Manisha Sonee

Tissue Kim-1 and Urinary Clusterin as Early Indicators of Cisplatin-Induced Acute Kidney Injury in Rats

The kidney is one of the main targets of drug toxicity, and early detection of renal damage is critical in preclinical drug development. A model of cisplatin-induced nephrotoxicity in male Sprague Dawley rats treated for 1, 3, 5, 7, or 14 days at 1 mg/kg/day was used to monitor the spatial and temporal expression of various indicators of kidney toxicity during the progression of acute kidney injury (AKI). As early as 1 day after cisplatin treatment, positive kidney injury molecule-1 (Kim-1) immunostaining, observed in the outer medulla of the kidney, and changes in urinary clusterin indicated the onset of proximal tubular injury in the absence of functional effects. After 3 days of treatment, Kim-1 protein levels in urine increased more than 20-fold concomitant with a positive clusterin immunostaining and an increase in urinary osteopontin. Tubular basophilia was also noted, while serum creatinine and blood urea nitrogen levels were elevated only after 5 days, together with tubular degeneration. In conclusion, tissue Kim-1 and urinary clusterin were the most sensitive biomarkers for detection of cisplatin-induced kidney damage. Thereafter, urinary Kim-1 and osteopontin, as well as clusterin immunostaining accurately correlated with the histopathological findings. When AKI is suspected in preclinical rat studies, Kim-1, clusterin, and osteopontin should be part of urinalysis and/or IHC can be performed.

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.

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.

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 Institutes 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.

Analytic Evaluation of a Human ELISA Kit for Measurement of Inhibin B in Rat Samples

BACKGROUND A cross-laboratory analytic evaluation of a commercially available human inhibin B ELISA for measuring inhibin B in rat serum and plasma has been undertaken. METHODS Dilution linearity, spiked recovery, intra- and inter-assay precision, functional sensitivity, matrix effects, and frozen stability were assessed across five laboratories. Reference ranges were generated for male Sprague Dawley and Han Wistar rats. RESULTS Acceptable performance was defined as an overall assay coefficient of variation ≤ 20% with an intraday LLOQ ≤ 20 pg/ml. Intra- and inter-assay precision and functional sensitivity (≤6.4 pg/ml) generally met these criteria, but with occasional evidence of greater variability, particularly at lower concentrations. Dilution linearity was acceptable with occasional low recovery. Acceptable recovery of kit calibrators from rat serum confirmed the absence of matrix effects. Matched serum and plasma samples gave comparable results. The signal increased on freezing, remained constant for ≥3 freeze-thaw cycles and was generally stable for at least 8 weeks. Mean inhibin B ranged from 33.5 to 140.6 pg/ml in adult rats across laboratories, with some evidence for a decline from 6 to 9 weeks of age. Power calculations using preliminary reference range data indicated 10 animals/group would generally detect a 40% decrease in inhibin B at AstraZeneca, but laboratories with lower control values would require larger groups. CONCLUSIONS The assay meets the analytical performance criteria; however, precision at the low end of the standard curve, biological variability, and low control values observed in some laboratories indicate that the utility of the assay may be limited in some laboratories.

MicroRNA-34c-3p is an early predictive biomarker for doxorubicin-induced glomerular injury progression in male Sprague-Dawley rats

Recently, eight urinary protein biomarkers were qualified for renal toxicity prediction in drug development; however, there are no biomarkers unique to glomerular toxicity. Albuminuria is a hallmark biomarker for primary glomerular injury but lacks specificity. MicroRNA species associated with genes that regulate kidney injury could potentially be used as biomarkers of nephrotoxicity. In this study, microRNA and protein expression changes in urine, blood, and/or kidneys, in addition to histopathology were evaluated in male Sprague-Dawley rats following weekly intravenous injections of doxorubicin (5 mg kg−1 per dose). Following the first administration, urinary miRNA-34c-3p was significantly increased on day 2 and remained elevated on day 7. Urinary osteopontin was significantly increased on day 2 only. Significant urinary albumin was detected on day 7, in the absence of histopathological findings. Following a second doxorubicin administration on day 7, significantly increased urinary kidney injury molecule 1, cystatin C, β2-microglobulin, total protein, and neutrophil gelatinase-associated lipocalin concentrations were detected on day 14. These alterations were concurrent to significant and progressive albuminuria, urinary miR-34c-3p, remarkable microscopic primary glomerular injury and secondary tubular alterations. Urinary miR-34c-3p elevations were predictive of histopathologic injury progression and outperformed the traditional renal biomarkers, serum creatinine and blood urea nitrogen, which did not increase with treatment. MiR-34c-3p was also significantly enriched in damaged glomeruli compared to adjacent nonglomerular tissue. Taken together, miR-34c-3p was identified as a highly sensitive candidate renal safety biomarker with relative specificity, particularly for early prediction of doxorubicin-induced glomerular injury progression in male Sprague-Dawley rats.

Summary of the HESI consortium studies exploring circulating inhibin B as a potential biomarker of testis damage in the rat.

