Secreted Proteins, Lipids and Low-Molecular-Weight Metabolites as Early Biomarkers of Human Proximal Tubular Cell Exposure to Nephrotoxic Agents.

Mitochondria and other organelles are early and sensitive targets for numerous nephrotoxic drugs and environmental contaminants. To prevent irreversible kidney injury from such chemical exposures it is critical to identify indicators (biomarkers) that correspond with the exposures before significant injury occurs. Primary cultures of human proximal tubular (hPT) cells were used as the model system to test the hypothesis that exposure to low, physiologically relevant concentrations of these agents causes release of proteins, lipids and metabolites into the extracellular space that correlate with specific exposures and cytotoxicity. Previous work with the nephrotoxic and mitochondrial-targeting toxicant S-(1,2-dichlorovinyl)-L-cysteine (DCVC) confirmed that a large proportion (43%) of proteins recovered in the extracellular medium was of mitochondrial origin. Three of the most abundant proteins detected, for which levels were increased by 5- to 10-fold by DCVC, were sulfite oxidase (mitochondrial), keratins (cytoskeletal), and HSP90 (cytoplasm). Comparisons of proteins detected in media from cells exposed to cisplatin (CDDP) vs. Control or polymyxin B (PmxB) vs. Control showed several commonly increased proteins. As with results from DCVC exposure, keratins and HSP90 were detected. The only mitochondrial protein that increased in abundance in supernatants from toxicant-treated cells was carbamoyl-phosphate synthase (1.4- and 2.3-fold higher in supernatants from CDDP- and PmxB-treated cells, respectively). Metabolomic analysis showed moderate changes (429 features at raw P < 0.05) for CDDP-treated cells vs. Control and moderate to high changes (619 features at raw P < 0.05) for PmxB-treated cells vs. Control. For CDDP. There was consistently lower release of 2-aminoadipate 6-semialdehyde and differences in purines, lipid species, and several amino acids. For PmxB, there was consistently lower release of aminoadipic acid and 2-aminoadipate 6-semialdehyde and differences in release of lipid species and lower release of phosphoenolpyruvate. These findings support the overall hypothesis and identify multiple candidate biomarkers of early exposure to diverse nephrotoxicants.