Comparative in vitro mechanisms of combined metal toxicity to renal proximal tubule cells and the role of the stress response

Renal proximal tubule cells are a primary target cell population in the kidney for arsenite (As), cadmium (Cd), and lead (Pb) toxicity following chronic in vivo exposures, but mixture interactions of these elements have received relatively little study. The combined effects of these elements on regulatory processes such as the stress protein response, protein phosphorylation, and the caspase system were studied. The relative in vitro toxicity of single and combined As and Cd in both the immortalized rat NRK-52E and human HK-2 kidney proximal tubule cell lines and in primary cultures of human kidney cells were evaluated by cell morphology, Alamar Blue metabolism and for cell density by the Cyquant assay. Western Immunoblot expression studies of the 25/27 kDa, 32 kDa, 60 kDa, 70 kDa and 90 kDa stress protein families, caspase-3, and tyrosine phosphorylation in both immortalized renal cell lines were examined in relation to cell toxicity. Results from toxicity assay studies showed the HK-2 cell line was generally more sensitive than NRK-52E cells to single As and Cd doses, but NRK-52E was more sensitive to the combination mixtures of these elements. Both kidney cell lines were more sensitive than primary human kidney cells to the toxic effects of As or Cd alone or in combination. Combinations of As plus Cd were more toxic in a synergistic manner as evaluated on a statistical basis using multiple linear regression. Caspase-3 expression was not altered at lower dose levels, but was inhibited at the highest dose levels where protein tyrosine phosphorylation was increased. General effects on altered stress protein expression patterns were observed at lower doses by combinations of As plus Cd and further enhanced in the As, Cd and Pb mixture combination for longer exposures. Results of DNA laddering and TUNEL assays showed only a minority of cells underwent cell death via apoptosis. Overall, results of these studies indicate that combinations of these elements alter the normal stress protein response patterns that occur for a single element as a function of dose, time, and cell type and act to determine cellular toxicity and cell death pathways.