ABSTRACT
The kidney is an important site of xenobiotic-induced toxicity. Because the traditional markers of renal injury indicate only severe renal damage, new biomarkers are needed for a more sensitive and reliable evaluation of renal toxicity. This study was designed to identify in vitro noninvasive biomarkers for efficient assessment of nephrotoxicity by using cisplatin as a model of nephrotoxic compounds. To this end, a comparative proteomic analysis of conditioned media from HK-2 human kidney epithelial cells treated with cisplatin was performed. Here, we identified pyruvate kinase M1/M2 isoform M2 (PKM2) and eukaryotic translation elongation factor 1 gamma (EF-1γ) as potential biomarker candidates for evaluation of nephrotoxicity. PKM2 and EF-1γ were increased by cisplatin in a kidney cell-specific manner, most likely due to cisplatin-induced apoptosis. The increase of PKM2 and EF-1γ levels in conditioned media was also observed in the presence of other nephrotoxic agents with different cytotoxic mechanisms such as CdCl2, HgCl2, and cyclosporine A. Rats treated with cisplatin, CdCl2, or HgCl2 presented increased levels of PKM2 and EF-1γ in the urine and kidney tissue. Taken together, this study identified two noninvasive biomarker candidates, PKM2 and EF-1γ, by comparative proteomic analysis. These new biomarkers may offer an alternative to traditional renal markers for efficient evaluation of nephrotoxicity.
Subject(s)
Biomarkers/metabolism , Cisplatin/toxicity , Kidney/drug effects , Base Sequence , Carrier Proteins/metabolism , Cell Line , DNA Primers , Enzyme-Linked Immunosorbent Assay , Epithelial Cells/drug effects , Humans , Kidney/cytology , Membrane Proteins/metabolism , Peptide Elongation Factor 1/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Thyroid Hormones/metabolism , Thyroid Hormone-Binding ProteinsABSTRACT
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), ß2-microglobulin (ß2-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 ß2-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.
