一氧化氮在肾缺血再灌注肾小球损伤中的作用

目的:探讨一氧化氮(NO)对肾缺血再灌注（ischemia-reperfusion injury，I-RI）时大鼠肾小球超微结构及负电荷位点的影响。 方法:SD大鼠15只，建立肾缺血再灌注模型，动物随机分为5组：（1）假手术（sham）组（n=6）；（2）I-RI组（n=6），缺血前20 min舌静脉注入生理盐水0.3 mL；（3）SNP+I-RI组（n=6），缺血前20 min舌静脉注入2.5 μg/kg硝普钠（SNP）；（4）AG+I-RI组（n=6），缺血前20 min舌静脉注入10 mg/kg氨基胍（AG）；（5）L-NNA+I-RI组（n=6），缺血前20 min舌静脉注入10 mg/kg L-硝基精氨酸（L-NNA）。以聚乙烯亚胺（PEI）为阳离子探针标记肾小球滤过膜负电荷位点，透射电镜观察肾I-RI对大鼠肾小球超微结构及负电荷位点的影响。 结果:(1) sham组电镜下见肾小球结构正常，肾小球基底膜（GBM）外透明层负电荷位点（AS）清晰，呈连续的规则点线状排列［（19.3±1.7）个/1 000 nm］。I-RI组肾小球足细胞足突有明显的融合现象；GBM外透明层AS排列稀疏［（16.6±1.0）个/1 000 nm，P<0.05］，PEI颗粒小。（2）与I-RI组相比，给予SNP使肾I-RI大鼠肾小球滤过膜上皮细胞足突融合现象加重，肾小球GBM的AS［（11.7±3.2）个/1 000 nm］显著少于假手术组（P<0.05），且PEI颗粒的电子致密度也明显低于假手术组；而AG的应用使I-RI大鼠肾小球滤过膜损伤减轻，可见清晰的足突间隙；L-NNA+I-RI组大鼠肾小球上皮细胞足突融合也明显加重，但和I-RI组相比，L-NNA+I-RI组大鼠GBM的AS数量［（14.7±0.9）个/1 000 nm］无显著差异（P>0.05）。 结论:肾I-RI时出现肾小球上皮细胞足突融合、肾小球滤过膜的负电荷位点减少等病理性损伤，NO可加重这些损伤；肾I-RI时肾小球滤过膜超微结构的损伤与NO的生成及其作用有关。

Role of nitric oxide in the development of glomerular ischemia reperfusion injury in rats

AIM:To investigate the effects of nitric oxide on ultrastructure and anionic sites of glomerular in renal ischemia reperfusion injured (I-RI) rats. METHODS:Animals were divided randomly into five groups:(1) sham group (n=6);(2) I-RI group (n=6), 0.3 mL normal saline was injected via venae lingualis 20 min before ischemia;(3) SNP+I-RI group (n=6), 2.5 μg/kg sodium nitroprusside (SNP) was injected via venae lingualis 20 min before ischemia;(4) AG+I-RI group (n=6), 10 mg/kg aminoguanidine (AG) was injected via venae lingualis 20 min before ischemia;(5) L-NNA+I-RI group (n=6), 10 mg/kg Nω-nitro-L-arginine (L-NNA) was injected via venae lingualis 20 min before ischemia. Anionic sites of glomerular were studied with a cationic probe-polyethyleneimine (PEI) and ultrastructure was observed under electron microscope in renal I-RI rats. RESULTS:(1) Ultrastructure of glomerular was normal and anionic sites (AS) was located clearly in lamina rare externa of GBM in sham rats. The PEI particles arranged regularly in line (19.3±1.7/1 000 nm) under electronic microscope. Obvious foot processes derangement and effacement were observed and the AS number in GBM of I-RI group was fewer (16.6±1.0/1 000 nm, P<0.05) and the particle was smaller than that in sham group. (2) Compared with I-RI group, the foot process effacement was aggravated in SNP+I-RI group and L-NNA+I-RI group. SNP caused the numbers of anionic sites reduced after renal I-RI (11.7±3.2/1 000 nm, P<0.05), and the electronic density of the PEI granule was also reduced. AG lead a increase in anionic site number (17.8±1.0/1 000 nm, P<0.05), but still fewer than that in sham group (P<0.05). The numbers of anionic sites was not changed in L-NNA+I-RI group (14.7±0.9/1 000 nm, P>0.05). CONCLUSION:Foot process effacement and reduction of anionic sites were present in glomerular filtration membrane in renal I-RI rats. NO aggravated those injuries, indicating that NO plays a role in the ultrastructure damages of glomerular filtration membrane in I-RI rats.