[Cd.sup.2+]-induced cytochrome c release in apoptotic proximal tubule cells: role of mitochondrial permeability transition pore and [Ca.sup.2+] uniporter

[Cd.sup.2+] induces apoptosis of kidney proximal tubule (PT) cells. Mitochondria play a pivotal role in toxic compound-induced apoptosis by releasing cytochrome c. Our objective was to investigate the mechanisms underlying [Cd.sup.2+]-induced cytochrome c release from mitochondria in rat PT cells. Using Hoechst 33342 or MTT assay, 10 [micro]M [Cd.sup.2+] induced ~5-10% apoptosis in PT cells at 6 and 24 h, which was associated with cytochrome c and apoptosis-inducing factor release at 24 h only. This correlated with previously described maximal intracellular [Cd.sup.2+] concentrations at 24 h, suggesting that elevated [Cd.sup.2+] may directly induce mitochondrial liberation of proapoptotic factors. Indeed, [Cd.sup.2+] caused swelling of energized isolated kidney cortex mitochondria (E[C.sub.50] ~9 [micro]M) and cytochrome c release, which were independent of permeability transition pore (PTP) opening since PTP inhibitors cyclosporin A or bongkrekic acid had no effect. On the contrary, [Cd.sup.2+] inhibited swelling and cytochrome c release induced by PTP openers (P[O.sup.3-.sub.4] or [H.sub.2][O.sub.2]+[Ca.sup.2+]). The mitochondrial [Ca.sup.2+] uniporter (MCU) played a key role in mitochondrial damage: 1) MCU inhibitors ([La.sup.3+], ruthenium red, Ru360) prevented swelling and cytochrome c release; and 2) ruthenium red attenuated [Cd.sup.2+] inhibition of P[O.sup.3-.sub.4]-induced swelling. Using the [Cd.sup.2+]-sensitive fluorescent indicator FluoZin-1, [Cd.sup.2+] was also taken up by mitoplasts. The aquaporin inhibitor AgN[O.sub.3] abolished [Cd.sup.2+]-induced swelling of mitoplasts. This could be partially mediated by activation of the mitoplast-enriched water channel aquaporin-8. Thus cytosolic [Cd.sup.2+] concentrations exceeding a certain threshold may directly cause mitochondrial damage and apoptotic development by interacting with MCU and water channels in the inner mitochondrial membrane. cadmium; aquaporin; apoptosis-inducing factor; cyclosporin A