Investigation of new biomarkers of kidney injury in renal transplant recipients undergoing graft biopsy.

AIM: Urinary and blood kidney biomarkers (BM) remain insufficient for early kidney injury detection. We aimed to compare new kidney BM with histopathological data in kidney allograft recipients. METHODS: Blood and urine samples were collected from consecutive adult patients just before graft biopsy. All kidney samples were classified according to the Banff 2007 classification. The diagnostic performance of 16 new BM was compared to those of urinary proteins, blood urea nitrogen, eGFR, and serum creatinine to identify histopathological groups. RESULTS: Two hundred and twenty-three patients were analyzed. Microalbuminuria and urinary proteins performed well to discriminate glomerular injury from slightly modified renal parenchyma (SMRP). Urinary neutrophil gelatinase-associated lipocalin (NGAL) had the best performance relative to SMRP (AUROC .93) for acute tubular necrosis (ATN) diagnosis. Other BM had a slightly lower AUROC (.89). For the comparison of ATN to acute rejection, several new urinary BM (NGAL, cystatin C, MCP1) and classical BM (eGFR, serum creatinine) gave similar AUROC values (from .80 to .85). Urinary NGAL values in patients with ATN were 10-time higher than those with acute rejection (P=.0004). CONCLUSION: The new BM did not outperform classical BM in the context of renal transplantation. Urinary NGAL may be useful for distinguishing between ATN and acute rejection.

Methodological considerations for measuring biofluid-based microRNA biomarkers.

MicroRNAs (miRNAs) are small non-coding RNA that regulate the expression of messenger RNA and are implicated in almost all cellular processes. Importantly, miRNAs can be released extracellularly and are stable in these matrices where they may serve as indicators of organ or cell-specific toxicity, disease, and biological status. There has thus been great enthusiasm for developing miRNAs as biomarkers of adverse outcomes for scientific, regulatory, and clinical purposes. Despite advances in measurement capabilities for miRNAs, miRNAs are still not routinely employed as noninvasive biomarkers. This is in part due to the lack of standard approaches for sample preparation and miRNA measurement and uncertainty in their biological interpretation. Members of the microRNA Biomarkers Workgroup within the Health and Environmental Sciences Institute's (HESI) Committee on Emerging Systems Toxicology for the Assessment of Risk (eSTAR) are a consortium of private- and public-sector scientists dedicated to developing miRNAs as applied biomarkers. Here, we explore major impediments to routine acceptance and use of miRNA biomarkers and case examples of successes and deficiencies in development. Finally, we provide insight on miRNA measurement, collection, and analysis tools to provide solid footing for addressing knowledge gaps toward routine biomarker use.

The use of emerging safety biomarkers in nonclinical and clinical safety assessment - The current and future state: An IQ DruSafe industry survey.

Pharmaceutical and biotechnology companies rarely disclose their use of translational emerging safety biomarkers (ESBs) during drug development, and the impact of ESB use on the speed of drug development remains unclear. A cross-industry survey of 20 companies of varying size was conducted to understand current trends in ESB use and future use prospects. The objectives were to: (1) determine current ESB use in nonclinical and clinical drug development and impact on asset advancement; (2) identify opportunities, gaps, and challenges to greater ESB implementation; and (3) benchmark perspectives on regulatory acceptance. Although ESBs were employed in only 5-50% of studies/programs, most companies used ESBs to some extent, with larger companies demonstrating greater nonclinical use. Inclusion of ESBs in investigational new drug applications (INDs) was similar across all companies; however, differences in clinical trial usage could vary among the prevailing health authority (HA). Broader implementation of ESBs requires resource support, cross-industry partnerships, and collaboration with HAs. This includes generating sufficient foundational data, demonstrating nonclinical to clinical translatability and practical utility, and clearly written criteria by HAs to enable qualification. If achieved, ESBs will play a critical role in the development of next-generation, translationally-tailored standard laboratory tests for drug development.

