Serum and urinary biomarkers of vancomycin-induced acute kidney injury: A prospective, observational analysis.

Vancomycin, a first-line drug for treating methicillin-resistant Staphylococcus aureus infections, is associated with acute kidney injury (AKI). This study involved an evaluation of biomarkers for AKI detection and their comparison with traditional serum creatinine (SCr). We prospectively enrolled patients scheduled to receive intravenous vancomycin for methicillin-resistant S aureus infection. Blood samples for pharmacokinetic assessment and SCr and cystatin C (CysC) measurements were collected at baseline and on days 3, 7, and 10 from the initiation of vancomycin administration (day 1). Urinary biomarkers, including kidney injury molecule 1 (KIM-1), neutrophil gelatinase-associated lipocalin, and clusterin, were collected from days 1 to 7 and adjusted for urinary creatinine levels. The estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation. Of the 42 patients, 6 experienced vancomycin-induced AKI. On day 7, the change from baseline eGFR using CysC (ΔeGFRCysC) showed a stronger correlation with vancomycin area under the curve (r = -0.634, P < .001) than that using SCr (ΔeGFRSCr; r = -0.437, P = .020). ΔeGFRSCr showed no significant correlation with vancomycin pharmacokinetic in patients with body mass index ≥23. The median (interquartile range) level of KIM-1 (µg/mg) was significantly higher in the AKI group (0.006 [0.005-0.008]) than in the non-AKI group (0.004 [0.001-0.005]) (P = .039, Mann-Whitney U test), with area under the receiver operating characteristic curve (95% confidence interval) of 0.788 (0.587-0.990). Serum CysC, particularly in overweight individuals or those with obesity, along with urinary KIM-1 are important predictors of vancomycin-induced AKI. These results may aid in selecting better biomarkers than traditional SCr for detecting vancomycin-induced AKI.

Evaluation of Novel Urinary Biomarkers in Beagle Dogs With Amphotericin B-Induced Kidney Injury.

Next-generation urinary protein biomarkers have been qualified to enable monitoring for drug-induced kidney injury in toxicology studies conducted in rats. However, there is limited literature on the utility of these biomarkers in dogs. To add to the existing body of knowledge on the utility of the next-generation drug-induced kidney injury (DIKI) biomarkers, we evaluated the value of these biomarkers for the early detection of DIKI in Beagle dogs using a differentiated nephrotoxicant, Amphotericin B (AmpB). In dogs with AmpB-induced kidney injury, we monitored the response of urinary albumin, total protein, clusterin, kidney injury molecule 1, neutrophil gelatinase-associated lipocalin and N-acetyl-beta-D-glucosaminidase. We also measured blood urea nitrogen, serum creatinine and cystatin C. The results showed that urinary clusterin (up to ∼ 112x) was much more sensitive to AmpB-induced kidney injury relative to other biomarkers. Moreover, other than urinary clusterin and to a much lesser extent urinary albumin and total protein, none of the other biomarkers analyzed in this study were more sensitive than blood urea nitrogen and serum creatinine. The AmpB related tubular alterations were characterized by minimal to mild, multifocal necrosis, degeneration, regeneration, dilatation and mineralization. The mild nature of these histopathologic findings further attested to the sensitivity of urinary clusterin to AmpB-induced kidney injury in dogs. These results will help drug developers make informed decisions when selecting urinary biomarkers for monitoring DIKI in dogs for toxicology studies.

Development of Parallel Reaction Monitoring Assays for the Detection of Aggressive Prostate Cancer Using Urinary Glycoproteins.

