Effect of nitric oxide on iron-mediated cytotoxicity in primary cultured renal proximal tubules.
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 NO2 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 NO2(-) production in a dose-dependent manner (P < 0.001). The level of iNOS mRNA induced by LPS was enhanced by 500 microM NTA-Fe (P < 0.01), lower or higher concentrations had no effect. However, the supernatantNO2(-) 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 NO2(-) production was only significant in the LPS-treated group. In contrast, SOD was partially effective in the LPS group (P < 0.05) whereas the NO2(-) level in the supernatant was inversely raised (P < 0.05). GSH had no effect on either iron toxicity or NO2(-) 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.
Wu
Z
Z
Division of Nephrology, Zhong Shan Hospital, Shanghai Medical University, Shanghai 200032, PR China.
Qiu
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L
D000818:Animals; D001120:Arginine / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology; D002478:Cells, Cultured; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005290:Ferric Compounds / Q000494:pharmacology / Q000633:toxicity*; D016166:Free Radical Scavengers / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000633:toxicity*; D009573:Nitrites / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013482:Superoxide Dismutase / Q000494:pharmacology; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology
D002273:Carcinogens; D004791:Enzyme Inhibitors; D005290:Ferric Compounds; D016166:Free Radical Scavengers; D008070:Lipopolysaccharides; D009573:Nitrites; D009569:Nitric Oxide; C038983:1,3-dimethylthiourea; D001120:Arginine; D007770:L-Lactate Dehydrogenase; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D013482:Superoxide Dismutase; D005978:Glutathione; D013890:Thiourea; D009571:Nitrilotriacetic Acid; D019331:NG-Nitroarginine Methyl Ester; C020326:ferric nitrilotriacetate