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.

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.

Quantitative Mass Spectrometry Imaging to Study Drug Distribution in the Intestine Following Oral Dosing.

Local delivery to the lower gut to treat diseases of the colon has become a topic of special attention. Tissue exposure of locally acting agents is not represented by plasma concentrations. Therefore, reliable methods to measure tissue uptake at the primary site of action (e.g., epithelial layer or lamina propria) are vital. This work investigates the suitability of mass spectrometry imaging (MSI) in quantitatively visualizing intestinal transmural drug distribution. Tofacitinib (Tofa), a drug approved for the treatment of several autoimmune diseases, including ulcerative colitis, was selected as a tool compound for feasibility studies. One- and 7-h postdose sections of the ileum, proximal- and distal-colon from rats that received an oral solution of Tofa were subjected to matrix-assisted laser desorption ionization (MALDI)-MSI. A dilution series of individual concentrations sprayed over an entire tissue section allowed for tissue type-specific quantitation. At 1 h (systemic Tmax), the signal was highest in the ileum, whereas at 7 h, the signal was highest in the colon, when the unabsorbed fraction of the compound reached the colon. A combination of three-dimensional (3D) intensity plots and hematoxylin and eosin (H&E) stains showed a visually observable gradual decrease in Tofa concentration from the lumen toward the muscular layer of the proximal colon. The high luminal concentration of Tofa indicated that flushing of the intestines with saline does not result in complete removal of the drug material from the lumen. This could cause an overestimation of drug concentration in gut tissue homogenates by conventional liquid chromatography-mass spectrometry (LC-MS) methods. This study demonstrates the utility of MSI to differentiate between the lumen and intestinal wall layers and enables proper interpretation of tissue distribution data.

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.

Investigating the Value of Urine Volume, Creatinine, and Cystatin C for Urinary Biomarkers Normalization 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 seems to be no standardized normalization method for analyzing these urinary biomarkers, as some users normalize with urinary creatinine (uCr), urine volume (uVol), or leave biomarker un-normalized. More recently, urinary cystatin C is also emerging as a urinary biomarker normalizer, given some of its characteristics as a glomerular filtration marker. The purpose of this study was to identify an optimal drug-induced kidney injury biomarker normalization method that can be adopted more uniformly in the field. To this end, we compared the variability of uVol, urinary cystatin C, and Cr in healthy rats; we evaluated the sensitivity of the renal biomarkers to renal injury after normalization with uVol, uCr, and cystatin C in rats with cisplatin-induced renal injury. We showed that, over time, uCr was less variable than urinary cystatin C and uVol. When the renal biomarkers were normalized with the 3 normalizing end points, the biomarkers showed (1) least variability following normalization with Cr in healthy animals and (2) poor sensitivity when normalized with urinary cystatin C in animals with renal injury. Overall, the results suggested that uCr is better than urinary cystatin C and uVol for normalizing renal biomarkers in rats under controlled preclinical conditions. To our knowledge, this is the first report that compared the variability of uVol, cystatin C, and Cr in the context of renal biomarkers' normalization.

Brief Overview: Assessment of Compound-induced Acute Kidney Injury Using Animal Models, Biomarkers, and In Vitro Platforms.

The inability to unequivocally predict translatable drug-induced kidney injury in nonclinical studies during pharmacological development is evidenced by drug attrition in human clinical trials. Eight urinary proteins have been qualified as renal safety biomarkers for limited context of use in nonclinical drug development studies in rats. Formal qualification of human renal safety biomarkers is pending the submission of data from prospective clinical trials and analyses of biomarker performance to the Food and Drug Administration and European Medicines Agency by the Foundation for the National Institutes of Health and Predictive Safety Testing Consortium's Nephrotoxicity Working Group. In vitro kidney platforms may be leveraged to investigate the potential risk of compound-induced acute kidney injury and/or dysfunction. The early assessment of drug-related kidney safety profiles using biomarker-level changes in animal models and in vitro platforms could significantly reduce renal safety-related drug attrition; yet, there are no well-validated in vitro systems to enable comprehensive investigations of compound-induced nephrotoxicity. Thus, histopathology remains the gold standard for diagnosing nephron-specific damage. Traditional and emerging biomarker panels should be combined with histopathology and/or cytopathology to enable early identification of compound-induced kidney injury.

High-resolution laser ablation-inductively coupled plasma-mass spectrometry imaging of cisplatin-induced nephrotoxic side effects.

Two-dimensional elemental mapping (bioimaging) via laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was performed on 5 µm thick formalin-fixed, paraffin-embedded kidney tissue sections from Cynomolgus monkeys administered with increasing pharmacological doses of cisplatin. Laterally resolved pixels of 1 µm were achieved, enabling elemental analysis on a (sub-)cellular level. Zones of high Pt response were observed in the renal cortex, where proximal tubules are present, the epithelium of which is responsible for partial reabsorption of cisplatin. Histopathological evaluation, of hematoxylin and eosin-stained serial sections, adjacent to the sections probed via LA-ICP-MS, revealed minimal to mild cisplatin-related lesions (<100 µm) in the renal cortex. Necrotic proximal tubules with sloughed epithelial cells in their lumen could be linked directly to the areas with the highest accumulation of cisplatin, indicating a direct link between cellular concentration and toxicity, thereby providing more insight into the mechanisms through which renal damage occurs.

