Cadmium-induced ceramide formation triggers calpain-dependent apoptosis in cultured kidney proximal tubule cells.
A major target of cadmium (Cd(2+)) toxicity is the kidney proximal tubule (PT) cell. Cd(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(2+). Upstream processes underlying Cd(2+)-induced calpain activation remain unclear. We describe for the first time that 10-50 microM Cd(2+) causes a significant increase in ceramide formation by approximately 22% (3 h) and approximately 72% (24 h), as measured by diacylglycerol kinase assay. Inhibition of ceramide synthase with fumonisin B(1) (3 microM) 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(2+) 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 Ca(2+) 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 Cd(2+)-induced apoptosis of PT cells entails endogenous ceramide elevation and subsequent Ca(2+)-dependent calpain activation, which propagates kidney damage by Cd(2+).
Lee
Wing-Kee
WK
Dept. of Physiology & Pathophysiology, Univ. of Witten/Herdecke, Faculty of Medicine, D-58448 Witten, Germany.
Torchalski
Blazej
B
Thévenod
Frank
F
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002104:Cadmium / Q000633:toxicity*; D002154:Calpain / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D002461:Cell Line, Transformed; D002518:Ceramides / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D037341:Fumonisins / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000166:cytology*; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D011921:Rats, Inbred WKY; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
D002518:Ceramides; D004791:Enzyme Inhibitors; D037341:Fumonisins; D002104:Cadmium; C101954:N-caproylsphingosine; C056933:fumonisin B1; D010088:Oxidoreductases; C108138:dihydroceramide desaturase; D002154:Calpain; D053148:Caspase 3