Use of flow cytometry and confocal microscopy techniques to investigate early CdCl(2)-induced nephrotoxicity in vitro

CdCl(2) is a well-known toxic compound for the kidney in vivo and in vitro. We report here part of the results of an ECVAM (European Centre for the Validation of Alternative Methods) contract study, aimed at establishing and assessing several flow cytometric and confocal microscopic endpoints for use in an in vitro nephrotoxicity model. Three renal tubule cell lines, OK (opossum, proximal tubule origin), LLC-PK1 (pig, proximal tubule origin) and MDCK (dog, distal tubule origin) were exposed for 1, 5 and 24 h to 25 microM and 100 microM CdCl(2). The results obtained for mitochondrial membrane potential showed a decrease in all the cell lines after 5 h of treatment with both CdCl(2) concentrations. In some cases, this decrease was detected by flow cytometry after a 1-h exposure. On the contrary, intracellular Ca(2+) increased in a time-dependent and concentration-dependent fashion. This increase was especially high in the MDCK cell line after a 24-h exposure to 100 microM CdCl(2). However, cell viability was not affected by 25 microM CdCl(2). Our results demonstrate early changes in mitochondrial membrane potential and cytoplasmic Ca(2+) levels in renal tubular epithelial cell lines treated with CdCl(2).