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

A major target of cadmium ( Cd2+) toxicity is the kidney proximal tubule ( PT) cell. Cd2+-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 Cd2+. Upstream processes underlying Cd2+-induced calpain activation remain unclear. We describe for the first time that 10-50 mu M Cd2+ causes a significant increase in ceramide formation by similar to 22% ( 3 h) and similar to 72% ( 24 h), as measured by diacylglycerol kinase assay. Inhibition of ceramide synthase with fumonisin B-1 ( 3 mu 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 Cd2+ enhances de novo ceramide synthesis and that calpains are a downstream target of ceramides in apoptosis execution. Moreover, addition of C-6-ceramide to PT cells increases cytosolic Ca2+ and activates calpains. Apoptosis mediated by C-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 Cd2+-induced apoptosis of PT cells entails endogenous ceramide elevation and subsequent Ca2+ -dependent calpain activation, which propagates kidney damage by Cd2+.