NGAL production at the site of injury. However, no NGAL illumination was seen following maneuvers that lead to significant pre-renal disease. Thus, NGAL may potentially be useful in differentiating pre-renal disease from ATN. [19]

This was further studied in post surgical patients. In a prospective study, urinary NGAL was sampled at 2-3 hours after general surgery and increased levels were found only in patients with sustained AKI (mostly likely ATN), while remaining normal in those with transient AKI (hemodynamic, pre-renal). [20]

NGAL has now been studied in various populations including pediatric and adult cardiac surgery patients, critically ill patients, emergency department patients, and kidney transplant recipients. A meta-analysis of 16 studies with a total of 2906 adult patients after cardiac surgery showed an area under receiver operating curve (AUC) of 0.72. [21]

NGAL is one of the most promising biomarker currently under investigation. Determining the appropriate cut-off values in different clinical situations appears to be next step in validating this biomarker.

Interleukin-18

A candidate biomarker for renal parenchymal injury, the cytokine IL-18 is formed in the proximal tubules and can be detected in the urine. [22] In animal models, IL-18 has been shown to exacerbate tubular necrosis; neutralizing antibodies formed against IL-18 were found to reduce renal injury in mice.

Parikh et al determined that patients with ATN had significantly higher levels of IL-18 in their urine than did control subjects or persons with other forms of kidney disease. [22] In renal transplant recipients, those patients with delayed graft function in the immediate post-transplantation period had higher urinary levels of IL-18 than did patients who had immediate graft function. Furthermore, patients with ischemia-reperfusion injury, glycerol injection, and cisplatin-induced renal injury have likewise been noted to have elevated levels of this pro-inflammatory cytokine. [23, 24, 11]

Evaluation of the potential use of urinary NGAL and IL-18 in patients with AKI (post–cardiopulmonary bypass) has led to the suggestion that the two may be sequential markers: NGAL levels peak within the first 2-4 hours following AKI, while IL-18 peaks at the 12th hour. [25]

A potential limitation of IL-18 is that it may be a more generalized marker of inflammation rather than a specific marker of AKI. This is particularly the case in the elderly population, who may have baseline decreased kidney function.

A meta-analysis of 11 studies with 2796 patients showed reasonable results in post-cardiac surgery AKI in pediatric patients, while other studies failed to demonstrate a strong predictive accuracy. Disagreement about a cut-off level of IL-18 may partly be responsible for the heterogeneity. [26]

Kidney Injury Molecule 1

KIM-1 is a type 1 transmembrane protein that contains extracellular mucin and immunoglobulin domains. [27] It has low basal expression in the normal kidney but is up-regulated in post–ischemic injury in the proximal tubule. The extracellular domain of KIM-1 appears in the urine shortly after ischemic injury and can be readily detected by a KIM-1 urinary dipstick, potentially making it a convenient and readily measured marker of AKI. [28, 29]

Apart from its potential as a biomarker for ATN, KIM-1 may have a role in determining risk for the development of AKI. In a prospective study that included 123 patients undergoing cardiac surgery, preoperative KIM-1, along with alpha glutathione-s-transferase (GST), was able to predict the future development of stage 1 and stage 3 AKI. [30]

Moreover, a study by Ichimura et al demonstrated how KIM-1 is able to specifically recognize apoptotic cell surface–specific epitopes expressed by apoptotic tubular epithelial cells and subsequently phagocytose apopototic bodies and necrotic debris. [13] It has been proposed, therefore, that KIM-1 may play a role in renal remodeling after AKI and may be a target for pharmacological intervention.

In more recent cardiac studies , KIM-1 elevation correlated with increased risk of death or hospitalization independent of eGFR. In a study of biomarkers of AKI after cardiac surgery both KIM-1 and IL-18 had an AUC of 0.92 while the combination of both biomarkers together was a better predictor among 32 urinary biomarkers. [31, 32]

Liver-type Fatty Acid–Binding Protein

L-FABP is a 14-kD protein that is localized predominantly in the proximal tubule. [14] Its level in the urine has been noted to be elevated in patients who sustained AKI following cardiac surgery. The L-FABP gene is expressed in the renal cortex and is induced by hypoxia. [15] In renal transplant recipients, urinary L-FABP has been noted to strongly correlate with ischemic time and has thus been proposed to be a marker of renal hypoxia. [15]

Several studies have shown that L-FABP can predict a patient’s susceptibility to AKI and determine the implications of the injury. For example, a study in critically ill patients with early AKI found that L-FABP improved the prediction model for AKI progression, dialysis, and death within 7 days. [33]

In a meta-analysis of seven prospective cohort studies, L-FABP was shown to detect AKI and predict the need for renal replacement therapy and in-hospital mortality in hospital-based cohorts of patients at risk of AKI. [34] In a more recent systematic review of 1700 patients the AUC of L-FABP for predicting AKI was 0.72. [21] Further validation of its potential value is needed in large cohort studies.

