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.

MicroRNA-122: a novel hepatocyte-enriched in vitro marker of drug-induced cellular toxicity.

Emerging hepatic models for the study of drug-induced toxicity include pluripotent stem cell-derived hepatocyte-like cells (HLCs) and complex hepatocyte-non-parenchymal cellular coculture to mimic the complex multicellular interactions that recapitulate the niche environment in the human liver. However, a specific marker of hepatocyte perturbation, required to discriminate hepatocyte damage from non-specific cellular toxicity contributed by non-hepatocyte cell types or immature differentiated cells is currently lacking, as the cytotoxicity assays routinely used in in vitro toxicology research depend on intracellular molecules which are ubiquitously present in all eukaryotic cell types. In this study, we demonstrate that microRNA-122 (miR-122) detection in cell culture media can be used as a hepatocyte-enriched in vitro marker of drug-induced toxicity in homogeneous cultures of hepatic cells, and a cell-specific marker of toxicity of hepatic cells in heterogeneous cultures such as HLCs generated from various differentiation protocols and pluripotent stem cell lines, where conventional cytotoxicity assays using generic cellular markers may not be appropriate. We show that the sensitivity of the miR-122 cytotoxicity assay is similar to conventional assays that measure lactate dehydrogenase activity and intracellular adenosine triphosphate when applied in hepatic models with high levels of intracellular miR-122, and can be multiplexed with other assays. MiR-122 as a biomarker also has the potential to bridge results in in vitro experiments to in vivo animal models and human samples using the same assay, and to link findings from clinical studies in determining the relevance of in vitro models being developed for the study of drug-induced liver injury.

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.

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.

Qualified kidney biomarkers and their potential significance in drug safety evaluation and prediction.

The kidney is one of the major organs drug toxicity may target. Some renal safety biomarkers have been proposed to measure kidney injury and function accordingly. Despite the widespread use for diagnosis and monitoring of renal injury and function for decades, serum creatinine and blood urea nitrogen are nonspecific biomarkers with insensitive and delayed response in the clinical setting. There is an urgent need to identify and qualify novel kidney safety biomarkers that would be used to detect and predict drug-induced nephrotoxicity in preclinical toxicological studies, clinical trials and patient care in sequence. To do that, eight novel renal safety biomarkers have been well characterized and qualified for preclinical drug safety screening, and their clinical bridging validation is underway as well. Of them, some are used to detect or predict proximal tubular injury, and others are used to diagnose and monitor glomerular damage. Thus, measurement of a panel of kidney safety biomarkers in parallel would help maximally capture all potential safety signals for a more informative decision to be made in drug research and development as well as for optimal selection of the drug and its dose in clinical practice.

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.

Biological qualification of biomarkers of chemical-induced renal toxicity in two strains of male rat.

This study reports the evaluation of four urinary biomarkers of renal toxicity, α-glutathione-S-transferase (α-GST), µ-GST, clusterin, and renal papillary antigen-1 (RPA-1), in male Sprague-Dawley and Han-Wistar rats given cisplatin, gentamicin, or N-phenylanthranilic acid (NPAA). Kidney injury was diagnosed histopathologically, according to site/nature of renal injury, and graded for severity. The area under the receiver operating characteristic (ROC) curve was used to compare the diagnostic accuracy of each exploratory renal biomarker with traditional indicators of renal function and injury (blood urea nitrogen [BUN], serum creatinine [sCr] as well as urinary N-acetyl-ß-D-glucosaminidase [NAG] and protein). These analyses showed that increased urinary α-GST was superior to BUN, sCr, and NAG for diagnosis of proximal tubular (PT) degeneration/necrosis. Paradoxically, urinary α-GST was decreased in the presence of collecting duct (CD) injury without PT injury (NPAA administration). RPA-1 demonstrated high specificity for CD injury, superior to all of the reference biomarkers. The clusterin response correlated well with tubular injury, whatever the location, particularly when regeneration was present (superior to all of the reference markers for cortical tubular regeneration). There was no conclusive evidence for the diagnostic utility of µ-GST. The data were submitted for qualification review by the European Medicines Agency and the US Food and Drug Administration. Both agencies concluded that the data justified the qualification of RPA-1 and increased the level of evidence for, and clarified the context of use of, the previously qualified clusterin for use in male rats. These biomarkers can be used in conjunction with traditional clinical chemistry markers and histopathology in Good Laboratory Practice rodent toxicology studies used to support renal safety studies in clinical trials. Qualification of α-GST must await further explanation of the differences in response to PT and CD injury.