Urinary miRNA Biomarkers of Drug-Induced Kidney Injury and Their Site Specificity Within the Nephron.

Drug-induced kidney injury (DIKI) is a major concern in both drug development and clinical practice. There is an unmet need for biomarkers of glomerular damage and more distal renal injury in the loop of Henle and the collecting duct (CD). A cross-laboratory program to identify and characterize urinary microRNA (miRNA) patterns reflecting tissue- or pathology-specific DIKI was conducted. The overall goal was to propose miRNA biomarker candidates for DIKI that could supplement information provided by protein kidney biomarkers in urine. Rats were treated with nephrotoxicants causing injury to distinct nephron segments: the glomerulus, proximal tubule, thick ascending limb (TAL) of the loop of Henle and CD. Meta-analysis identified miR-192-5p as a potential proximal tubule-specific urinary miRNA candidate. This result was supported by data obtained in laser capture microdissection nephron segments showing that miR-192-5p expression was enriched in the proximal tubule. Discriminative miRNAs including miR-221-3p and -222-3p were increased in urine from rats treated with TAL versus proximal tubule toxicants in accordance with their expression localization in the kidney. Urinary miR-210-3p increased up to 40-fold upon treatment with TAL toxicants and was also enriched in laser capture microdissection samples containing TAL and/or CD versus proximal tubule. miR-23a-3p was enriched in the glomerulus and was increased in urine from rats treated with doxorubicin, a glomerular toxicant, but not with toxicants affecting other nephron segments. Taken together these results suggest that urinary miRNA panels sourced from specific nephron regions may be useful to discriminate the pathology of toxicant-induced lesions in the kidney, thereby contributing to DIKI biomarker development needs for industry, clinical, and regulatory use.

The Utility of Novel Urinary Biomarkers in Mice for Drug Development Studies.

Novel urinary protein biomarkers have recently been identified and qualified in rats for the early detection of renal injury in drug development studies. However, there are few reports on the utility of these renal biomarkers in mice, another important and widely used preclinical animal species for drug development studies. The purpose of this study was to assess the value of these recently qualified biomarkers for the early detection of drug-induced kidney injury (DIKI) in different strains of mice using multiple assay panels. To this end, we evaluated biomarker response to kidney injury induced by several nephrotoxic agents including amphotericin B, compound X, and compound Y. Several of the biomarkers were shown to be sensitive to DIKI in mice. When measured, urinary albumin and neutrophil gelatinase-associated lipocalin were highly sensitive to renal tubular injury, regardless of the assay platforms, mouse strain, and nephrotoxic agents. Depending on the type of renal tubular injury, kidney injury molecule-1 was also highly sensitive, regardless of the assay platforms and mouse strain. Osteopontin and cystatin C were modestly to highly sensitive to renal tubular injury, but the assay type and/or the mouse strain should be considered before using these biomarkers. Calbindin D28 was highly sensitive to injury to the distal nephron in mice. To our knowledge, this is the first report that demonstrates the utility of novel urinary biomarkers evaluated across multiple assay platforms and nephrotoxicants in different mice strains with DIKI. These results will help drug developers make informed decisions when selecting urinary biomarkers for monitoring DIKI in mice for toxicology studies.

A pharmaceutical industry perspective on microphysiological kidney systems for evaluation of safety for new therapies.

The human kidney contains approximately one million nephrons. As the functional unit of the kidney, the nephron affords an opportunity to approximate the kidney at a microphysiological scale. Recent emergence of physiologically accurate human tissue models has radically advanced the possibilities of mimicking organ biology and multi-organ combinations in vitro. Anatomically, the nephron is one of the most complex, sequentially integrated microfluidic units in the body making the miniaturized microfluidic systems excellent candidates for capturing the kidney biology in vitro. While these models are promising, there are a number of considerations for practical implementation into a drug development paradigm. Opportunities for pharmaceutical industry applications of new MPS models often start with drug safety testing. As such, the intent of this article is to focus on safety and ADME applications. This article reviews biological functions of the kidney and options for characterizing known roles in nephrotoxicity. The concept of "context-of-use" is introduced as a framework for describing and verifying the specific features of an MPS platform for use in drug development. Overall, we present a perspective on key attributes of microphysiological kidney models, which the pharmaceutical industry could leverage to improve confident safety and ADME evaluations of experimental therapies.

