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.