Urinary Insulinlike Growth Factor–Binding Protein 7 and Tissue Inhibitor of Metalloproteinases–2

After sepsis or ischemic injury, renal tubular cells enter a brief period of cell cycle arrest, a key mechanism implicated in acute kidney injury. [35] In the Sapphire validation study of more than 700 critically ill patients identified in multicenter cohorts, insulinlike growth factor–binding protein 7 (IGFBP-7) and tissue inhibitor of metalloproteinases–2 (TIMP-2), both of which are inducers of G1 cell cycle arrest, have been shown to be predictive of AKI, with areas under the curve (AUCs) of 0.76 and 0.79, respectively. Taken together, urinary TIMP-2 and IGFBP-7 levels were significantly superior to all previously described markers of AKI (P <0.002), none of which achieved an AUC greater than 0.72. [36]

In the long term follow up of the SAPPHIRE validation study, the authors showed that [TIMP-2] x [IGFBP7] levels at the time of ICU admission were predictive of a composite of death or RRT during the next 9 months in patients who developed AKI. [37]

In another study of 50 patients at high risk for AKI who were undergoing cardiac surgery with cardiopulmonary bypass, urinary TIMP-2 and IGFBP7 was shown to be a sensitive and specific biomarker to predict AKI early after surgery and to predict renal recovery. [38]

In September 2014, the US Food and Drug Administration (FDA) allowed the marketing of a commercial test, NephroCheck, which detects the presence of IGFBP-7 and TIMP-2, to help determine whether certain critically ill hospitalized patients are at risk of developing moderate to severe acute AKI in the 12 hours after testing. [39]

Calprotectin

Calprotectin is a 24-kDa heterodimer, present intracelullarly associated with cytoskeleton. It is secreted by immune cells as a danger-associated molecular pattern protein. [40]

In a study of nephron sparing surgery for kidney tumors with transient clamping of the renal artery, calprotectin levels rose significantly within 2 h and reached a peak in 48 h, remaining elevated for 5 days post-op. [41]

Several studies have shown a promising role for calprotectin in differentiating pre-renal from intrinsic AKI. A recent multicenter study revealed an AUC of 0.94 differentiating patients from prerenal allograft AKI vs intrinsic AKI. [42]

Calprotectin levels need to be interpreted with caution as it is increased in several other conditions e.g. rheumatoid arthritis, inflammatory bowel disease, prostate cancer, etc. Calprotectin role in AKI is as yet incompletedly defined. [43]

 

Urine Angiotensinogen

AGT is 453 amino acid long protein cleaved by renin to form angiotensin 1. In recent studies increase in urine AGT has been shown as a promising biomarker for AKI progression in acute decompensated heart failure. [44, 45] Chen et al studies AGT along with other biomarkers like NGAL, KIM-1 and IL-18 and AGT outperformed the other biomarkers with an AUC 0.78 for AKI progression and 0.85 for progression with death.

Further studies for the role of AGT are awaited.

Urine microRNA

microRNA are endogenous noncoding RNA molecules containing 18-22 nucleotides. Recently there is increaing interest in their role in predicting AKI. Urine and serum miR-21 can predict AKI progression with an AUC of 0.81-0.83. [46] Various other sets of microRNA have been evaluated and their levels were found to be altered several days before serum creatinine increase, thus suggesting a role in predicting AKI in ICU patients. [47] Other micro RNA have been demonstrated to be an independent predictor of mortality in AKI patients requiring RRT. [48]

 

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Biomarkers of Chronic Kidney Disease

Serum creatinine and albuminuria form the core of most predictive models of CKD and risk of progression. However both these biomarkers show alterations relatively late in the disease trajectory and thus are suitable for very early diagnosis of CKD. [49]

Biomarkers of chronic kidney disease (CKD) include the following:

  • Cystatin C
  • β-trace protein (BTP)
  • Neutrophil gelatinase-associated lipocalin (NGAL)
  • Kidney injury molecule 1 (KIM-1)
  • Liver-type fatty acid–binding protein (L-FABP)
  • Asymmetric dimethylarginine (ADMA)
  • Uromodulin
  • micro RNA

Cystatin C

Cystatin C is a 13-kD cysteine protease inhibitor that has gained popularity in the measurement of renal function and determination of the estimated glomerular filtration rate (eGFR). As with serum creatinine, higher cystatin C levels have been associated with male sex, greater height and weight, and higher lean body mass. [50]

However, evidence supports the addition of cystatin C measurements to creatinine measurements in calculating the eGFR as a confirmatory test for CKD. The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) reported that using both creatinine and cystatin C to calculate the eGFR provided greater precision and accuracy and resulted in more accurate classification of measured GFR at less than 60 ml/min/1.73 m2 — the threshold for the diagnosis of CKD. [51]

Shlipak et al reported that adding cystatin C levels in calculations of eGFR in patients with CKD provides a more accurate estimate of the risk of death from any cause, and to a lesser extent the risks of death from cardiovascular causes and end-stage renal disease (ESRD). [52]

Most notably, when eGFR is calculated using both creatinine and cystatin C, these investigators found a consistent linear association with increased risks of death from any cause and from cardiovascular causes for all eGFR levels below approximately 85 ml/min/1.73 m2, which is well above the threshold of 60 ml/min/1.73 m2 for the detection of CKD with a creatinine-based eGFR. [52]

Equations combining cystatin C and serum creatinine perform better than either measurement alone in diagnosis of CKD. Combining serum creatinine, cystatin C, and urine albumin-to-creatinine ratio also improves risk stratification and assessment of CKD progression and mortality. [53]

β-Trace Protein

BTP, also known as lipocalin prostaglandin D2 synthase, is a lipocalin glycoprotein that has been studied in the evaluation of kidney function. In a report from the mild to moderate kidney disease (MMKD) study group, BTP provided reliable risk prediction for CKD progression. [54] In a study of more than 800 African Americans with hypertensive CKD, higher BPT level was strongly associated with progression to ESRD, compared with traditional markers of kidney function such as measured GFR. [55] Although promising, BTP requires validation in large CKD populations.

Inker et al studied BTP in a pooled database of 3 populations with CKD and concluded that BTP is less influenced by age, sex, and race than creatinine and is less influenced by race than cystatin C. However, BTP provides less accurate GFR estimates than the CKD-EPI creatinine and cystatin C equations. [56]

Neutrophil Gelatinase-associated Lipocalin

In addition to its potential role in the diagnosis of AKI, NGAL may also be a useful biomarker in patients with CKD. Studies in a strain of mice that develop severe renal lesions upon nephron reduction have shown NGAL to be an active player in kidney disease progression. [57]

In a cross-sectional study of 80 non-diabetic patients with CKD stages 2–4, serum NGAL was found to be elevated in those with the most advanced CKD.{ref 29} Moreover, elevation of urinary and serum NGAL levels has been noted in a wide range of kidney diseases, including IgA nephropathy, autosomal polycystic kidney disease, and diabetic nephropathy. [58, 59]

NGAL was shown to be a promising biomarker for identifying CKD of uncertain etiology, which is becoming epidemic in agricultural communities in Sri Lanka. [60]

Kidney Injury Molecule 1

When KIM-1 is expressed in renal epithelial cells of mice, these animals develop spontaneous and progressive interstitial kidney inflammation with fibrosis leading to renal failure with anemia, proteinuria, hyperphosphatemia, hypertension, cardiac hypertrophy, and death—findings that are comparable to progressive kidney disease in humans. [61] Consequently, sustained KIM-1 expression has been proposed to promote kidney fibrosis and provide a mechanistic link between acute and recurrent injury with progressive CKD.