Recently, we have found that two urinary glycoproteins, prostatic acid phosphatase (ACPP) and clusterin (CLU), combined with serum prostate-specific antigen (PSA) can serve as a three-signature panel for detecting aggressive prostate cancer (PCa) based on a quantitative glycoproteomic study. To facilitate the translation of candidates into clinically applicable tests, robust and accurate targeted parallel reaction monitoring (PRM) assays that can be widely adopted in multiple labs were developed in this study. The developed PRM assays for the urinary glycopeptides, FLN*ESYK from ACPP and EDALN*ETR from CLU, demonstrated good repeatability and a sufficient working range covering three to four orders of magnitude, and their performance in differentiating aggressive PCa was assessed by the quantitative analysis of urine specimens collected from 69 nonaggressive (Gleason score = 6) and 73 aggressive (Gleason ≥ 8) PCa patients. When ACPP combined with CLU, the discrimination power was improved from an area under a curve (AUC) of 0.66 to 0.78. By combining ACPP, CLU, and serum PSA to form a three-signature panel, the AUC was further improved to 0.83 (sensitivity: 84.9%, specificity: 66.7%). Since the serum PSA test alone had an AUC of 0.68, our results demonstrated that the new urinary glycopeptide PRM assays can serve as an adjunct to the serum PSA test to achieve better predictive power toward aggressive PCa. In summary, our developed PRM assays for urinary glycopeptides were successfully applied to clinical PCa urine samples with a promising performance in aggressive PCa detection.

Netrin-1 and clusterin: Innovative potential diagnostic biomarkers for early renal damage in ß-thalassemia major children.

BACKGROUND: Children with ß-thalassemia major (ß-TM) suffer from tubular dysfunction even before the onset of any renal impairment symptoms and/or clinical signs. Therefore, identifying innovative biomarkers allowing early renal damage detection has focused attention. AIM: This study aims to preliminary assess Netrin-1(NTN-1) and clusterin (CLU) in ß-TM children and explore their possible roles as surrogate noninvasive biomarkers of renal tubular dysfunction. SUBJECTS AND METHODS: In this study, 40 ß-TM children and 30 healthy children were enrolled. Routine serum and urinary biochemical variables were determined. Urinary NTN-1 and CLU levels were measured using ELISA and their mRNA expression in PBMCs were assayed using real-time PCR. Serum TNF-α, MDA levels and GST activity were measured. RESULTS: Urinary NTN-1 and CLU concentrations and mRNA relative expression levels in PBMCs were significantly increased in ß-TM children relative to controls. Oxidative stress and inflammatory markers revealed significant elevation in ß-TM children compared to controls. The change in these parameters correlated significantly with other renal parameters. ROC curves analysis showed that urinary NTN-1 and CLU levels are of promising diagnostic performance. CONCLUSION: Our results suggest that NTN-1 and CLU are qualified as new noninvasive biomarker panels for early detection of renal injury in ß-TM children. Moreover, urinary NTN-1 is recommended as a precise one during the clinical practices.

Urinary clusterin and cystatin B as biomarkers of tubular injury in dogs following envenomation by the European adder.

Diagnosing acute kidney injury remains a challenge since the established renal biomarkers, serum creatinine (sCr) and symmetric dimethylarginine (SDMA) reflect glomerular function and not tubular injury. Sensitive tubular markers such as urinary clusterin (uClust) and cystatin B (uCysB) have been proposed to detect AKI at an earlier stage. Since envenomation by the European adder (Vipera berus berus) could serve as a spontaneous disease model of AKI we investigated these new biomarkers in affected dogs. Concentrations of uClust and uCysB as well as sCr and SDMA were analyzed retrospectively in stored samples from 26 dogs with snake envenomation and 13 healthy controls. Higher concentrations of uClust (P < 0.012) and uCysB (P < 0.001) were observed in the snake-envenomed group. Normalization of uClust and uCysB to urinary creatinine did not alter the results. No differences were observed in sCr and SDMA between the snake-envenomed group and the healthy control group. Spearman rank correlation analysis revealed a strong association of uClust with uCysB in the snake-envenomed dogs (r = 0.75 P < 0.001) but not in the healthy controls. The high percentage of snake-envenomed dogs with increased uClust and uCysB concentrations in the absence of increased sCr and SDMA suggests renal tubular injury in the affected dogs. Larger prospective case-controlled studies are warranted to evaluate the clinical utility and prognostic value of these biomarkers.

Chronic treatment with the (iso-)glutaminyl cyclase inhibitor PQ529 is a novel and effective approach for glomerulonephritis in chronic kidney disease.