Direct Detection of miR-122 in Hepatotoxicity Using Dynamic Chemical Labeling Overcomes Stability and isomiR Challenges.

Circulating microRNAs are biomarkers reported to be stable and translational across species. MicroRNA-122 (miR-122) is a hepatocyte-specific microRNA biomarker for drug-induced liver injury (DILI). We developed a single molecule, dynamic chemical labeling (DCL) assay to directly detect miR-122 in blood. The DCL assay specifically measured miR-122 directly from 10 µL of serum or plasma without any extraction steps, with a limit of detection of 1.32 pM that enabled the identification of DILI. Testing of 192 human serum samples showed that DCL accurately identified patients at risk of DILI after acetaminophen overdose (area under ROC curve 0.98 (95% CI; 0.96-1), P < 0.0001). The DCL assay also identified liver injury in rats and dogs. The use of specific captured beads had the additional benefit of stabilizing miR-122 after sample collection, with no signal loss after 14 days at room temperature, in contrast to PCR that showed significant loss of signal. RNA sequencing demonstrated the presence of multiple miR-122 isomiRs in the serum of patients with DILI that were at low concentration or not present in healthy individuals. Sample degradation over time produced more isomiRs, particularly rapidly with DILI. PCR was inaccurate when analyzing miR-122 isomiRs, whereas the DCL assay demonstrated accurate quantification. We conclude that the DCL assay can accurately measure miR-122 to diagnose liver injury in humans and other species and can overcome microRNA stability and isomiR challenges.

Next-Generation Sequencing to Investigate Urinary microRNAs from Macaca fascicularis (Cynomolgus Monkey).

Advanced sequencing technologies like next-generation sequencing (NGS) not only detect microRNAs (miRNAs) in biological samples but also facilitate de novo identification of miRNAs. Using an Ion Torrent's Ion Proton System, here we described miRNAs sequencing of urine samples collected from Macaca fascicularis (Cynomolgus monkey) to investigate miRNAs as potential novel biomarkers of nephrotoxicity in this species. Urinary miRNA sequencing methodologies described here include (a) urinary exosomal RNA isolation, (b) sequencing library preparation, (c) sequencing template preparation, and (d) template library sequencing using Ion Proton System. The sequencing method presented in this study serves as a valuable resource in the identification of novel urinary miRNAs in M. fascicularis.

Correction: Identification of microRNAs in Macaca fascicularis (Cynomolgus Monkey) by Homology Search and Experimental Validation by Small RNA-Seq and RT-qPCR Using Kidney Cortex Tissues.

[This corrects the article DOI: 10.1371/journal.pone.0142708.].

Evaluation of novel biomarkers of nephrotoxicity in Cynomolgus monkeys treated with gentamicin.

