血管紧张素Ⅱ灌注诱导nephrin表达改变与足细胞凋亡

Angiotensin Ⅱ infusion induces nephrin expression change and podocyte apoptosis

目的研究血管紧张素Ⅱ（AngⅡ）灌注对大鼠足细胞裂隙膜分子nephrin表达及足细胞凋亡的影响，以及探讨AngⅡ引起蛋白尿及肾小球硬化的机制。方法36只雄性Sprague Dawley大鼠分为AngⅡ灌注组（400ng·kg^-1·min^-1）、生理盐水灌注组和正常对照组，测定28d内大鼠血压及尿蛋白。分别于14、28d处死动物取肾，观察组织学改变，并用免疫荧光、免疫电镜检测nephrin分布。RT-PCR及Western印迹法分别检测nephfin mRNA及蛋白表达。TUNEL法检测足细胞凋亡。结果（11AngⅡ灌注组大鼠血压升高，14d达峰值并维持该水平至28d；AngⅡ灌注7d即出现蛋白尿，并持续增加。（2）AngⅡ灌注14d时，足细胞裂隙膜变窄；灌注28d时，足突增宽及节段性融合，部分足细胞有凋亡小体形成，少数肾小球出现节段性硬化。TUNEL法检测发现足细胞凋亡[（2．7±1．6）个／肾小球切面]，凋亡数与蛋白尿量呈正相关（r=0．86，P〈0．01）。（3）AngⅡ灌注14d时，肾皮质nephrin mRNA及蛋白表达上调（P〈0．05）。nephfin由正常的沿毛细血管袢线状分布向粗颗粒、团块状分布模式转变。AngⅡ灌注28d时，肾皮质nephrin mRNA及蛋白表达下降（P〈0．05），且nephfin蛋白表达与足细胞凋亡数呈负相关（r=-0．63，P〈0．01）。结论AngⅡ灌注诱导的nephrin表达及分布改变可能导致了足细胞凋亡及肾小球硬化的发生与发展。

Objective To evaluate the effects of angiotensin Ⅱ （Ang Ⅱ） infusion on nephrin expression and podocyte apoptosis in vivo, and investigate the mechanism of proteinuria and glomerulosclerosis induced by Ang Ⅱ infusion. Methods Thirty-six male Sprague-Dawley rats were randomly assigned to receive either normal saline or Ang Ⅱ （400 mg·kg^-1·min^-1） by osmotic mini-pump, or to be used as normal control. After the systolic blood pressure was measured, the 24 h urine sample was collected for analysis of proteinuria at day 7, 14, 21, 28. Animals were sacrificed at day 14, 28 respectively. Serum creatinine, renal pathological change, podocyte apoptosis were observed. Expression of nephrin mRNA and protein was examined by RT-PCR, Western blot, and immunofluorescence staining. Results （1） Ang Ⅱ-infused rats developed significant hypertension associated with marked proteinuria. Significantly positive correlation was found between blood pressure and proteinuria （r = 0.80,P 〈 0.01）. （2）At day 14, Ang Ⅱ infusion induced the narrowing of slit diaphragm. At day 28, apoptotic podocytes were detected by electren microscopy and TUNEL assay [（2.7±1.6） apoptotic podocytes per glomerular cross section] in Ang Ⅱ-infused rats. （3） Distribution of nephrin expression was changed from linear to granular pattern in Ang Ⅱ-infused rats at day 14 and expression of nephrin mRNA and protein increased （P 〈 0.05）.But at day 28, expression of nephrin mRNA and protein was decreased, and significantly negative correlation was found between the expression of nephrin and the number of apoptotic podocytes （r = -0.63,P 〈 0.01）. Coonclusion Change in nephrin expression may play a critical role in the pathogenesis of Ang Ⅱ-induced podocyte apoptosis.