Role of Mitochondrial Ca2+ in the Oxidative Stress-induced Dissipation of the Mitochondrial Membrane Potential

The relationship between mitochondrial Ca2+, oxidative stress, and a dissipation of the mitochondrial membrane potential (A+) was investigated in proximal tubular kidney cells. Freshly isolated proximal tubular cells from rat kidney were exposed to the nephrotoxin 1,2dichlorovinyl-L-cysteine (DCVC). DCVC stimulated the formation of hydroperoxides as determined by flow cytometry using the hydroperoxide-sensitive compound dichlorofluorescin. This was prevented by the antioxidant diphenylphenylenediamine (DPPD) and the iron chelator desferrioxamine. Studies in individual cells with video-intensified fluorescence microscopy showed that a DCVC-induced increase in the intracellular free calcium concentration ([Ca2+li) was accompanied by an increase in the mitochondrial free calcium concentration ([Ca2+1,). The latter increase was selectively prevented by an inhibitor of the mitochondrial calcium uniporter, ruthenium red (RR). Chelation of cellular Ca2+ with EGTA acetoxymethyl ester (EGTNAM) completely prevented the formation of hydroperoxides, whereas inhibition of the uptake of Ca2+ by the mitochondria with RR reduced it. This indicates that the increase in [Ca2+l, is important for the induction of oxidative stress by DCVC. DPPD and desferrioxamine did not protect against a DCVC-induced increase in [Ca2+], and [Ca2+],, indicating that oxidative stress is the consequence rather than the cause of the cellular calcium perturbations. DCVC decreased A+ and caused cell death; both effects were clearly delayed by EGTNAM and RR, although they could not prevent a decrease in A+. The latter decrease was completely prevented by inhibition of the P-lyase-mediated metabolism of DCVC with aminooxyacetic acid. Like EGTNAM, inhibition of oxidative stress with DPPD and desferrioxamine delayed the decrease in A+. This strongly suggests hat the decrease in A+ caused by metabolites of DCVC directly is potentiated by Ca2+-dependent DCVC-induced hydroperoxide formation. The importance of both hydroperoxide formation and mitochondrial damage in DCVC-induced cell killing is discussed.