Most studies to evaluate kidney safety biomarkers have been performed in rats. This study was conducted in Cynomolgus monkeys in order to evaluate the potential usefulness of novel biomarkers of nephrotoxicity in this species. Groups of 3 males were given daily intramuscular injections of gentamicin, a nephrotoxic agent known to produce lesions in proximal tubules, at dose-levels of 10, 25, or 50mg/kg/day for 10days. Blood and 16-h urine samples were collected on Days -7, -3, 2, 4, 7, and at the end of the dosing period. Several novel kidney safety biomarkers were evaluated, with single- and multiplex immunoassays and in immunoprecipitation-LC/MS assays, in parallel to histopathology and conventional clinical pathology parameters. Treatment with gentamicin induced a dose-dependent increase in kidney tubular cell degeneration/necrosis, ranging from minimal to mild severity at 10mg/kg/day, moderate at 25mg/kg/day, and to severe at 50mg/kg/day. The results showed that the novel urinary biomarkers, microalbumin, α1-microglobulin, clusterin, and osteopontin, together with the more traditional clinical pathology parameters, urinary total protein and N-acetyl-ß-D-glucosaminidase (NAG), were more sensitive than blood urea nitrogen (BUN) and serum creatinine (sCr) to detect kidney injury in the monkeys given 10mg/kg/day gentamicin for 10days, a dose leading to an exposure which is slightly higher than the desired therapeutic exposure in clinics. Therefore, these urinary biomarkers represent non-invasive biomarkers of proximal tubule injury in Cynomolgus monkeys which may be potentially useful in humans.

Rat Urinary Osteopontin and Neutrophil Gelatinase-Associated Lipocalin Improve Certainty of Detecting Drug-Induced Kidney Injury.

Traditional kidney biomarkers are insensitive indicators of acute kidney injury, with meaningful changes occurring late in the course of injury. The aim of this work was to demonstrate the diagnostic potential of urinary osteopontin (OPN) and neutrophil gelatinase-associated lipocalin (NGAL) for drug-induced kidney injury (DIKI) in rats using data from a recent regulatory qualification submission of translational DIKI biomarkers and to compare performance of NGAL and OPN to five previously qualified DIKI urinary biomarkers. Data were compiled from 15 studies of 11 different pharmaceuticals contributed by Critical Path Institute's Predictive Safety Testing Consortium (PSTC) Nephrotoxicity Working Group (NWG). Rats were given doses known to cause DIKI or other target organ toxicity, and urinary levels of the candidate biomarkers were assessed relative to kidney histopathology and serum creatinine (sCr) and blood urea nitrogen (BUN).OPN and NGAL outperformed sCr and BUN in identifying DIKI manifested as renal tubular epithelial degeneration or necrosis. In addition, urinary OPN and NGAL, when used with sCr and BUN, increased the ability to detect renal tubular epithelial degeneration or necrosis. NGAL and OPN had comparable or improved performance relative to Kim-1, clusterin, albumin, total protein, and beta-2 microglobulin. Given these data, both urinary OPN and NGAL are appropriate for use with current methods for assessing nephrotoxicity to identify and monitor DIKI in regulatory toxicology studies in rats. These data also support exploratory use of urinary OPN and NGAL in safety monitoring strategies of early clinical trials to aid in the assurance of patient safety.

Identification of microRNAs in Macaca fascicularis (Cynomolgus Monkey) by Homology Search and Experimental Validation by Small RNA-Seq and RT-qPCR Using Kidney Cortex Tissues.

MicroRNAs (miRNAs) present in tissues and biofluids are emerging as sensitive and specific safety biomarkers. MiRNAs have not been thoroughly described in M. fascicularis, an animal model used in pharmaceutical industry especially in drug safety evaluation. Here we investigated the miRNAs in M. fascicularis. For Macaca mulatta, a closely related species of M. fascicularis, 619 stem-loop precursor miRNAs (pre-miRNAs) and 914 mature miRNAs are available in miRBase version 21. Using M. mulatta miRNAs as a reference list and homology search tools, we identified 604 pre-miRNAs and 913 mature miRNAs in the genome of M. fascicularis. In order to validate the miRNAs identified by homology search we attempted to sequence miRNAs expressed in kidney cortex from M. fascicularis. MiRNAs expressed in kidney cortex may indeed be released in urine upon kidney cortex damage and be potentially used to monitor drug induced kidney injury. Hence small RNA sequencing libraries were prepared using kidney cortex tissues obtained from three naive M. fascicularis and sequenced. Analysis of sequencing data indicated that 432 out of 913 mature miRNAs were expressed in kidney cortex tissues. Assigning these 432 miRNAs to pre-miRNAs revealed that 273 were expressed from both the -5p and -3p arms of 150 pre-miRNAs and 159 miRNAs expressed from either the -5p or -3p arm of 176 pre-miRNAs. Mapping sequencing reads to pre-miRNAs also facilitated the detection of twenty-two new miRNAs. To substantiate miRNAs identified by small RNA sequencing, 313 miRNAs were examined by RT-qPCR. Expression of 262 miRNAs in kidney cortex tissues ware confirmed by TaqMan microRNA RT-qPCR assays. Analysis of kidney cortex miRNA targeted genes suggested that they play important role in kidney development and function. Data presented in this study may serve as a valuable resource to assess the renal safety biomarker potential of miRNAs in Cynomolgus monkeys.