The Developmental and Reproductive Toxicity Technical Committee of the Health and Environmental Sciences Institute hosted a working consortium of companies to evaluate a new commercially available analytic assay for Inhibin B in rat serum or plasma. After demonstrating that the kit was stable and robust, the group performed a series of independent pathogenesis studies (23 different compound/investigator combinations) designed to examine the correlation between the appearance of lesions in the testis and changes in circulating levels of Inhibin B. These studies were reported individually in the previous articles in this series (this issue), and are discussed in this paper. For roughly half of these exposures, lesions appeared well before Inhibin B changed. A few of the studies showed a good correlation between seminiferous tubule damage and reduced circulating Inhibin B levels, while for seven exposures, circulating Inhibin B was reduced with no detectable alteration in testis histology. Whether this indicates a prodromal response or a false-positive signal will require further investigation. These exceptions could plausibly suggest some value of circulating Inhibin B as a useful biomarker in some circumstances. However, for roughly half of these exposures, Inhibin B appeared to be a lagging biomarker, requiring significant damage to the seminiferous tubules before a consistent and credible reduction in circulating levels of Inhibin B was observed.

The Inhibin B Response to the Testicular Toxicant 1, 3 Dinitrobenzene in Male Rats

BACKGROUND This study was conducted as part of an ILSI-HESI International Life Sciences Institute-Health & Environmental Sciences Institute consortium effort to assess the utility of circulating Inhibin B as an early biomarker of Sertoli cell-specific testicular toxicity in rats. 1, 3-Dinitrobenzene (1,3-DNB) was selected as a testicular toxicant in this study as it is known to target Sertoli cells. METHODS 1,3-DNB (2 and 6 mg/kg/day) or control (corn oil) was administered orally to male rats for two or five consecutive days. Blood was collected from rats treated for 2 days on days 1 and 2 and from rats treated for 5 days on days 1, 3, and 5. The resulting serum was evaluated for Inhibin B and follicle stimulating hormone. At the end of the treatment periods, the testes were removed, weighed, and examined histopathologically. RESULTS Daily administration of 1,3-DNB resulted in decreased testis weight only on day 5 and only at the high dose (6 mg/kg/day). There was a time-dependent increase in incidence and severity of testicular findings characterized by degeneration of the germinal epithelium with loss of pachytene spermatocytes and vacuolization of the Sertoli cells in the seminiferous tubules at the high dose. Inhibin B levels in 1,3-DNB-treated animals were decreased with treatment only on day 5 at the high dose; there were no associated changes in follicle stimulating hormone. CONCLUSIONS Changes in serum Inhibin B levels were detected only in association with moderate or severe testicular toxicity as evidenced by histopathology and is therefore considered to be of limited value as a biomarker for Sertoli cell toxicity.

Immunolocalization of novel corticomedullary tubule injury markers in Cynomolgus monkeys treated with amphotericin B

Amphotericin B (AmpB) nephrotoxicity was used to assess the utility of drug‑induced kidney injury (DIKI) biomarkers in an exploratory study in male cynomolgus monkeys. All animals had quantifiable levels of AmpB in plasma on days 1 and 4. There were no clinical signs of AmpB‑induced toxicity in this study. The gold standard method used to confirm AmpB‑induced DIKI was anatomic pathology which revealed microscopic lesions with varying grades of severity. Immunolocalization of alpha‑1 microglobulin (α‑1M), kidney injury molecule 1 (KIM‑1), osteopontin (OPN) and neutrophil gelatinase‑associated lipocalin (NGAL) proteins was evaluated in formalin‑fixed, paraffin‑embedded monkey kidney tissue sections. AmpB related immunoreactivities were identified in distinct nephron segments of treated monkeys including α‑1M in damaged proximal tubule epithelium, KIM‑1 in damaged medullary tubule epithelium, OPN mostly in the infiltrating cells of cortical tubule interstitium, and NGAL in the granular and cellular cast in dilatated cortical tubules. Variations in α‑1M, KIM‑1, OPN and NGAL immunolocalization appear as promising DIKI protein biomarkers when monitoring for AmpB‑induced corticomedullary tubule injury in male cynomolgus monkeys.

Feasibility of Protein Biomarkers in the Prediction of Subclinical Doxorubicin Nephrotoxicity in Male Sprague-Dawley Rat

The feasibility of proteinbiomarkers in the prediction of subclinical doxorubicin nephrotoxicity was evaluated in male Sprague-Dawley rats during a 2-week study with once‑weekly dosing. Doxorubicin (5, 7.5, and 10 mg/kg/ dose) or 0.9% Saline was intravenously administered on days 1 and 8. Urine and serum were collected at various time points. Surviving animals were euthanized on day 14, and tissues were collected for microscopic examination. Severe clinical signs were observed in the 7.5 and 10 mg/kg/dose groups. Biomarker data are not reported for these groups because the objective of this study was to evaluate biomarkers at doses not associated with clinical signs. In the 5 mg/kg group, increased serum concentrations of urea nitrogen were observed on day 14 with concurrent renal histopathology findings which were primarily characterized as slight renal glomerular and tubular injury with mild multi-focal intratubular hyaline casts consistent with protein leakage from damaged glomeruli. Of the various urinary protein biomarkers examined, increased urinary concentrations of albumin was observed on day 7 and increased total protein, albumin and lipocalin‑2 were observed on day 14. Taken together, these findings showed that urinary albumin was more sensitive and selective than urinary total protein, lipocalin-2, kidney injury molecule 1 and/or osteopontin in the prediction of progressive doxorubicin‑induced glomerular toxicity with secondary renal tubular toxicity in male Sprague‑Dawley rats.