Glomeruli and renal tubule injury in chronic kidney disease (CKD) is reported to involve induction of macrophage activation through the CCL2/CCR2 axis. The effects of inhibitors of the CCL2/CCR2 axis, such as anti-CCL2 antibody and CCR2 antagonist, on kidney function in animal models or humans with kidney dysfunction have been demonstrated. The N-terminal glutamine on immature CCL2 is replaced with pyroglutamate (pE) by glutaminyl cyclase (QC) and isoQC. pE-CCL2 is stable and resistant to peptidases. We hypothesized that inhibiting QC/isoQC activity would lead to the degradation of CCL2, thereby ameliorating CKD and reducing kidney inflammation. To test this hypothesis, we investigated the renoprotective properties of the QC/isoQC inhibitor PQ529 in anti-glomerular basement membrane (GBM) antibody-induced glomerulonephritis Wistar Kyoto (WKY) rats. Three-week repeated administration of PQ529 (30 and 100 mg/kg, twice daily) significantly reduced the serum and urine CCL2 and urinary protein excretion in a dose-dependent manner. Correlations between the urinary protein level and serum or urinary CCL2 levels were confirmed in tested animals. Repeated administration of PQ529 significantly reduced the expression of CD68, a macrophage marker, in the kidney cortex and mononuclear infiltration into the tubulointerstitium. In addition, decreased levels of urinary KIM-1, ß2 microglobulin, and clusterin were detected, suggesting the inhibition of inflammation in both the proximal and distal tubules. These results suggest that PQ529 suppresses the progression of inflammation-induced renal dysfunction by inhibiting the CCL2/CCR2 axis. Inhibition of QC/isoQC may thus be a viable alternative therapeutic approach for treating glomerulonephritis and CKD patients.

Urine kidney safety biomarkers improve understanding of indirect intra-renal injury potential in dogs with a drug-induced prerenal azotemia.

Drug-induced kidney injury (DIKI) is a frequent occurrence in nonclinical drug development. It is well established that novel urine kidney safety biomarkers will outperform urea nitrogen (BUN) and serum creatinine (sCr) for monitoring direct drug injury to the kidney across numerous compounds spanning diverse mechanisms and efforts are underway for a formal regulatory clinical qualification. However, it remains unclear how these novel biomarkers will perform under prerenal azotemia when BUN and sCr are elevated but no intra-renal injury is suspected. This lack of knowledge is largely due to the dearth of such nonclinical animal models. We report here that treatment of dogs with a potent antihypertensive compound MK-5478 at a suprapharmacologic dose for up to 9 days results in the development of prerenal azotemia and, in some dogs, kidney toxicity through the dual sustained effects of MK-5478 as a nitric oxide donor and an angiotensin II receptor blocker (ARB). While conventional serum biomarkers BUN, and often sCr as well, were highly elevated in these dogs with or without kidney damage, urine kidney biomarkers clusterin (CLU) and neutrophil gelatinase-associated lipocalin (NGAL) showed increases only in dogs with kidney histopathologic changes following the sustained period of prerenal azotemia. Urine albumin (ALB) and total protein also tracked with kidney lesions but with less sensitivity. Thus, we present evidence for the first time that urine kidney safety biomarkers used together with BUN and sCr can distinguish intra-renal injury among dogs with prerenal azotemia while the conventional serum biomarkers alone are ambiguous, either being interpreted as false positives of kidney injury, or dismissed under circumstances as benign without appreciation for a threshold of impending injury.

Measurements of echocardiographic indices and biomarkers of kidney injury in dogs with chronic kidney disease.

Pathophysiological cardiac and renal interactions are termed cardiovascular-renal disorder (CvRD). Cardiovascular disease/dysfunction secondary to kidney disease (CvRDK), is a leading cause of death in human chronic kidney disease (CKD) patients. The presence and clinical impact of CvRDK in dogs with CKD is unknown. We hypothesized that echocardiographic measurements, and cardiac and renal biomarkers, will be altered in dogs with CKD and associated with survival. Eleven dogs with CKD (n = 6 IRIS stage 2, n = 5 IRIS stage 3) and without primary cardiac disease, plus 12 healthy age-matched control dogs, were recruited to this prospective observational study. Dogs underwent standard echocardiography, glomerular filtration rate (GFR) estimation by iohexol clearance, and measurement of plasma cardiac troponin I and N-terminal pro-B-type natriuretic peptide (NT-proBNP), plasma and urinary cystatin B, and urinary clusterin and neutrophil gelatinase-associated lipocalin (NGAL). Values were compared between groups, and their association with all-cause mortality explored. Dogs with CKD had significantly lower GFR and higher NT-proBNP, urinary cystatin B, clusterin, and NGAL, compared to controls (P < 0.05). Echocardiographic measurements were similar between dogs with CKD and controls. Median follow-up time was 666 days, during which six dogs with CKD died. Risk of death was associated with increasing age, serum total protein, and normalized left ventricular posterior wall thickness (LVPWDN) and decreasing bodyweight and packed cell volume. Although baseline differences in echocardiographic measurements were not evident between dogs with moderate CKD and controls, the presence of CvRDK was suggested by the association between LVPWDN and survival.

