Role and regulation of activation of caspases in cisplatin-induced injury to renal tubular epithelial cells

Background. Cellular and molecular mechanisms responsible for cisplatin-ind uced nephrotoxicity to renal tubular epithelial cells are not well understo od. Although caspases play a critical role in the execution of the cell dea th pathway, their specific role in toxic injury to renal tubular epithelial cells has not been elucidated previously. Methods. The role of caspases in cisplatin-induced injury was determined us ing caspase inhibitors and p35 transfected LLC-PK1 cells. The Akt/PKB phosp horylation pathway was studied for the regulation of caspase activation in these cells. Results. The activation of initiator caspases-8, -9 and -2, and executioner caspase-3 began after eight hours of cisplatin treatment, thereafter marke dly increased in a time (8 to 24 hours) and dose-dependent manner (0 to 200 mu mol/L). Proinflammatory caspase-1 did not show cisplatin-induced activa tion. Inhibition of caspase-3 by over expressing cowpox virus p35 protein o r alternatively by the peptide inhibitor DEVD-CHO provided marked protectio n against cell death and partial protection against DNA damage. We then exa mined the role of the Akt/PKB phosphorylation pathway in regulation of cisp latin-induced caspase activation. There was a marked induction of Akt/PKB p hosphorylation in a time (0 to 8 hours) and dose-dependent (0 to 200 mu mol /L) manner during the course of cisplatin injury. Cisplatin-induced Akt/PKB activation was associated with Bad phosphorylation, suggesting induction o f a cell survival signal mediated by the Bcl-2 family member, Bad. Wortmann in or LY294002, two structurally dissimilar inhibitors of phosphatidylinosi tol 3'-kinase (PI-3 kinase), abolished both cisplatin-induced Akt phosphory lation and Bad phosphorylation, and promoted cisplatin-induced early and ac celerated activation of caspase-3 and caspase-9, but not of caspase-8 and c aspase-1, indicating that inhibition of the Akt/PKB phosphorylation pathway enhances the mitochondrial-dependent activation of caspases. The impact of enhanced activation of caspases by wortmannin or LY294002 was reflected on accelerated cisplatin-induced cell death. Conclusions. These studies demonstrate differential activation and role of caspases in cisplatin injury, and provide the first evidence of cisplatin-i nduced induction of the Akt/PKB phosphorylation pathway, inhibition of whic h enhances activation of caspase-3 and caspase-9.