Evaluation of novel biomarkers of nephrotoxicity in two strains of rat treated with Cisplatin.

Cisplatin is an anticancer agent that induces renal proximal tubule lesions in many species. Studies were conducted in Sprague-Dawley and Han-Wistar rats to evaluate the utility of novel preclinical biomarkers of nephrotoxicity for renal lesions caused by this compound. Groups of 10 males of each strain were given a single intraperitoneal injection of 0.3, 1, or 3 mg/kg cisplatin and were sacrificed on days 2, 3, and 5. The novel biomarkers α-glutathione-S-transferase (α-GST) (for proximal tubular injury), µ-glutathione-S-transferase (µ-GST) (for distal tubular injury), clusterin (for general kidney injury), and renal papillary antigen-1 (RPA-1) (for collecting duct injury) were measured in urine by enzyme immunoassay. Histologically, degeneration and necrosis of the S3 segment of the renal proximal tubule were observed on day 2 (Han-Wistar) and days 3 and 5 (both strains) at 1 and 3 mg/kg. Results showed that in both strains of rats, urinary α-GST and clusterin can be detected in urine soon after injury, are more sensitive than BUN and serum creatinine, and therefore are usable as noninvasive biomarkers of proximal tubule injury. Changes in both µ-GST or RPA-1 were considered to represent secondary minor effects of proximal tubular injury on distal segments of the nephron.

Towards consensus practices to qualify safety biomarkers for use in early drug development.

Application of any new biomarker to support safety-related decisions during regulated phases of drug development requires provision of a substantial data set that critically assesses analytical and biological performance of that biomarker. Such an approach enables stakeholders from industry and regulatory bodies to objectively evaluate whether superior standards of performance have been met and whether specific claims of fit-for-purpose use are supported. It is therefore important during the biomarker evaluation process that stakeholders seek agreement on which critical experiments are needed to test that a biomarker meets specific performance claims, how new biomarker and traditional comparators will be measured and how the resulting data will be merged, analyzed and interpreted.

Renal biomarker qualification submission: a dialog between the FDA-EMEA and Predictive Safety Testing Consortium.

The first formal qualification of safety biomarkers for regulatory decision making marks a milestone in the application of biomarkers to drug development. Following submission of drug toxicity studies and analyses of biomarker performance to the Food and Drug Administration (FDA) and European Medicines Agency (EMEA) by the Predictive Safety Testing Consortium's (PSTC) Nephrotoxicity Working Group, seven renal safety biomarkers have been qualified for limited use in nonclinical and clinical drug development to help guide safety assessments. This was a pilot process, and the experience gained will both facilitate better understanding of how the qualification process will probably evolve and clarify the minimal requirements necessary to evaluate the performance of biomarkers of organ injury within specific contexts.

A rationale for determining, testing, and controlling specific impurities in pharmaceuticals that possess potential for genotoxicity.

The synthesis of pharmaceutical products frequently involves the use of reactive reagents and the formation of intermediates and by-products. Low levels of some of these may be present in the final drug substance and drug product as impurities. Such chemically reactive impurities may have at the same time the potential for unwanted toxicities including genotoxicity and carcinogenicity and hence can have an impact on product risk assessment. This paper outlines a procedure for testing, classification, qualification, toxicological risk assessment, and control of impurities possessing genotoxic potential in pharmaceutical products. Referencing accepted principles of cancer risk assessment, this document proposes a staged threshold of toxicological concern (TTC) approach for the intake of genotoxic impurities over various periods of exposure. This staged TTC is based on knowledge about tumorigenic potency of a wide range of genotoxic carcinogens and can be used for genotoxic compounds, for which cancer data are limited or not available. The delineated acceptable daily intake values of between approximately 1.5 microg/day for approximately lifetime intake and approximately 120 microg/day for < or = 1 month are virtually safe doses. Based on sound scientific reasoning, these virtually safe intake values do not pose an unacceptable risk to either human volunteers or patients at any stage of clinical development and marketing of a pharmaceutical product. The intake levels are estimated to give an excess cancer risk of 1 in 100,000 to 1 in a million over a lifetime, and are extremely conservative given the current lifetime cancer risk in the population of over 1 in 4 (http://seer.cancer.gov/statfacts/html.all.html). The proposals in this document apply to all clinical routes of administration and to compounds at all stages of clinical development. It is important to note that certain types of products, such as those for life-threatening indications for which there are no safer alternatives, allow for special considerations using adaptations of the principles outlined in this paper.