The kidney is one of the main targets of xenobiotic-induced toxicity, but early detection of renal damage is difficult. Recently, several novel biomarkers of nephrotoxicity have been identified by transcription profiling, including kidney injury molecule-1 (Kim-1), lipocalin-2, tissue inhibitor of metalloproteinases-1 (Timp-1), clusterin, osteopontin (OPN), and vimentin, and suggested as sensitive endpoints for acute kidney injury in vivo. However, it is not known if these cellular marker molecules may also be useful to predict chronic nephrotoxicity or to detect nephrotoxic effects in vitro. In this study, a panel of new biomarkers of renal toxicity was assessed via quantitative real-time PCR, immunohistochemistry, and immunoblotting in rats treated with the nephrotoxin ochratoxin A (OTA) for up to 90 days and in rat proximal tubule cells (NRK-52E) treated with OTA in vitro. Repeated administration of OTA to male F344/N rats for 14, 28, or 90 days resulted in a dose- and time-dependent increase in the expression of Kim-1, Timp-1, lipocalin-2, OPN, clusterin, and vimentin. Changes in gene expression were found to correlate with the progressive histopathological alterations and preceded effects on traditional clinical parameters indicative of impaired kidney function. Induction of Kim-1 messenger RNA expression was the earliest and most prominent response observed, supporting the use of this marker as sensitive indicator of chronic kidney injury. In contrast, no significant increase in the expression of putative marker genes and proteins were evident in NRK-52E cells after exposure to OTA for up to 48 h, suggesting that they may not be suitable endpoints for sensitive detection of nephrotoxic effects in vitro.

Ochratoxin A (OTA) is nephrotoxic and a potent renal carcinogen. Male rats are most susceptible to OTA toxicity, and chronic administration of OTA (70 and 210 microg/kg bw) for 2 years has been shown to induce high incidences of adenomas and carcinomas arising from the straight segment of the proximal tubule epithelium. In contrast, treatment with a lower dose of 21 microg/kg bw did not result in increased tumor rates, suggesting a nonlinear dose response for renal tumor formation by OTA. Since the mechanism of OTA carcinogenicity is still largely unknown, this study was conducted to investigate early functional and pathological effects of OTA and to determine if sustained stimulation of renal cell proliferation plays a role. Male F344/N rats were treated with OTA for up to 13 weeks under conditions of the National Toxicology Program (NTP) bioassay. Cell proliferation in the renal cortex and outer stripe of the outer medulla (OSOM) was determined using bromodeoxyuridine incorporation and immunohistochemistry. Histopathological examination showed renal alterations in mid- and high-dose-treated animals involving single-cell death and prominent nuclear enlargement within the straight proximal tubules. Treatment with OTA at doses of 70 and 210 microg/kg bw led to a marked dose- and time-dependent increase in renal cell proliferation, extending from the medullary rays into the OSOM. No effects were evident in kidneys of low-dose-treated animals or in the liver, which is not a target for OTA carcinogenicity. A no observed effect level in this study was established at 21 microg/kg bw, correlating with the dose in the NTP 2-year bioassay that did not produce renal tumors. The apparent correlation between enhanced cell turnover and tumor formation induced by OTA indicates that stimulation of cell proliferation may play an important role in OTA carcinogenicity and provides further evidence for an epigenetic, thresholded mechanism.

The discussion on whether the Sprague Dawley (SD), the Fischer F344, or the Hannover Wistar rat is the most appropriate model for toxicity studies in rodents is ongoing. A substantial quantity of data on these strains concerning their source, diet, and housing conditions have been published. Generally, before starting a toxicology program in rodents, it should be taken into account that oncogenicity studies will be required for the majority of compounds successfully completing development. Survival, body weight development, incidence, type, time of onset of age-dependent lesions and neoplasms, as well as some special considerations of the rat model selected may be decisive. Therefore, an understanding of the historical background data is essential. These aspects demonstrate why the use of a specific rat model should be carefully considered at the beginning of the toxicology program.

Many variables may affect the outcome of continuous infusion studies. The results largely depend on the experience of the laboratory performing these studies, the technical equipment used, the choice of blood vessels and hence the surgical technique as well the quality of pathological evaluation. The latter is of major interest due to the fact that the pathologist is not involved until necropsy in most cases, i.e. not dealing with the complicated surgical or in-life procedures of this study type. The technique of tissue sampling during necropsy and the histology processing procedures may influence the tissues presented for evaluation, hence the pathologist may be a source of misinterpretation. Therefore, ITO proposes a tissue sampling procedure and a standard nomenclature for pathological lesions for all sites and tissues in contact with the port-access and/or catheter system.

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