Serial Quantification of Urinary Protein Biomarkers to Predict Drug-induced Acute Kidney Injury.

BACKGROUND: Drug-induced Acute Kidney Injury (AKI) develops in 10-15% of patients who receive nephrotoxic medications. Urinary biomarkers of renal tubular dysfunction may detect nephrotoxicity early and predict AKI. METHODS: We prospectively studied patients who received aminoglycosides, vancomycin, amphotericin, or calcineurin inhibitors, and collected their serial urine while on therapy. Patients who developed drug-induced AKI (fulfilling KDIGO criteria) were matched with non-AKI controls in a 1:2 ratio. Their urine samples were batch-analyzed at time-intervals leading up to AKI onset; the latter benchmarked against the final day of nephrotoxic therapy in non- AKI controls. Biomarkers examined include clusterin, beta-2-microglobulin, KIM1, MCP1, cystatin-C, trefoil-factor- 3, NGAL, interleukin-18, GST-Pi, calbindin, and osteopontin; biomarkers were normalized with corresponding urine creatinine. RESULTS: Nine of 84 (11%) patients developed drug-induced AKI. Biomarkers from 7 AKI cases with pre-AKI samples were compared with those from 14 non-AKI controls. Corresponding mean ages were 55(±17) and 52(±16) years; baseline eGFR were 99(±21) and 101(±24) mL/min/1.73m2 (all p=NS). Most biomarker levels peaked before the onset of AKI. Median levels of 5 biomarkers were significantly higher in AKI cases than controls at 1-3 days before AKI onset (all µg/mmol): clusterin [58(8-411) versus 7(3-17)], beta-2-microglobulin [1632(913-3823) versus 253(61-791)], KIM1 [0.16(0.13-0.76) versus 0.07(0.05-0.15)], MCP1 [0.40(0.16-1.90) versus 0.07(0.04-0.17)], and cystatin-C [33(27-2990) versus 11(7-19)], all p<0.05; their AUROC for AKI prediction were >0.80 (confidence intervals >0.50), with average accuracy highest for clusterin (86%), followed by beta-2-microglobulin, cystatin-C, MCP1, and KIM1 (57%) after cross-validation. CONCLUSION: Serial surveillance of these biomarkers could improve the lead time for nephrotoxicity detection by days.

Twenty-four hour pharmacokinetic relationships for intravenous vancomycin and novel urinary biomarkers of acute kidney injury in a rat model.

OBJECTIVES: To identify the pharmacokinetic (PK) and toxicodynamic (TD) relationship for vancomycin-induced kidney injury. METHODS: Male Sprague-Dawley rats received intravenous (iv) vancomycin. Doses ranging from 150 mg/kg/day to 400 mg/kg/day were administered as a single or twice-daily injection over 24 h (total protocol duration). Controls received iv saline. Plasma was sampled with up to eight samples in 24 h per rat. Twenty-four hour urine was collected and assayed for kidney injury molecule 1 (KIM-1), osteopontin and clusterin. Vancomycin in plasma was quantified via LC-MS/MS. PK analyses were conducted using Pmetrics for R. PK exposures during the first 24 h (i.e. AUC0-24h, Cmax 0-24h and Cmin 0-24h) were calculated. PK/TD relationships were assessed with Spearman's rank coefficient (rs) and the best-fit mathematical model. RESULTS: PK/TD data were generated from 45 vancomycin-treated and 5 control rats. A two-compartment model fit the data well (Bayesian: observed versus predicted R2 = 0.97). Exposure-response relationships were found between AUC0-24h versus KIM-1 and osteopontin (R2 = 0.61 and 0.66) and Cmax 0-24h versus KIM-1 and osteopontin (R2 = 0.50 and 0.56) using a four-parameter Hill fit. Conversely, Cmin 0-24h was less predictive of KIM-1 and osteopontin (R2 = 0.46 and 0.53). A vancomycin AUC0-24h of 482.2 corresponded to a 90% of maximal rise in KIM-1. CONCLUSIONS: Vancomycin-induced kidney injury as defined by urinary biomarkers is driven by vancomycin AUC or Cmax rather than Cmin. Further, an identified PK/TD target AUC0-24h of 482.2 mg·h/L may have direct relevance to human outcomes.

