温阳活血利水方对被动型Heymann肾炎大鼠足细胞的影响

Effects of Wenyang Huoxue Lishui recipe on podocytes in rats with passive Heymann nephritis

目的:观察温阳活血利水方药对被动型Heymann肾炎(PHN)模型大鼠的干预作用,并探讨该方药的疗效机制。方法:雄性SD大鼠60只,随机分为正常对照组、PHN模型组、温阳活血利水组、他克莫司组,每组15只。以尾静脉注射Anti-Fx1A血清法构建IMN模型。检测各组大鼠24h尿蛋白定量、血清总蛋白、血清白蛋白、谷丙转氨酶、谷草转氨酶、血肌酐、胆固醇及甘油三酯水平。采用免疫荧光法检测肾小球Klotho、瞬时受体电位通道6(TRPC6)、组织蛋白酶L(CatL)及其底物Synaptopodin表达;采用酶联免疫吸附(ELISA)法检测肾皮质Ras同源基因家族成员(Rho)A、Rho关联含卷曲螺旋蛋白激酶(ROCK)1与ROCK2的活性;采用Western blot方法检测肾皮质磷酸化LIM结构域激酶p-LIMK1、磷酸化后p-cofilin蛋白的表达。光镜下观察肾脏病理形态学变化,电子显微镜下观察肾小球足细胞足突及电子致密物变化情况。结果:与正常对照组比较,干预4周后PHN模型组24h尿蛋白定量、血清胆固醇与甘油三酯水平显著升高(P<0.01),血清白蛋白水平显著下降(P<0.01);肾小球Klotho及Synaptopodin的表达显著下调(P<0.01),RhoA、ROCK1、ROCK2活性显著升高(P<0.01),TRPC6、CatL、p-LIMK1/LIMK1及p-cofilin/cofilin表达显著上调(P<0.01);足细胞足突弥漫性融合,足细胞下电子致密物沉积。与PHN模型组比较,干预4周后温阳活血利水组血清白蛋白显著升高(P<0.05),24h尿蛋白定量与血清甘油三酯水平显著下降(P<0.05,P<0.01);肾小球Klotho及Synaptopodin表达显著上调(P<0.01),RhoA、ROCK1、ROCK2活性显著下降(P<0.01),TRPC6、CatL、p-LIMK1/LIMK1与p-cofilin/cofilin表达显著下调(P<0.01);足突融合改善,足细胞下电子致密物沉积减轻。结论:Klotho/TRPC6/CatL/Synaptopodin/RhoA/ROCK/LIMK/cofilin信号通路表达异常可能参与了IMN的发病过程;温阳活血利水方药能够减少IMN大鼠蛋白尿,调整足细胞形态,其作用机制或与调控该通路有关。

Objective: To observe the interventional effect of Wenyang Huoxue Lishui recipe(WHLR) on passive heymann nephritis(PHN) model and to explore the therapeutic mechanism. Methods: Sixty male SD rats were randomly divided into normal control group,PHN model group,WHLR group,tacrolimus group,15 rats in each group. PHN rats were modeled by injecting anti-Fx1A serum. The levels of 24 h urine total protein and serum total protein,albumin,alanine aminotransferase,aspartate aminotransferase,creatinine,cholesterol,triglyceride were detected in each group of rats. Immunofluorescence methods were used to detect the expression of glomerular Klotho,transient receptor potential channel 6(TRPC6),cathepsin L(CatL) and its substrate synaptopodin in each group of podocytes. Enzyme-linked immunosorbent assay(ELISA) was adopted to measure the activities of Ras homologgene family member(Rho) A and Rho-activated kinase(ROCK) 1 and ROCK2 of the renal cortex.And Western blot method was used to detect the expression of phosphorylated-LIMK1(p-LIMK1),p-cofilin protein of the renal cortex. The pathological changes of rat kidney tissue were observed under ordinary light microscope,and the changes of glomerular podocyte foot processes and electron dense deposits were observed under electron microscope. Results: Corresponding drugs were given by gavage for 4 weeks,compared with normal control group,the level of 24 h urine total protein,serum cholesterol and triglycerides significantly increased(P<0.01) as well as the level of serum albumin decreased(P<0.01);expression of glomerular Klotho and Synaptopodin were down-regulated(P<0.01) as well as expression of TRPC6,CatL,RhoA,ROCK1,ROCK2,p-LIMK1/LIMK1 and p-cofilin/cofilin were up-regulated in the PHN model group(P<0.01). The effacement of the podocyte foot processes was diffuse,and electron dense deposits were deposited under the podocytes. Corresponding drugs were given by gavage for 4 weeks,compared with the PHN model group,the level of serum albumin significantly increased(P<0.05)as well as the level of 24 h urine total protein,serum triglycerides decreased(P<0.05,P<0.01) in WHLR group;expression of glomerular Klotho and Synaptopodin were up-regulated(P<0.01) as well as expression of TRPC6,CatL,RhoA,ROCK1,ROCK2,p-LIMK1/LIMK1 and p-cofilin/cofilin significantly were down-regulated in WHLR group(P<0.01). The fusion of the podocyte foot processes was relieved,and the deposition of electron dense under the podocytes was reduced. Conclusion: Abnormalities of Klotho/TRPC6/CatL/Synaptopodin/RhoA/ROCK/LIMK/cofilin signal pathway may be involved in the pathogenesis of IMN.WHLR can decrease proteinuria,and adjust podocyte morphology in IMN rat,whose mechanism may be related to the regulation of the above-mentioned signal pathway.