In a retrospective study of patients with non-diabetic proteinuric kidney disease, KIM-1 levels in urine were found to be elevated, but subsequently decreased when patients received treatment with angiotensin-converting enzyme inhibitors or a low-sodium diet. Those findings suggest a potential role for KIM-1 as a measure of therapeutic efficacy. [62]

In a study of patients with type 1 diabetes and proteinuria, serum KIM-1 level at baseline strongly predicted the rate of estimated GFR loss and risk of ESRD during 5-15 years of follow-up, identifying KIM-1 as a marker for CKD and predictor of CKD progression. [63]

Like urinary NGAL, KIM-1 has shown some promise as a potential biomarker in identification of CKD of uncertain etiology in agricultural communities in Sri Lanka. [60]

Liver-type Fatty Acid–Binding Protein

In a study of 50 patients with CKD, the level of L-FABP in urine correlated with the degree of tubulointerstitial damage and urinary protein excretion. [64] In a prospective study, urinary L-FABP was found to be more sensitive than proteinuria in predicting the progression of CKD. [65] In 165 patients without albuminuria from a cohort of 227 patients with type 1 diabetes, baseline urinary L-FABP levels predicted the development of micro- and macro-albuminuria, suggesting a potential role in distinguishing patients who may benefit from early preventive therapies. [66]

Asymmetric Dimethylarginine (ADMA)

ADMA is an endogenously generated methylated arginine that reversibly inhibits nitric oxide synthase. If present in increased quantities, ADMA results in decreased nitric oxide production, which in turn is associated with endothelial dysfunction and kidney damage. [67]

ADMA has been investigated as a biomarker in CKD and its progression. In a study of early CKD in type 2 diabetics, Hanai et al found that increased plasma levels of ADMA were predictive of the development and progression of nephropathy. [68] A prospective study by Ravani et al in patients with CKD found that plasma ADMA levels were inversely correlated with GFR and predicted progression to ESRD. [69] ADMA seems to be relevant biomarker for CKD. [53]

Uromodulin

Uromodulin (also known as the Tamm–Horsfall protein) is a glycoprotein produced in the tubular cells of the thick ascending limb and the early distal tubule and released into the tubular lumen. Patients with CKD characterized by interstitial fibrosis and tubular atrophy have lower levels of uromodulin. Thus, uromodulin may represent intact renal mass rather than renal function. [70]

Steubl et al showed that plasma concentration of uromodulin was a robust marker for intact renal mass and allowed for identification of early stages of CKD. [71]

microRNA

There is increasing interest in the role of microRNA in the pathogenesis and progression of CKD. MicroRNA may be a marker of impaired filtration (as microRNAs are cleared renally) and also indicate tubular function (levels change with tubular dysfunction). [72]

Khurana et al analyzed several other noncoding RNA classes, such as transfer RNAs (tRNAs), tRNA fragments (tRFs), mitochondrial tRNAs, or long intergenic noncoding RNAs (lincRNAs), and identified nearly 30 differentially expressed noncoding RNAs in CKD patients as suitable biomarkers for early diagnosis. Of these, miRNA-181a appeared to be the most robust biomarker for CKD. [73]

 

 

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Biomarkers of Nephrotoxicity

Novel biomarkers of nephrotoxicity include the following:

  • N-acetyl-glucosaminidase (NAG)
  • Glutathione-s-transferase (GST)
  • Gamma-glutamyl transpeptidase (GGT)
  • Alanine aminopeptidase (AAP)
  • Lactate dehydrogenase (LDH)
  • Kidney injury molecule 1 (KIM-1)

N-acetyl-glucosaminidase (NAG)

NAG, which is an enzyme found predominantly in lysosomes of the proximal tubular cells, has reemerged as an important biomarker in recent studies. [74] Multiple experiments have shown that NAG is a sensitive marker of acute ischemic and oxidative stress within the kidney. For example, elevations in urinary concentration of NAG have been demonstrated in mice exposed to gentamicin [75] and in rats exposed to cisplatin [76] or lithium [77] ; in the lithium study, antioxidant treatment attenuated the nephrotoxicity. [77]

Glutathione-s-transferase (GST)

GST represents a family of cytosolic, microsomal, and membrane-bound enzymes. The GST alpha isoform is localized in the proximal tubular cells, whereas the pi isoform is confined to distal tubular cells. Increased levels of GST in the urine after nephrotoxic injury are attributed to leakage from the tubular epithelial cells into the tubular lumen secondary to cell damage. [78]

In two strains of rats, GST alpha exhibited its biomarker potential by detecting the presence of proximal tubular necrosis as early as 48 hours after cisplatin-induced injury. [79] In patients with rheumatoid arthritis, acute tubular injury from methotrexate and disease-modifying anti-rheumatic drugs was excluded by normal activity of GST alpha. [80] KIM-1 and GST alpha proved the most sensitive means of predicting polymyxin-induced nephrotoxicity, outperforming conventional biomarkers such as serum creatinine and blood urea nitrogen. [81]

