摘要：目的探讨一氧化氮(NO)参与铁负荷肾小管细胞毒作用的可能机制，评价氧自由基清除剂在铁肾小管细胞毒中的防治作用及其与NO的关系。方法在建立原代鼠肾近端小管上皮细胞培养体系的基础上，以孵育液中NO2-产量(Griess反应)和小管细胞乳酸脱氢酶(LDH)释放率改变为线索，比较不同剂量铁剂、各氧自由基清除剂、L精氨酸(L-Arg)和CO合成酶抑制剂，左旋硝基精氨酸甲酯(L-NAME)分别作用12h后在非脂多糖(LIS)组和LPS组(10μg／ml)中对小管细胞毒作用及与NO代谢的影响；同时用半定量逆转录－聚合酶链反应(RT-PCR)法检测不同剂量铁剂与LPS的联用下诱导型一氧化氮合酶(iNOS)mRNA的表达改变。结果NTA-Fe可以剂量依赖的方式同步增加LDH释放(P＜0.001)及孵育液中NO2－的产量(P＜0.001)；但在LPS组中细胞毒作用的增加(P＜0.001)并不伴随N02－的明显改变(P＞0.05)。LPS诱导的iNOS转录上调可为NTA-Fe增强，但仅限于500μmol／L(P＜0.01)。在LPS组中，加入L-精氨酸及不同剂量L-NAME可分别加重和缓解铁的细胞毒作用；然而，L-NAME的防护作用在铁剂组中并不明显；OH清除剂可防护铁的毒作用(P＜0.001)，但NO2－水平的相应下降仅见于二甲基硫脲(DMTU)作用的LPS组中(P＜0.05)。结论在肾小管上皮细胞中，NO可促进NTA-Fe的细胞毒作用；一定浓度范围内铁可增强LPS诱导的iNOS转录上调；OH是NTA-Fe细胞毒作用的主要介质；NO的参与很可能是通过与O2-和H2O2的相互作用及促进Fenton反应，生成高反应活性的OH及或ONOO-。

Effect of nitric oxide on iron-mediated cytotoxicity in primary cultured renal proximal tubules

qIU Lianqun,WU Zhaolong,XU Xunhui(Division of Nephrology, Zhongshan Hospital, Shanghai Medical University, Shanghai 200032, China)

Abstract：Objective To explore the possible mechanism of nitric oxide(NO) involved in iron-mediated cytotoxicity on renal tubular cells, meanwhile to estimate the effect of reactive oxygen sepcies scavenger on iron-mediated cytotoxicity and its relation to nitric oxide. Methods In this study, the relationship between NO production and lactate dehydrogenase(LDH) release were observed in primary subconfiuent proximal tubular cells coincubated with different doses of nTA-Fe and lipopolysaccharide(LPS) alone or in combination. NO production was monitored by NO2- concentration in supernatant based on Griess reaction. Meanwhile, 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 exposed to reactive oxygen species(ROS) scavengers to determine the impact of the interaction between NO and ROS on iron-mediated cytotoxlcity. Results After 12-hour coincubation, NTA-Fe could increase both LDH release and NO2- production in a dose-dependent manner (P＜0.001). Only 500 μmol/L nTA-Fe could enhance the level of iNOS mRNA induced by LPS (P＜0.01) . However, the supernatant NO2- 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 and L-NAME could respectively exacerbate and mitigate iron-mediated cytotoxicity in LPS additive group. Hydroxyl scavengers provided complete protection against iron-mediated cytotoxicity (P＜0.001), but the NO2- production decrease was only significant in LPS additive group. In contrast, SOD was partially effective in LPS group (P＜0.05), while the NO2- level in supernatant was inversely raised (P＜0.05) . GSH had no effect on both iron toxicity and NO2- production. Conclusions NO may exacerbate the cytotoxicity caused by nTA-Fe in primary cultured proximal tubular epithelium. NTA-Fe may enhance the up-regulation of iNOS transcription induced by LPS in a specific concentration range. Hydroxyl group was the major mediator in our model and the pro-oxidant role of NO was due to its ability to promote Fenton reaction and the form of both oNOO- and OH via its interaction with ROS such as O2-.

Keywords：Cytotoxicity; Nitric oxide; Reactive oxygen species; Renal proximal tubular cell参考文献：

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收稿日期：1999-09-17