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.

Exposure to Radiocontrast Agents Induces Pancreatic Inflammation by Activation of Nuclear Factor-κB, Calcium Signaling, and Calcineurin

“…Further, the shape of the Ca 2+ signal in most cells mimicked a high amplitude peak plateau, which is a characteristic pathological signal that precedes acinar cell injury and pancreatitis 25, 26, 48–53 and which would favor calcineurin activation 54 . A recent report demonstrated that a long incubation (of 4 hr) with ioversol, a RC that is similar to the iohexol and iopamidol we used, caused a slow, globalized increase in baseline cytosolic Ca 2+ levels in the NRK-52E renal cell line 55 . It was suggested that the RC triggered voltage-gated Ca 2+ channels or caused reversal of the plasma membrane Na + /Ca 2+ exchanger (NCX), so as to favor Ca 2+ influx.…”

Role of Reactive Oxygen Species-Mediated Endoplasmic Reticulum Stress in Contrast-Induced Renal Tubular Cell Apoptosis

“…It has been proven that the ER stress-induced apoptosis can be mediated by ROS [22] and that oxidative stress plays an important role in the pathogenesis of CI-AKI [12,23]. In the present study, the role of ROS in the ER stress-induced apoptosis in iopromide-treated NRK-52E cells was investigated.…”

“…Cells were subcultured when the cell monolayer reached 80% confluence. Cells were exposed to increasing concentration [50, 100, 150 mg iodine (I)/ml] of iopromide for 4 h based on our previous experiment [12]. In a separate experiment, cells were divided into four groups: (A) control (cells without any treatment); (B) cells exposed to NAC (10 mmol/l); (C) cells exposed to iopromide (100 mg I/ml, 4 h), and (D) NAC (10 mmol/l) added 1 h before iopromide treatment (100 mg I/ml, 4 h).…”

“…Intracellular ROS were measured based on our previous report [12]. Briefly, cells were incubated for 40 min in the presence of 10 μ M CM-H2DCFDA at 37°C, followed by washing in Hanks' balanced salt solution three times.…”

A Voltage Dependent Non-Inactivating Na+ Channel Activated during Apoptosis in Xenopus Oocytes

“…Another possibility is that the increased intracellular Na + concentration leads to an increased intracellular Ca 2+ which consequently affects the apoptotic process. Increased intracellular Ca 2+ has been suggested to trigger apoptosis in different cell types [40], [41]. Normally intracellular Ca 2+ is pumped out through a Na + /Ca 2+ exchanger (NCX exchangers) [42].…”