Gamma-glutamyl Transpeptidase (GGT), Alanine Aminopeptidase (AAP) and Lactate Dehydrogenase (LDH)

GGT, AAP, and LDH are brush border enzymes that are present in the proximal renal tubule and are normally present in urine as a consequence of tubular cell shedding. Following gentamicin treatment in rats, increased levels of AAP and GGT were noted at all time points tested: days 4, 7, 10, and 14. The results suggest that increased levels of AAP and GGT in urine reflect loss of brush border integrity while an increased urinary NAG level is consistent with the autophagic response of the kidney to acute injury. [82]

Vancomycin-induced acute tubular necrosis in rats was associated with dose-dependent renal injury by pathological assessment and increased urinary excretion of AAP, GGT, and LDH. However, of these, LDH was the most sensitive indicator of acute kidney injury (AKI) and correlated most closely with the extent of acute tubular injury. [83]

Kidney Injury Molecule 1

In a dose-response study in rats, KIM-1 and the KIM-1/hepatitis A viral cellular receptor-1 (KIM-1/Havcr1) were found to be more sensitive markers of AKI from nephrotoxic chemicals and drugs than were serum urea and creatinine concentrations. In a time course study, urinary KIM-1 was elevated within 24 hours after exposure to gentamicin, mercury, and chromium and remained elevated through 72 hours. [84] In cases where acute drug exposure caused necrosis of around half of all proximal tubules, urinary KIM-1 levels increased but serum urea and creatinine and urinary NAG activity did not differ from controls, indicating that these were too insensitive to detect tubular injury.{ref 45}

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Biomarkers of Glomerular Diseases

Novel biomarkers may have a role in the following glomerular disorders:

  • IgA nephropathy (IgAN)
  • Membranous nephropathy (MN)
  • Focal segmental glomerulosclerosis (FSGS)
  • Lupus nephritis (LN)

IgA Nephropathy

Serum levels of galactose-deficient (Gd)-IgA1 and glycan-specific antibodies directed against the hinge region of Gd-IgA1 represent the most promising candidate biomarkers for IgA nephropathy. [85] These immune complexes deposit in the glomerular mesangium and induce the mesangioproliferative glomerulonephritis characteristic of IgA nephropathy.

A lectin-based enzyme-linked immunosorbent assay (ELISA) for circulating Gd-IgA1 demonstrates 90% specificity and 76% sensitivity in the diagnosis of IgA nephropathy and thus appears to be one of the best candidates for a new, noninvasive biomarker. [85] In a study from China, higher levels of Gd-IgA1 were shown to be independently associated with a greater risk of deterioration in renal function and thus with a poor prognosis in IgA nephropathy. [86] These observations are promising, but require replication in independent cohorts.

The combination of serum creatinine and normalized fractional excretion of IgG can be used to stratify patients with  IgA nephropathy into high and low risk for progression. [87] The presence of granule membrane protein of 17 kDa (GMP-17)–positive T-lymphocytes has proved predictive of progression of IgA nephropathy. [88]

Torres et al reported that the ratio of epidermal growth factor (EGF) to monocyte chemotactic peptide-1 (MCP-1) in the urine can be used to predict renal prognosis in IgA nephropathy. Patients were divided into tertiles based on the ratio of EGF to MCP-1. Patients in the lowest tertile had a significant decline in renal function while those in the highest tertile had a 100% renal survival at 48 and 84 months of follow-up. [89]

Membranous Nephropathy

In 2009, Beck and colleagues identified the M-type phospholipase A2 receptor (PLA2R), a transmembrane receptor that is highly expressed in glomerular podocytes, as a target podocyte antigen that triggers an antibody response in membranous nephropathy.  In this study, the levels of anti-PLA2R antibodies, primarily of the IgG4 subclass, were elevated in approximately 60% to 70% of patients with primary membranous nephropathy, and a clear correlation between the antibody titers, clinical disease activity, and response to treatment was  demonstrated. In contrast, PLA2R antibodies were not found in serum from patients with secondary membranous nephropathy due to lupus or hepatitis B, from those with proteinuric conditions other than membranous nephropathy, or from healthy controls. [90]

Seropositivity develops after the development of clinical membranous nephropathy., and spontaneous or treatment-induced decline or disappearance of circulating anti-PLA2R antibodies precedes clinical remission by months. In kidney transplant recipients, anti-PLA2R antibodies could be used to diagnose relapsing membranous nephropathy. [91] Treatment with rituximab has been shown to reduce the antibody titers as well as proteinuria. [92]