Biomarkers of Drug-Induced Kidney Toxicity.

Blood urea nitrogen and serum creatinine are imperfect markers of kidney function because they are influenced by many renal and nonrenal factors independent of kidney function. A biomarker that is released directly into the blood or urine by the kidney in response to injury may be a better early marker of drug-induced kidney toxicity than blood urea nitrogen and serum creatinine. Urine albumin and urine protein, as well as urinary markers kidney injury molecule-1 (KIM-1), ß2-microglobulin (B2M), cystatin C, clusterin, and trefoil factor-3 (TFF-3) have been accepted by the Food and Drug Administration and European Medicines Agency as highly sensitive and specific urinary biomarkers to monitor drug-induced kidney injury in preclinical studies and on a case-by-case basis in clinical trials. Other biomarkers of drug-induced kidney toxicity that have been detected in the urine of rodents or patients include IL-18, neutrophil gelatinase-associated lipocalin, netrin-1, liver-type fatty acid-binding protein (L-FABP), urinary exosomes, and TIMP2 (insulin-like growth factor-binding protein 7)/IGFBP7 (insulin-like growth factor-binding protein 7), also known as NephroCheck, the first Food and Drug Administration-approved biomarker testing platform to detect acute kidney injury in patients. In the future, a combined use of functional and damage markers may advance the field of biomarkers of drug-induced kidney toxicity. Earlier detection of drug-induced kidney toxicity with a kidney-specific biomarker may result in the avoidance of nephrotoxic agents in clinical studies and may allow for earlier intervention to repair damaged kidneys.

Urinary clusterin and glutathione-s-transferase levels in HIV positive normotensive and preeclamptic pregnancies.

OBJECTIVE: To determine the level and effect of urinary clusterin (CLU) and glutathione-s-transferase (GST) proteins in normotensive and preeclamptic pregnant women with HIV infection. METHODS: The urine concentration of CLU and GST in normotensive (n = 38) and preeclamptic pregnant (n = 38) women stratified by HIV status were estimated using the Bio-Plex® ProTM immunoassay. RESULTS: Across the group, a significant down-regulation of CLU (p = 0.039) with a reduced trend in GST was shown in HIV positive preeclampsia. CONCLUSION: HIV infection affects the activity of urinary CLU protein in HIV positive preeclampsia. However, the cytoprotective role of these proteins neutralizes the oxidative radicals associated with preeclampsia development through complement response in HIV infection.

Evaluation of kidney injury biomarkers in an adult Mexican population environmentally exposed to fluoride and low arsenic levels.

Fluoride (F) is a toxicant widely distributed in the environment. Experimental studies have shown kidney toxicity from F exposure. However, co-exposure to arsenic (As) has not been considered, and epidemiological information remains limited. We evaluated the association between F exposure and urinary kidney injury biomarkers and assessed As co-exposure interactions. A cross-sectional study was conducted in 239 adults (18-77 years old) from three communities in Chihuahua, Mexico. Exposure to F was assessed in urine and drinking water, and As in urine samples. We evaluated the urinary concentrations of albumin (ALB), cystatin-C (Cys-C), kidney injury molecule 1 (KIM-1), clusterin (CLU), osteopontin (OPN), and trefoil factor 3 (TFF-3). The estimated glomerular filtration rate (eGFR) was calculated using serum creatinine (Creat) levels. We observed a positive correlation between water and urine F concentrations (ρ = 0.7419, p < 0.0001), with median values of 1.5 mg/L and 2 µg/mL, respectively, suggesting that drinking water was the main source of F exposure. The geometric mean of urinary As was 18.55 ng/mL, approximately 39% of the urine samples had As concentrations above the human biomonitoring value (15 ng/mL). Multiple linear regression models demonstrated a positive association between urinary F and ALB (ß = 0.56, p < 0.001), Cys-C (ß = 0.022, p = 0.001), KIM-1 (ß = 0.048, p = 0.008), OPN (ß = 0.38, p = 0.041), and eGFR (ß = 0.49, p = 0.03); however, CLU (ß = 0.07, p = 0.100) and TFF-3 (ß = 1.14, p = 0.115) did not show significant associations. No interaction with As exposure was observed. In conclusion, F exposure was related to the urinary excretion of early kidney injury biomarkers, supporting the hypothesis of the nephrotoxic role of F exposure.

