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Effect of nitric oxide on iron‐mediated cytotoxicity in primary cultured renal proximal tubules
Author(s) -
Wu Zhaolong,
Qiu Lianqun
Publication year - 2001
Publication title -
cell biochemistry and function
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.933
H-Index - 61
eISSN - 1099-0844
pISSN - 0263-6484
DOI - 10.1002/cbf.921
Subject(s) - nitric oxide , cytotoxicity , chemistry , lipopolysaccharide , lactate dehydrogenase , reactive oxygen species , nitric oxide synthase , incubation , nitrite , griess test , biochemistry , pharmacology , endocrinology , enzyme , in vitro , biology , nitrate , organic chemistry
Nitric oxide (NO) has been proved to be a mediator of hypoxic injury in renal proximal tubules (PT), but its effect on iron‐induced cytotoxicity has remained little known. In this study, we observed the relationship between NO production and lactate dehydrogenase (LDH) release in primary proximal tubular epithelia co‐incubated with different doses of NTA‐Fe and lipopolysaccharide (LPS) alone or in combination. NO production was monitored by NO 2 concentration in supernatants based on the Griess reaction; while the semi‐quantitative RT‐PCR was applied to detect the inducible nitric oxide synthase (iNOS) mRNA level induced by NTA‐Fe and LPS together. In addition, experimental groups were subjected to reactive oxygen species (ROS) scavengers to determine the impact of the interaction between NO and ROS on iron‐mediated cytotoxicity. After a 12‐h co‐incubation, we found that NTA‐Fe increased both LDH release and 2 − production in a dose‐dependent manner ( P < 0.001). The level of iNOS mRNA induced by LPS was enhanced by 500 μ m NTA‐Fe ( P < 0.01), lower or higher concentrations had no effect. However, the supernatant 2 − level in the same group did not change significantly ( P > 0.05) although tubular injury was aggravated ( P < 0.001). The addition of l ‐arginine increased LDH release from 25.05 ± 8.36% in the iron group to 38.67 ± 7.67% in iron plus LPS group ( P < 0.05); concomitantly, l ‐NAME mitigated iron toxicity in LPS‐treated PT ( P < 0.05). Hydroxyl scavengers provided complete protection against iron‐mediated cytotoxicity ( P < 0.001), but the decrease of 2 − production was only significant in the LPS‐treated group. In contrast, SOD was partially effective in the LPS group ( P < 0.05) whereas the 2 − level in the supernatant was inversely raised ( P < 0.05). GSH had no effect on either iron toxicity or 2 − production. Thus, we conclude that NO can exacerbate the cytotoxicity caused by NTA‐Fe in cultured proximal tubular epithelia, but NO is not the only factor. NTA‐Fe could enhance the upregulation of iNOS transcription induced by LPS in a specific concentration range, and its regulation of NO production might also involve a post‐transcription mechanism. The hydroxyl group is the major mediator in our model and the pro‐oxidant role of NO is probably due to its ability to promote the Fenton reaction and form both ONOO − and ˙ OH via its interaction with ROS. Copyright © 2001 John Wiley & Sons, Ltd.