一氧化氮参与铁负荷肾小管细胞毒作用的机制及其防治

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

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 １２ ｈ后在非脂多糖（ＬＰＳ）组和 ＬＰＳ组（１０ ｕｇ／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Ｎ０２－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５００ｕｍｏｌ／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。

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 subconfluent 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 cytotoxicity. 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 umol/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 ), hut 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-.