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 µM Cd²⁺ 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 B₁ (3 µM) 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 C₆-ceramide to PT cells increases cytosolic Ca²⁺ and activates calpains. Apoptosis mediated by C₆-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⁺. nephrotoxicity; cell signaling; cell biology and structure