Background: Cellular and molecular mechanisms responsible for cisplatin-induced nephrotoxicity to renal tubular epithelial cells are not well understood. Although caspases play a critical role in the execution of the cell death 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 using caspase inhibitors and p35 transfected LLC-PK1 cells. The Akt/PKB phosphorylation 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 markedly increased in a time (8 to 24 hours) and dose-dependent manner (0 to 200 micromol/L). Proinflammatory caspase-1 did not show cisplatin-induced activation. Inhibition of caspase-3 by over expressing cowpox virus p35 protein or alternatively by the peptide inhibitor DEVD-CHO provided marked protection against cell death and partial protection against DNA damage. We then examined the role of the Akt/PKB phosphorylation pathway in regulation of cisplatin-induced caspase activation. There was a marked induction of Akt/PKB phosphorylation in a time (0 to 8 hours) and dose-dependent (0 to 200 micromol/L) manner during the course of cisplatin injury. Cisplatin-induced Akt/PKB activation was associated with Bad phosphorylation, suggesting induction of a cell survival signal mediated by the Bcl-2 family member, Bad. Wortmannin or LY294002, two structurally dissimilar inhibitors of phosphatidylinositol 3'-kinase (PI-3 kinase), abolished both cisplatin-induced Akt phosphorylation and Bad phosphorylation, and promoted cisplatin-induced early and accelerated activation of caspase-3 and caspase-9, but not of caspase-8 and caspase-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-induced induction of the Akt/PKB phosphorylation pathway, inhibition of which enhances activation of caspase-3 and caspase-9.