Urinary Clusterin Is Upregulated in Nephropathia Epidemica.

Kidney insufficiency is a hallmark of nephropathia epidemica (NE). Little is known about the mechanisms of the NE kidney pathology, with current knowledge mainly based on findings in postmortem tissue. We have analyzed kidney damage biomarkers in urine collected from early- and late-phase NE using Bio-Plex kidney toxicity panels 1 and 2. To determine the disease specificity, kidney damage biomarkers were also analyzed in urine samples from patients diagnosed with gout, type 2 diabetes, systemic lupus erythematosus, and chronic kidney insufficiency. Analysis of 12 biomarkers suggests damage to the kidney proximal tubule at the onset of NE. Also, upregulation of biomarkers of inflammation and leukocyte chemotaxis were detected in NE urine. Furthermore, increased clusterin levels were found in early- and late-phase NE urine. Comparative analysis revealed that clusterin is a biomarker, upregulated in NE urine.

Urinary clusterin-a novel urinary biomarker associated with pediatric lupus renal histopathologic features and renal survival.

BACKGROUND: Lupus nephritis (LN) is a major risk factor for systemic lupus erythematous (SLE)-related morbidity and mortality. With the aim of bypassing renal biopsy, we analyzed urinary biomarkers for their ability to predict renal histopathologic features and end-stage kidney disease (ESKD). METHODS: Urinary albumin, ß2-microglobulin (B2M), cystatin C, kidney injury molecule-1 (KIM-1), monocyte chemoattractant protein 1 (MCP-1), clusterin, calbindin, interleukin-18 (IL-18), neutrophil gelatinase-associated lipocalin (NGAL), trefoil factor 3 (TFF3), osteopontin, and glutathione S-transferase π (GST-π) levels were measured at time of renal biopsy. Renal histopathologies were carefully reviewed. RESULTS: Urine from 60 pediatric SLE cases with LN, 29 without and 22 healthy controls were collected. Median age at SLE diagnosis was 12.92 years (range = 4.27-17.30 years) and 10 cases progressed to ESKD during a period of 4.12 ± 2.17 years. Urinary albumin and clusterin were significantly elevated (p = 0.035 and 0.048, respectively) in patients with tubulointerstitial renal lesions. Urinary clusterin among all urinary markers, performed best at predicting ESKD with cutoff of 0.61 × 10-4 (AUC = 0.804; p = 0.002). Interestingly, elevation of urinary clusterin likely resulted from local over-expression in tubulointerstitial tissue since the level of serum clusterin was not concomitantly higher (p = 0.424). CONCLUSION: Urinary biomarkers are emerging as non-invasive indicators for lupus-related renal histopathology and renal outcome prediction in pediatric SLE patients. Urinary clusterin, a newly identified biomarker, is an indicator that shows an association with tubulointerstitial renal lesions and demonstrates the best ability to predict ESKD.

Perspectives on using a multiplex human kidney safety biomarker panel to detect cisplatin-induced tubular toxicity in male and female Cynomolgus monkeys.

