Cadmium-induced ceramide formation triggers calpain-dependent apoptosis in cultured kidney proximal tubule cells

A major target of cadmium ([Cd.sup.2+]) toxicity is the kidney proximal tubule (PT) cell. [Cd.sup.2+]-induced apoptosis of PT cells is mediated by sequential activation of calpains at 3-6 h and caspases-9 and -3 after 24-h exposure. Calpains also partly contribute to caspase activation, which emphasizes the importance of calpains for PT apoptosis by [Cd.sup.2+]. Upstream processes underlying [Cd.sup.2+]-induced calpain activation remain unclear. We describe for the first time that 10-50 [micro] M [Cd.sup.2+] causes a significant increase in ceramide formation by ~22% (3 h) and ~72% (24 h), as measured by diacylglycerol kinase assay. Inhibition of ceramide synthase with fumonisin [B.sub.1] (3 [micro] M) prevents ceramide formation at 3 h and abolishes calpain activation at 6 h, which is associated with significant attenuation of apoptosis at 3-6 h with Hoechst 33342 nuclear staining and/or 3(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) death assays. This indicates that [Cd.sup.2+] enhances de novo ceramide synthesis and that calpains are a downstream target of ceramides in apoptosis execution. Moreover, addition of [C.sub.6]-ceramide to PT cells increases cytosolic [Ca.sup.2+] and activates calpains. Apoptosis mediated by [C.sup.6]-ceramide at 24 h is significantly reduced by caspase-3 inhibition, which supports cross talk between calpain- and caspase-dependent apoptotic pathways. We conclude that [Cd.sup.2+]-induced apoptosis of PT cells entails endogenous ceramide elevation and subsequent [Ca.sup.2+] -dependent calpain activation, which propagates kidney damage by [Cd.sup.2+]. nephrotoxicity; cell signaling; cell biology and structure