Selective inhibition of the reverse mode of Na(+)/Ca(2+) exchanger attenuates contrast-induced cell injury

Background: The precise mechanisms underlying radiocontrast nephropathy (RCN) are not well understood. Intracellular CA(2+) overload is considered to be a key factor in RCN. The Na(+)/CA(2+) exchanger (NCX) system is one of the main pathways of intracellular CA(2+) overload. We investigated whether intracellular CA(2+) overload via the NCX system was involved in contrast-induced renal tubular cytotoxicity.

Methods: NRK-52E cells were exposed to ioversol (100 mg iodine/ml) for 4 h. KB-R7943 (inhibitor of reverse mode of NCX, 4 × 10(-5), 4 × 10(-6)M) was added 1 h before incubation with ioversol. Cell viability and permeability were determined by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide and Lactate Dehydrogenase assay. Apoptosis was determined by flow cytometry. Intracellular CA(2+) concentration ([CA(2+)](i)] and Reactive Oxygen Species (ROS) were detected by confocal microscopy. The expression of NCX1 mRNA and Caspase-3 protein was evaluated by reverse transcription-polymerase chain reaction and Western blot, respectively.

Results: Ioversol exposure induced significantly increased Lactate Dehydrogenase release and decreased 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide conversion in NRK-52E cells. Significantly increased Apoptosis and Caspase-3 protein expression were observed in the NRK-52E cells exposed to ioversol for 4 h. Ioversol treatment induced a significant increase in [CA(2+)](i) and intracellular ROS. KB-R7943 dose-dependently and significantly suppressed the increase in [CA(2+)](i), intracellular ROS and Caspase-3 overexpression induced by ioversol and attenuated the contrast-induced NRK-52E cell Apoptosis. No significant changes in NCX1 mRNA expression were observed following contrast exposure.

Conclusion: Intracellular CA(2+) overload via the reverse mode of NCX, followed by ROS overproduction and Caspase-3 overexpression played an important role in the contrast-induced renal tubular cytotoxicity. The reverse mode of the NCX inhibitor KB-R7943 attenuated contrast-induced renal tubular cytotoxicity.