Acute biomarker panel changes associated with amphotericin B nephrotoxicity in female Sprague-Dawley rats.

To date, eight next­generation urinary protein kidney safety biomarkers have been qualified to enable monitoring for subclinical drug­induced kidney injury (DIKI) in rat preclinical studies; however, most DIKI biomarker studies have included only male rats. The objective of this study was to assess the utility of novel DIKI biomarkers, including but not limited to urinary total protein, albumin, cystatin C and osteopontin in female Sprague­Dawley rats (8/group) that received repeated intravenous injections of amphotericin B (AmpB, 3 mg/kg/day) or vehicle for 10 consecutive days. Serial serum/urine samples were collected on study day (D)4, D8, and D11. Surviving animals were necropsied on D11. The AmpB­induced kidney histopathology findings were characterized by cortical and medullary tubular alterations, interstitial inflammation, intratubular granular and inflammatory cell casts, acute pelvic inflammation and tubular mineralization. Significant elevations in urinary clusterin on D4, D8 and D11 (3.5 fold, 2.2 fold and 3.3 fold respectively) were observed (versus concurrent controls) following repeated injections of AmpB. In addition, significantly elevated (fold changes) in biomarkers, neutrophil gelatinase­associated lipocalin (14.6 fold), albumin (13.5 fold), cystatin C (13.5 fold), total protein (3.5 fold), kidney injury molecule 1 (3.0 fold) and osteopontin (2.3 fold) were detected in urine as early as D4. These findings demonstrate the value of early elevations in nephron­specific DIKI biomarkers for detecting subclinical AmpB nephrotoxicity in female Sprague­Dawley rats. These findings are anticipated to provide the basis for inclusion of female rats on a case­by­case basis in preclinical toxicology studies designed to detect DIKI.

Beyond miR-122: Identification of MicroRNA Alterations in Blood During a Time Course of Hepatobiliary Injury and Biliary Hyperplasia in Rats.

Identification of circulating microRNAs for the diagnosis of liver injury and as an indicator of underlying pathology has been the subject of recent investigations. While several studies have been conducted, with particular emphasis on miR-122, the timing of miRNA release into the circulation and anchoring to tissue pathology has not been systematically evaluated. In this study, miRNA profiling was conducted over a time course of hepatobiliary injury and repair using alpha-naphthylisothiocyanate (ANIT) and a proprietary compound, FP004BA. ANIT administration (50 mg/kg) to rats caused significant biliary epithelial cell and hepatocellular necrosis between 24 and 72 h, followed by resolution and progression to biliary hyperplasia by 120 h which was associated with miRNA release into the blood. FP004BA (100 mg/kg) was used to confirm associations of miRNA along a time course with similar hepatic pathology to ANIT. Treatment with ANIT or FP004BA resulted in significant alterations of overlapping miRNAs during the early and peak injury phases. In addition to well-characterized liver injury markers miR-122-5p and miR-192-5p, multiple members of the 200 family and the 101 family along with miR-802-5p and miR-30d-5p were consistently elevated during hepatobiliary injury caused by both toxicants, suggesting that these species may be potential biomarker candidates for hepatobiliary injury. After 14 days of dosing with 4BA, miR-182-5p remained elevated-while miR-122-5p and miR-192-5p had returned to baseline-suggesting that miR-182-5p may have added utility to monitor for hepatobiliary injury in the repair phases when there remains histological evidence of ongoing cellular injury.

Time course of renal proximal tubule injury, reversal, and related biomarker changes in rats following cisplatin administration.

Cisplatin (CDDP) is known to produce renal proximal tubule injury. Various renal biomarkers have been related to CDDP nephrotoxicity in previous research, but the temporal and spatial relationship of these biomarkers to injury reversal has not been well defined. In this study, the progression and reversal of renal histopathology findings relative to serum and urinary biomarker changes were examined during a 4-week postdose period following single intraperitoneal administration of CDDP (1 mg/kg) or 0.9% saline. Degeneration, vacuolation, inflammation, and regeneration of the S3 segment of proximal tubules were evident 72 hours following CDDP administration. Tubular degeneration and regeneration were also observed at 1 and 1.5 weeks but at lower incidences and/or severity indicating partial reversal. Complete histologic reversal was observed by 2 weeks following CDDP administration. Urinary kidney injury molecule 1 (KIM-1), α-glutathione-S-transferase (α-GST), and albumin levels increased at 72 hours postdosing, concurrently with the earliest histologic evidence of tubule injury. Changes in urinary KIM-1 correlated with KIM-1 immunostaining in the proximal tubular epithelial cells. No significant changes in serum biomarkers occurred except for a minimal increase in urea nitrogen at 1.5 weeks postdosing. Of the novel renal biomarkers examined, urinary KIM-1, α-GST, and albumin showed excellent concordance with CDDP-induced renal injury progression and reversal; and these biomarkers were more sensitive than traditional serum biomarkers in detecting early, acute renal tubular damage confirmed by histopathology. Furthermore, urinary KIM-1, α-GST, and albumin outperformed other biomarkers in correlating with the time of maximum histologic injury.

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.