Multiplex biomarker panel assays would enable early de-risking of discovery compound related kidney safety liabilities. The objective of this study was to evaluate the usefulness of the Myriad RBM Human KidneyMAP (Multi-Analyte Profile)® v.1.0 panel to detect experimental nephrotoxicity in Cynomolgus monkeys following a single intravenous administration of cisplatin (2.5mg/kg). Urine samples were collected at baseline on day -2; at approximately 4hr post-dose on day 1; and on days 4, 9, 15 and/or 20. Blood samples were collected at predose on day -2; at 4hr post-dose on day 1; and on days 2, 5, 10 and/or 21. Changes in toxicokinetic and biochemistry parameters in plasma, qualitative/quantitative urinalysis parameters, and urinary kidney safety biomarkers were assessed. Kidney tissues were collected on days 2, 5, 10 and 21 for routine microscopy. Cisplatin-induced tubular alterations were characterized by acute and progressive cortical tubular degeneration/necrosis, regeneration, tubular dilation and proteinaceous cast in the absence of statistically significant changes in traditional plasma biochemistry and urinalysis parameters. When normalized to urinary creatinine, cisplatin-induced significant increases in urinary levels of kidney injury molecule 1 (females on day 4), increases in calbindin D28k (males and females on day 4), decreases in Tamm-Horsfall glycoprotein (males on days 1, 4 and 9), and increases in clusterin (females and males on days 15 and 20, respectively), when compared to concurrent controls. This study revealed the usefulness of the Human KidneyMAP® multiplex panel when measuring changes in urine-based biomarkers to reliably detect cisplatin-induced acute/progressive cortical tubular injury in male and female Cynomolgus monkeys.

Usefulness of urinary biomarkers for nephrotoxicity in cynomolgus monkeys treated with gentamicin, cisplatin, and puromycin aminonucleoside.

The objective of this study was to investigate the availability of novel urinary biomarkers (BMs) such as total protein, albumin, ß2-microglobulin, clusterin, cystatin C, neutrophil gelatinase-associated lipocalin (NGAL) for the detection of acute nephrotoxicity in cynomolgus monkeys. Animals (total 9 males/3 groups) were administered gentamicin (GM) subcutaneously at 40 mg/kg for 7 days, cisplatin (CDDP) intravenously at 3 mg/kg once and puromycin aminonucleoside (PAN) intravenously at 20 mg/kg for 7 days. Two-hr urine on Days 0, 3, and 6, and 16-hr urine and blood on Days 1, 4, and 7 were collected. Novel urinary BMs and conventional clinical pathology parameters were evaluated in parallel to histopathological and electron microscopic examinations on the kidneys at termination. Urinary BMs and enzymes increased earlier than serum creatinine and blood urea nitrogen, particularly in 2-hr urine after dosing on Day 0, urinary albumin was increased in all groups and urinary NGAL with the highest magnitude of change rate among urinary BMs was observed in the GM and CDDP groups. Degeneration/necrosis and hyaline droplet of renal tubule, cellular cast and dilatation of renal tubule, and hypertrophy of podocytes were observed in the GEN, CDDP, and PAN groups, respectively. These results showed that the increases of urinary BMs reflected the agent-specific renal damages and these urinary BMs could be useful for the detection of segment-specific nephrotoxicity. Urinary albumin and NGAL are the most useful BMs to estimate glomerular and distal tubular damages, respectively, as well as proximal tubular damage in cynomolgus monkeys.

Performance of urinary neutrophil gelatinase-associated lipocalin, clusterin, and cystatin C in predicting diabetic kidney disease and diabetic microalbuminuria: a consecutive cohort study.

BACKGROUND: Tubular biomarkers have been regarded as emerging and promising markers for early diagnosis of diabetic kidney disease (DKD). The study was to determine the diagnostic capabilities of tubular biomarkers (urinary neutrophil gelatinase-associated lipocalin [NGAL], clusterin, and cystatin C) for DKD and diabetic microalbuminuria, and whether or not the tubular biomarkers appear earlier than microalbuminuria. METHODS: In this consecutive cohort study, 146 type 2 diabetes mellitus (T2DM) patients with a disease duration of ≥6 years were enrolled. Thirty age- and gender-matched subjects without any systemic diseases were recruited as the control group. Urinary samples collected before treatment were tested for NGAL, clusterin, and cystatin C. RESULTS: The levels of biomarkers were higher in patients with DKD (p < 0.001); and positively correlated with the urinary albumin creatinine ratio (UACR; p < 0.001). With respect to the diagnosis of DKD, the areas under the receiver operating characteristic curve (AUCs) for urinary NGAL, clusterin, and cystatin C were 0.816 (95% confidence interval [CI], 0.741-0.891), 0.775 (95% CI: 0.694-0.857), and 0.803 (95% CI: 0.722-0.884), respectively. The levels of urinary NGAL and cystatin C in the normoalbuminuria group (UACR <30 mg /g•Cr) were elevated compared with the control group, unlike urinary clusterin. There was no statistical difference in the levels of the three biomarkers between groups with different levels of haemoglobin A1C (HbA1c). The diagnostic AUCs for urinary NGAL, clusterin, and cystatin C in patients with diabetic microalbuminuria were 0.841 (95% CI: 0.775-0.907), 0.783(95% CI: 0.710-0.856), and 0.805 (95% CI: 0.733-0.877), respectively. CONCLUSIONS: Urinary NGAL, clusterin, and cystatin C may be promising biomarkers for diagnosing DKD and diabetic microalbuminuria. It is possible that urinary NGAL and cystatin C increase before the onset of microalbuminuria in T2DM patients.

