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Alternatives to Laboratory Animals - ATLA

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Evaluation of a Short-term Rat Proximal Tubule Incubation System for the Detection of Nephrotoxicants

Rebecca C. Elton, Pauline Rhodes and Jeffrey R. Fry

Many nephrotoxic agents act primarily on proximal tubule cells. Accordingly, the optimal conditions for isolating rat proximal tubule fragments by a collagenase digestion technique and their short-term maintenance have been defined, and the viability of the preparation and its sensitivity to toxicants have been determined. Tubular fragment viability was maintained in incubation for up to 6 hours, as assessed by measuring lactate dehydrogenase leakage, levels of intracellular glutathione, and ATP content. In addition, tubular transport function was maintained, as determined by measuring the uptake of p-aminohippuric acid and α-methylglucose, which could be blocked by the selective inhibitors, probenecid and phloridzin, respectively. In this study, the toxicities of allyl alcohol, cephalosporins and cisplatin to proximal tubular fragments were investigated. Allyl alcohol toxicity was greater in tubular fragments isolated from female rats than in those from male rats. Cephaloridine was toxic, while cephalexin and cephalothin were not. These features were consistent with the known in vivo responses to these agents. Toxicity was evident after exposure to cisplatin, with an early reduction in tubular transport being noted. The results highlight the potential of the system described for the isolation and incubation of rat proximal tubule fragments to study xenobiotic-mediated nephrotoxicity. This system could be of benefit in the high-throughput toxicity screening of novel therapeutic agents.