A second podocyte antigen, the thrombospondin type 1 domain-containing 7A (THSD7A) has gained prominence in recent years. THSD7A is also deposited in the subepithelial region and it accounts for up to 5% of cases of idiopathic membranous nephropathy. in Western countries. [93]

Both PLA2R and THSD7A are now being used to guide diagnosis and treatment of membranous nephropathy. [94]

Focal Segmental Glomerulosclerosis (FSGS)

Support for the role of soluble urokinase receptor (suPAR) in primary FSGS was provided by the selective expression suPAR in mice. [95] Mice exposed to certain forms of suPAR developed foot process effacement, proteinuria, and FSGS-like glomerulopathy. Experimental data indicate that suPAR acts through binding to the podocyte β3 integrin, one of the principal proteins that anchor podocytes to the glomerular basement membrane. The interaction of suPAR and β3 integrin produces structural changes in podocytes, altering the permeability of the glomerular filtration membrane. [96]

In addition, plasmapheresis, which is commonly used to treat recurrent FSGS following kidney transplantation, has been found to induce remission and decrease both serum suPAR levels and beta3 integrin activity in a subset of patients with recurrent FSGS.

Unfortunately, subsequent studies cast doubts on the usefulness of serum suPAR as a diagnostic biomarker for FSGS and its ability to distinguish FSGS from other nephrotic syndrome (eg, minimal change disease) or primary FSGS from secondary FSGS. [96, 97, 98, 99, 100]

Lupus Nephritis

Avihingsanon and colleagues reported that measurement of urinary messenger RNA (mRNA) levels of chemokine and growth factor genes could identify active class IV LN more accurately than the current available clinical markers, and could be used to follow response to therapy. In their evaluation of urinary mRNA levels of interferon gamma–induced protein 10 (IP-10), its receptor (CXCR3), transforming growth factor–β (TGF-β), and vascular endothelial growth factor (VEGF), the receiver operating characteristic (ROC) curve analysis showed that IP-10 had the best discriminative power, with an area under the curve of 0.89. [101]

β1-integrin has also been studied as a highly specific marker for class IV LN. Initial studies have shown some correlation. [102]

Several other biomarkers have been studied for LN, including the following:

  • Urinary FOXP3 mRNA
  • Urinary tumor necrosis factor–like weak inducer of apoptosis (TWEAK)
  • Urinary neutrophil gelatinase-associated lipocalin (NGAL)
  • Urinary MCP-1
  • Interleukin-6 (IL-6)
  • Vascular cell adhesion molecule 1 (VCAM-1)
  • Osteoprotegerin

Of those, urinary NGAL seems to be most promising. However, multiple biomarker panels may have better predictive value for LN than measurement of individual biomarkers. [103, 104]

 

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Biomarkers of ADPKD

The identification of biomarkers for autosomal dominant polycystic kidney disease (ADPKD) is still in its infancy. In an exploratory study in 2009, Kistler et al identified 197 proteins and peptides whose urinary excretion was significantly different in ADPKD subjects than in healthy controls and patients with other renal diseases. [105] Using those findings, they derived a score that could differentiate patients with ADPKD with good sensitivity and specificity; this score was subsequently validated in another cohort.

In a more recent study, Hogan et al examined differential protein abundance on urinary exosome-like vesicles (ELVs) from subjects with ADPKD compared with controls. Among more than 2000 ELV proteins studied, eight showed reduced levels in ADPKD patients (among them, polycystin-1 and polycystin-2), whereas levels of one, transmembrane protein 2 [TMEM2]), were consistently increased. These authors also determined that the ratio of polycystin-1 (PC1) or polycystin-2 (PC2) to MEM2 may have utility in diagnosis and monitoring of polycystic kidney disease. [106]

The following proteins are also under investigation [107, 108] :

  • Fetuin-A
  • Plasma copeptin
  • Secreted frizzled-related protein 4 [109]

 

 

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Conclusion

Biomarker discovery in nephrology is increasingly being revitalized, as advances in the fields of proteomics, genomics, and metabolomics improve the ability of researchers to study various proteins, and these techniques become widely available. Given the significant limitations of current established biomarkers (eg, creatinine and urinary albumin), the results from biomarker discovery studies are much anticipated.

 

 

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