Subclinical chronic kidney disease modifies the diagnosis of experimental acute kidney injury.

Extensive structural damage within the kidney must be present before serum creatinine increases. However, a subclinical phase of chronic kidney disease (CKD) usually goes undetected. Here we tested whether experimental subclinical CKD would modify functional and damage biomarker profiles of acute kidney injury (AKI). Subclinical CKD was induced in rats by adenine or aristolochic acid models but without increasing serum creatinine. After prolonged recovery (three to six weeks), AKI was induced with a subnephrotoxic dose of cisplatin. Urinary levels of kidney injury molecule-1 (KIM-1), cytochrome C, monocyte chemotactic protein-1 (MCP-1), clusterin, and interleukin-18 increased during CKD induction, without an increase in serum creatinine. After AKI in adenine-induced CKD, serum creatinine increased more rapidly, while increased urinary KIM-1, clusterin, and MCP-1 were delayed and reduced. Increased serum creatinine and biomarker excretion were associated with diffuse tubulointerstitial injury in the outer stripe of outer medulla coupled with over 50% cortical damage. Following AKI in aristolochic acid-induced CKD, increased serum creatinine, urinary KIM-1, clusterin, MCP-1, cytochrome C, and interleukin-18 concentrations and excretion were greater at day 21 than day 42 and inversely correlated with cortical injury. Subclinical CKD modified functional and damage biomarker profiles in diametrically opposite ways. Functional biomarker profiles were more sensitive, while damage biomarker diagnostic thresholds and increases were diminished and delayed. Damage biomarker concentrations and excretion were inversely linked to the extent of prior cortical damage. Thus, thresholds for AKI biomarkers may need to be lower or sampling delayed in the known presence of CKD.

Nephrotoxicity-induced proteinuria increases biomarker diagnostic thresholds in acute kidney injury.

BACKGROUND: Paraquat ingestion is frequently fatal. While biomarkers of kidney damage increase during paraquat-induced acute kidney injury (AKI), significant concurrent proteinuria may alter diagnostic thresholds for diagnosis and prognosis to an unknown extent. This study evaluated the effect of albuminuria on biomarker cutoffs for diagnosis and outcome prediction. METHODS: This was a multi-centre prospective clinical study of patients following acute paraquat self-poisoning in 5 Sri Lankan hospitals. Biomarker concentrations were quantified using ELISA and microbead assays and correlated with urinary albumin. Functional-AKI was defined by the Acute Kidney Injury Network serum creatinine definition and alternatively by a ≥50% increase in serum cystatin C. Albuminuria was defined as albumin-creatinine ratio >30 mg/g. The study outcomes were compared with a retrospective analysis of a pre-clinical study of paraquat-induced nephrotoxicity with appropriate controls. RESULTS: Albuminuria was detected in 34 of 50 patients, and increased with functional-AKI severity. The concentrations of uNGAL, uCysC, uClusterin, uß2M, and uKIM-1 were higher in albuminuric compared to non-albuminuric patients (p < 0.001). Albuminuria correlated with biomarker concentration (r > 0.6, p < 0.01) and was associated with death (p = 0.006). Optimal biomarker cutoffs for prediction of death were higher in the albuminuric group. Similar outcomes with more detailed analysis were obtained in experimental paraquat nephrotoxicity. CONCLUSION: Albuminuria was associated with paraquat-induced nephrotoxicity and increased excretion of low-molecular weight protein biomarkers. AKI biomarker cutoffs for diagnosis, outcome prediction and AKI stratification increased in the presence of albuminuria. This may lead to over-diagnosis of AKI in conditions independently associated with proteinuria.