血管紧张素Ⅱ输注诱导大鼠足突滤孔膜改变增加蛋白尿产生

Podoyte Foot Process Broadening in Experimental Angiotensin Ⅱ Infusion Rats Causes Proteinuria and Changes in Nephrin Expression

目的:通过直接输注血管紧张素Ⅱ(AngⅡ)的方法观察大鼠尿蛋白及足细胞足突和裂孔结构损伤的变化,分析这些改变与足细胞裂孔膜蛋白nephrin表达改变的相互关系。方法:Wistar大鼠随机分为两组,AngⅡ组通过皮下埋置渗透性微泵(osmotic minipump)以400 ng/(kg.min)持续给予AngⅡ,对照组由生理盐水代替AngⅡ。大鼠每周检测24 h尿蛋白并收集肾组织标本,连续4周。电镜观察并计算肾小球足突宽度。Nephrin蛋白和mRNA检测分别应用免疫荧光及RT-PCR的方法。结果:AngⅡ组大鼠在第1周即出现蛋白尿,且与对照组相比具有显著性差异(P<0.05),并且随试验的进展蛋白尿逐渐增加。肾小球形态学显示在第1周时肾小球基底膜被拉伸变薄,裂孔膜宽度增加,nephrin表达增加;至第2周可见足细胞足突数量明显增多,单位长度基底膜上的裂孔数量增加,同时观察到nephrin的表达较对照组明显增加(P<0.05);到第3,4周时,随着足细胞进一步损伤,基底膜出现有不规则增厚和裸露,足突出现部分的融合和足突裂孔膜的消失,nephrin的表达逐渐下降至低于对照组水平。结论:AngⅡ可以直接导致足细胞结构完整性的破坏,引起蛋白尿,并与肾小球内nephrin的表达密切相关。表明在某些肾小球疾病或损伤中,足突裂孔膜结构和蛋白表达异常是蛋白尿产生的主要原因。

Objective： To evaluated the association between the development of proteinuria and angiotensin Ⅱ infusion and investigate the morphological changes in podocyte foot process and the expression of the glomerular podocyte molecule, nephrin. Methods： Wistar rats were randomly assigned into twogroups according to receiving either normal saline （control） or Ang Ⅱ at a dose of 400 ng/（kg · min） via subcutaneous osmotic mini-pumps. Proteinuria was measured before rats were sacrificed at each week. Renal morphological changes were evaluated by light and transmission electron microscopy. Semiquantitative reverse transcription-polymerase chain reaction （RT-PCR） and immunofluorescence （IF） staining were performed to study the glomerular expression of nephrin. Results： Rats with Ang Ⅱ infusion developed proteinuria markedly were compared with control rats. The capsules of glomeruli in Ang Ⅱ group became thinner at the 7th day and electron microscopy revealed flourish podocyte foot processes. The intensity of nephrin expression increased significantly and the negative expression was localized at glomerular epithelial cells in Ang Ⅱ group after 14 days （by 58.9% more than in control, P〈0.01） and no linear staining could be observed. During the increasing of proteinuria, the foot process became wider at 21 d and nephrin decreased significantly both in protein intensity and at mRNA level. Conclusion： During the onset of AngⅡ , the first response of podocyte is the flourish foot process as well as the distribution change of nephrin. Ang Ⅱ increases the expression of nephrin. There was a close relationship between nephrin distribution pattern with the development of proteinuria and with the podocyte foot process changes. Proteinuria might occur after series of events of nephrin distribution changes and morphologic changes of podocyte foot process.