Absolute quantification of podocalyxin, a potential biomarker of glomerular injury in human urine, by liquid chromatography-mass spectrometry.

Podocalyxin is a protein present in specialized glomerulus cells called podocytes and may be released in the urine in case of kidney injury. In this context, its quantification could be of great interest in order to monitor glomerular injury. Liquid chromatography tandem mass spectrometry (LC-MS/MS), in selected reaction monitoring (SRM) mode, has been demonstrated as a powerful technique that can be applied to protein quantification. This paper describes the development of a quantification method of human podocalyxin in urine by LC-MS/MS in SRM mode by monitoring one proteotypic peptide with an isotope-dilution standardization strategy employing (13)C/(15)N labelled peptides. Inter/intra assay precisions and accuracies of the assay were below 10% and between 90% and 106.1%, respectively. In addition, the method was linear between 0.78 and 100 ng/mL and could therefore be used to quantify endogenous level of podocalyxin that was estimated between 15.2 and 44.2 ng/mL in urine samples from healthy donor.

Comparison between male and female Sprague-Dawley rats in the response of urinary biomarkers to injury induced by gentamicin.

Differences were examined between male and female Sprague-Dawley rats in the response of 16 urinary biomarkers (measured using several assay platforms) to renal injury produced by gentamicin administered subcutaneously for 10 days at a dosage of 75 mg/kg. Urinary biomarkers expressed as fold difference from contemporaneous controls and renal histopathology were assessed after 3 and 10 doses. On day 4, minimal proximal tubular changes were observed microscopically in all males but no females; on day 11, more extensive and more severe injury was observed to a similar extent in all animals of both sexes. Modest increases (maximum 5-fold) in all urinary biomarkers (except epidermal growth factor [EGF], which was decreased) on day 4 and marked elevations (maximum 271-fold) on day 11 were seen consistently in both sexes. However, the magnitude of the increases differed between the sexes. On day 4, despite the lack of tubular injury, many biomarkers were more elevated in females than males but this rarely led to statistically significant sex differences; only 2 biomarkers (ß2-microglobulin and total protein) showed a greater increase in males than females in line with the histopathology. On day 11, there were many more biomarkers that showed a statistically significant difference between the sexes in fold change with treatment; in line with the results on day 4, the majority of biomarkers were more increased in females than males. It remains unresolved if sex differences in the magnitude of biomarker response at injury threshold would lead to any difference in diagnostic interpretation between the sexes. These data highlight the need for publication of more studies using animals of both sexes to fully explore the influence of sex on the diagnostic performance of the novel biomarkers.

Translation strategy for the qualification of drug-induced vascular injury biomarkers.

