In vitro evaluation of biomarkers for cisplatin-induced nephrotoxicity using HK-2 human kidney epithelial cells

The non-animal in vitro test methods, especially for assessment of kidney toxicity, have become invaluable tools due to the target organ-selective nature of many nephrotoxic xenobiotics. In vitro evaluation of biomarkers for nephrotoxicity assessment using human cell lines, which can provide more reliable information for toxicological risk evaluation in humans than animal cells, has not been well established to date. The present study investigated the potential use of biomarkers for cisplatin-induced nephrotoxicity assessment in vitro using HK-2 cells derived from human kidney proximal tubule epithelial cells. Cisplatin induced apoptosis of HK-2 cells in which down-regulation of Bcl-2 and activation of caspase-3 were possibly involved. We investigated the effect of cisplatin on the protein levels of kidney injury molecule (KIM)-1, clusterin, calbindin, tissue inhibitor of metalloproteinase (TIMP)-1, cystatin C (CysC), β₂-microglobulin (β₂-M) and neutrophil gelatinase associated lipocalin (NGAL), which have been recently identified as in vivo biomarkers of nephrotoxicity. The protein levels of KIM-1, calbindin and TIMP-1 were significantly increased in the conditioned media of HK-2 cells treated with cisplatin, while β₂-M, CysC, NGAL and clusterin were not affected by cisplatin treatment. The mRNA levels of KIM-1, calbindin and TIMP-1 were increased by cisplatin, indicating that cisplatin-induced up-regulation involves transcriptional activation. The levels of KIM-1, calbindin and TIMP-1 were significantly increased in urine of cisplatin-treated rats, providing in vivo validation of the in vitro results. Taken together, our results clearly demonstrate that among the known in vivo nephrotoxic biomarkers, KIM-1, calbindin and TIMP-1 can be effectively used as in vitro biomarkers for cisplatin-induced nephrotoxicity using a HK-2 human kidney cell system.