Selective Inhibition of the Reverse Mode of Na+/Ca2+ Exchanger Attenuates Contrast-Induced Cell Injury

Abstract

Background: The precise mechanisms underlying radiocontrast nephropathy (RCN) are not well understood. Intracellular Ca²⁺ overload is considered to be a key factor in RCN. The Na⁺/Ca²⁺ exchanger (NCX) system is one of the main pathways of intracellular Ca²⁺ overload. We investigated whether intracellular Ca²⁺ 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<smlcap>M</smlcap>) 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 Ca²⁺ concentration ([Ca²⁺]_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 [Ca²⁺]_i and intracellular ROS. KB-R7943 dose-dependently and significantly suppressed the increase in [Ca²⁺]_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 Ca²⁺ 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.