Drug-induced vascular injury (DIVI) is a common preclinical toxicity usually characterized by hemorrhage, vascular endothelial and smooth muscle damage, and inflammation. DIVI findings can cause delays or termination of drug candidates due to low safety margins. The situation is complicated by the absence of sensitive, noninvasive biomarkers for monitoring vascular injury and the uncertain relevance to humans. The Safer And Faster Evidence-based Translation (SAFE-T) consortium is a public-private partnership funded within the European Commission's Innovative Medicines Initiative (IMI) aiming to accelerate drug development by qualifying biomarkers for drug-induced organ injuries, including DIVI. The group is using patients with vascular diseases that have key histomorphologic features (endothelial damage, smooth muscle damage, and inflammation) in common with those observed in DIVI, and has selected candidate biomarkers associated with these features. Studied populations include healthy volunteers, patients with spontaneous vasculitides and other vascular disorders. Initial results from studies with healthy volunteers and patients with vasculitides show that a panel of biomarkers can successfully discriminate the population groups. The SAFE-T group plans to seek endorsement from health authorities (European Medicines Agency and Food and Drug Administration) to qualify the biomarkers for use in regulatory decision-making processes.

Normal ranges and variability of novel urinary renal biomarkers in Sprague-Dawley Rats: comparison of constitutive values between males and females and across assay platforms.

Differences were examined between male and female Sprague-Dawley rats in basal levels of a wide range of urinary biomarkers, including 7 recently qualified biomarkers. The data were generated from urine samples collected on 3 occasions from untreated rats included in a study of the effect of gentamicin nephrotoxicity on urinary renal biomarkers, reported in a companion article in this journal (Gautier et al. 2014). The performance of multiple assays (9 singleplex assays and 2 multiplex platforms from Rules Based Medicine [RBM] and Meso Scale Discovery [MSD]) was evaluated, and normal ranges and variability estimates were derived. While variability was generally greater on the RBM platform than other assays, the more striking difference in the results from different assays was in magnitude. Where differences were observed between assays for an individual biomarker, they were seen in both sexes and consistent across samples collected at different time points. Differences of up to 15-fold were observed for some biomarker values between assays indicating that results generated using different assays should not be compared. For 8 biomarkers, there was compelling evidence for a sex difference. Baseline values in males were significantly higher than in females for total protein, ß2-microglobulin, clusterin, cystatin-C, glutathione-S-transferase (GST-α), tissue inhibitor of metalloproteinases (TIMP-1), and vascular endothelial growth factor (VEGF); female values were significantly higher than that of males for albumin. The largest sex differences (male greater than female by 2- to 11-fold) were seen with ß2-microglobulin, GST-α, and TIMP-1. These data add substantially to the limited body of knowledge in this area and provide a useful framework for evaluation of the potential relevance of sex differences in the diagnostic performance of these biomarkers.

Absolute quantification of podocin, a potential biomarker of glomerular injury in human urine, by liquid chromatography-multiple reaction monitoring cubed mass spectrometry.

Glomeruli play a major role in the kidney function since they are involved in primary urine formation. It is then crucial to dispose of methods to monitor glomerular injury, especially in drug development. In this context, quantification of podocin could be of great interest since it is a protein exclusively present in highly specialized glomerulus cells called podocytes. Immunoassays are the most commonly used approach for protein assays. However, they rely on the availability of specific antibodies. When such antibodies are not available, liquid chromatography tandem mass spectrometry (LC-MS/MS), in selected reaction monitoring (SRM) or in multiple reaction monitoring cubed (MRM(3)) mode, has been demonstrated as a powerful alternative technique, and can be applied to multiple protein quantification. This paper describes the development of a quantification method of human podocin in urine by LC-MS/MS in MRM(3) mode. Inter assay precision and accuracy ranged from 7 to 20% and from 105 to 112% respectively and the lower limit of quantification (LLOQ) was 0.39ng/mL from only 1mL of urine which is compatible for endogenous level of podocin determination.

Overcoming biofluid protein complexity during targeted mass spectrometry detection and quantification of protein biomarkers by MRM cubed (MRM3).

