Daniel Wahl

Urinary clusterin, cystatin C, β2-microglobulin and total protein as markers to detect drug-induced kidney injury

Earlier and more reliable detection of drug-induced kidney injury would improve clinical care and help to streamline drug-development. As the current standards to monitor renal function, such as blood urea nitrogen (BUN) or serum creatinine (SCr), are late indicators of kidney injury, we conducted ten nonclinical studies to rigorously assess the potential of four previously described nephrotoxicity markers to detect drug-induced kidney and liver injury. Whereas urinary clusterin outperformed BUN and SCr for detecting proximal tubular injury, urinary total protein, cystatin C and β2-microglobulin showed a better diagnostic performance than BUN and SCr for detecting glomerular injury. Gene and protein expression analysis, in-situ hybridization and immunohistochemistry provide mechanistic evidence to support the use of these four markers for detecting kidney injury to guide regulatory decision making in drug development. The recognition of the qualification of these biomarkers by the EMEA and FDA will significantly enhance renal safety monitoring.

A panel of urinary biomarkers to monitor reversibility of renal injury and a serum marker with improved potential to assess renal function

The Predictive Safety Testing Consortiums first regulatory submission to qualify kidney safety biomarkers revealed two deficiencies. To address the need for biomarkers that monitor recovery from agent-induced renal damage, we scored changes in the levels of urinary biomarkers in rats during recovery from renal injury induced by exposure to carbapenem A or gentamicin. All biomarkers responded to histologic tubular toxicities to varied degrees and with different kinetics. After a recovery period, all biomarkers returned to levels approaching those observed in uninjured animals. We next addressed the need for a serum biomarker that reflects general kidney function regardless of the exact site of renal injury. Our assay for serum cystatin C is more sensitive and specific than serum creatinine (SCr) or blood urea nitrogen (BUN) in monitoring generalized renal function after exposure of rats to eight nephrotoxicants and two hepatotoxicants. This sensitive serum biomarker will enable testing of renal function in animal studies that do not involve urine collection.

CHANGES IN THE LIVER PROTEIN PATTERN OF FEMALE WISTAR RATS TREATED WITH THE HYPOGLYCEMIC AGENT SDZ PGU 693

SDZ PGU 693 acts as a hypoglycemic agent by stimulating glucose utilisation in insulin-sensitive peripheral tissues, such as skeletal muscle and fat. In a 28 day toxicity study the compound was found to induce hepatocellular hypertrophy in Wistar rats treated with 300 mg/kg/day. To gain insights into the pathomechanism of these alterations, aliquots of liver samples from control and treated female Wistar rats were separated by two-dimensional protein gel electrophoresis and the digitized images of the protein patterns were searched for protein abundance changes. Significant treatment-related quantitative changes (P < 0.001) were found in 29 liver proteins. Major increases were observed in several microsomal proteins, including NADPH cytochrome P-450 reductase, cytochrome b5 and serine protease inhibitor. The changes in the cytochrome related enzymes, both known co-factors of the P-450 enzyme system, strongly suggest that SDZ PGU 693 induces microsomal proliferation and induction of the P-450 enzyme system. Decreases were observed in a series of mitochondrial proteins, such as F1ATPase-delta subunit and ornithine aminotransferase precursor as well as in several cytosolic proteins such as the liver fatty acid binding protein, arylsulfotransferase and the senescence marker protein-30. The changes in F1ATPase-delta subunit and liver fatty acid binding protein together suggest a down-regulation of the mitochondrial liver fatty acid metabolism, likely reflecting the pharmacological action of the compound. These results show that SDZ PGU 693 produces a complex pattern of gene expression changes which give insights into the molecular mechanisms of both its pharmacological action and a toxic response.

Cyclosporine A-induced Decrease in Calbindin-D 28 kDa in Rat Kidney but not in Cerebral Cortex and Cerebellum

Recently, we reported that in rat, cyclosporine A (CsA) markedly decreases the levels of calbindin-D (CABP-D) 28 kDa in kidney. CABP-D 28 kDa is a calcium-binding protein which is highly expressed in calcium-transporting tissues such as kidney or brain. In this study, we investigated whether, in addition to the kidney, CsA also has an effect on CABP-D 28 kDa in rat brain. Three groups of male Wistar rats received 15 mg/kg/day or 50 mg/kg/day of CsA orally for 12 days, whereas controls received vehicle solution for the same period. CABP-D 28-kDa protein and CsA were quantified in homogenates of kidney, cerebral cortex and cerebellum, and the localization of CABP-D 28 kDa was assessed in the different tissue sections by immunohistochemistry. In kidney, CABP-D 28 kDa was strongly and dose dependently decreased, and was located in tubular epithelial cells. In brain, CABP-D 28 kDa was not changed and was mainly located in pyramidal cells of the cortex and in cerebellum exclusively in Purkinje cells. High CsA concentrations were measured in kidney, more than 17-fold greater than those found in cortex. In cerebellum, CsA was below the limit of detection. These data suggest that at clinically relevant doses, CsA may not affect CABP-D 28-kDa levels in brain.