Targeted mass spectrometry in the so-called multiple reaction monitoring mode (MRM) is certainly a promising way for the precise, accurate, and multiplexed measurement of proteins and their genetic or posttranslationally modified isoforms. MRM carried out on a low-resolution triple quadrupole instrument faces a lack of specificity when addressing the quantification of weakly concentrated proteins. In this case, extensive sample fractionation or immunoenrichment alleviates signal contamination by interferences, but in turn decreases assay performance and throughput. Recently, MRM(3) was introduced as an alternative to MRM to improve the limit of quantification of weakly concentrated protein biomarkers. In the present work, we compare MRM and MRM(3) modes for the detection of biomarkers in plasma and urine. Calibration curves drawn with MRM and MRM(3) showed a similar range of linearity (R(2) > 0.99 for both methods) with protein concentrations above 1 µg/mL in plasma and a few nanogram per milliliter in urine. In contrast, optimized MRM(3) methods improve the limits of quantification by a factor of 2 to 4 depending on the targeted peptide. This gain arises from the additional MS(3) fragmentation step, which significantly removes or decreases interfering signals within the targeted transition channels.

Optimization of liquid chromatography-multiple reaction monitoring cubed mass spectrometry assay for protein quantification: application to aquaporin-2 water channel in human urine.

Aquaporin-2 (AQP2) is a water channel protein located in the kidney collecting ducts that has been studied as a potential biomarker of a wide variety of water handling disorders and that could also be used to monitor lesions in the collecting ducts. Enzyme-linked immunosorbent assay (ELISA), the most commonly used approach for protein assay in biofluids, has a limited potential for biomarker verification due to the restricted possibility to perform multiplex assays, the cost and complexity of assay development for new candidates. Liquid chromatography tandem mass spectrometry (LC-MS/MS), in multiple reaction monitoring (MRM) mode, has been demonstrated as a powerful alternative technique, and applied to multiple protein quantification. An even more specific method, termed MRM cubed (MRM(3)), has recently been developed. This paper focuses on the development of an AQP2 assay in urine by LC-MS/MS, based on the MRM(3) strategy, and the influence of key MRM(3) parameters that enable to increase the method sensitivity by a factor of 10. Linearity is observed within the concentration range 0.5-50ng/mL, intra and inter assay precision ranged from 9 to 35% at the lower limit of quantification (LLOQ), and accuracy from 94 to 114%. This assay could therefore be used in the near future to evaluate human urinary AQP2 as a potential biomarker of kidney collecting duct injury.

Biomarkers of collecting duct injury in Han-Wistar and Sprague-Dawley rats treated with N-phenylanthranilic Acid.

N-phenylanthranilic acid is a chloride channel blocker that causes renal papillary necrosis in rats. Studies were conducted in two strains of male rats to evaluate novel biomarkers of nephrotoxicity. Han-Wistar rats were given daily oral doses of 50, 350, or up to 700 mg/kg/day of NPAA, and Sprague-Dawley rats were given 50 or 400 mg/kg/day of NPAA. Rats were euthanized on days 8 and 15. The candidate kidney injury biomarkers renal papillary antigen-1 (RPA-1, for collecting duct injury), clusterin (for general kidney injury), α-glutathione-S-transferase (a proximal tubular marker), and µ-glutathione-S-transferase (a distal tubular marker) were measured in urine by enzyme immunoassay. Characteristic degeneration and necrosis of the collecting duct and renal papilla were observed in Han-Wistar rats at the high dose on day 8 and at the mid and high doses on day 15, and in Sprague-Dawley rats given the high dose on days 8 and 15. Increases in urinary RPA-1, and to a lesser extent urine clusterin, were generally associated with the presence of collecting duct injury and were more sensitive than BUN and serum creatinine. On the other hand, decreases in α-glutathione-S-transferase without proximal tubule lesions in both strains and decreases in µ-glutathione-S-transferase in Sprague-Dawley rats only were not associated with morphological proximal or distal tubule abnormalities, so both were of less utility. It was concluded that RPA-1 is a new biomarker with utility in the detection of collecting duct injury in papillary necrosis in male rats.