来氟米特对高糖诱导的足细胞裂孔隔膜及细胞骨架的影响及机制  

Effects of leflunomide on high glucose-induced podocyte cytoskeleton and its mechanism

作　　者：柴蔚霞[1] 于为民[2] 李荣山[3] 薛晋杰[2] 赵星[1] 

机构地区：[1]山西医科大学研究生院,太原030001 [2]山西医科大学附属大医院肾内科 [3]山西医科大学第二附属医院肾内科

出　　处：《中华医学杂志》2013年第10期780-784,共5页National Medical Journal of China

基　　金：山西省科技攻关项目（20100311098-3）

摘　　要：目的研究来氟米特对高糖诱导的足细胞裂孔隔膜及细胞骨架的影响，并探讨其信号通路。方法条件永生的人肾小球足细胞系分为正常糖组、甘露醇高渗透压对照组、高糖组、高糖＋PDTC（pyrrolidinedithiocarbamate，为NF-κB通路特异性抑制剂）组及高糖＋A771726（来氟米特活性代谢产物）组。Western印迹法测定足细胞NF-κBp65途径的活化即P-NF-κB065／NF-κB065比值，Western印迹法、反转录PCR测定NF-κB0065、TRPC6、nephrin在肾小球足细胞的表达。免疫荧光染色法检测足细胞骨架的变化。结果（1）高糖刺激足细胞激活NF—κBp65信号通路，60min时P—NF—κBp65蛋白表达比0min时显著增加（1．20±0．04比0．79±0．02，P〈0．01）；正常糖组和甘露醇高渗透压对照组仅有少量NF-κBp65活化；PDTC及A771726可明显抑制P-NF-κBp65表达（均P〈0．05），NF-κBp65mRNA表达各组之间差异均无统计学意义（均P〉0．05）。（2）高糖刺激足细胞激活NF-κBp65信号通路后，TRPC6mRNA及蛋白表达较正常组高，nephrinmRNA及蛋白表达较正常组低；PDTC及A771726可抑制TRPC6高表达，使nephrin表达较高糖组高（均P〈0．05）。（3）免疫荧光法检测高糖刺激足细胞72h后可见细胞骨架蛋白微丝肌动蛋白紊乱及张力纤维消失。结论来氟米特活性代谢产物A771726可通过阻断高糖诱导的NF-κBp65信号通路的活化对足细胞起保护作用。Objective To explore the effects of leflunomide active metabolite A771726 on high glucose-induced podocyte cytoskeleton and its possible signaling pathway. Methods The conditionally immortal human glomeroular podoeytes were divided into normal glucose （ NG）, mannitol （ MA）, high glucose （HG）, high glucose with PDTC （pyrrolidine dithiocarbamate, a NF-κBp65 inhibitor） and high glucose with active leflunomide metabolite A771726 groups. Western blot was used to measure the ratio of p- NF-κBp65 to total NF-κBp65. And the protein and mRNA expressions of NF-κBp65, TRPC6 and nephrin were detected by Western blot and reverse transcription polymerase chain reaction （PCR）. Immunofluoreseence staining was used to detect the changes in the skeleton of podocyte. Results （ 1 ） Podocytes with high glucose could activate the NF-κBp65 signaling pathway. There was a significant increase of p-NF-κBp65 protein at 60 min versus 0 min （ 1.20 ±0. 04 vs 0. 79 ±0. 02, P 〈0. 01 ）. Little activation of the pathways was observed in groups NG and MA. The up-regulated protein expression of p-NF-κBp65 induced with high glucose was significantly inhibited by PDTC and A771726 （both P 〈 0. 05 ）. The difference of NF-κBp65 mRNA expression was not statistically significant between the groups （ all P 〉 0. 05 ）. （ 2 ） High glucose-induced podoeyte activated the NF-κBp65 signaling path. Its downstream TRPC6 mRNA and protein expression significantly increased than NG while nephrin became down-regulated more than NG. PDTC and A771726 inhibited the high expression of TRPC6 while the expression of nephrin was elevated （ all P 〈 0. 05 ）. （ 3 ） Immunofluoreseent assay of high glucose-induced podocyte cytoskeleton showed disorderly F-actin and a disappearance of tensile fiber after 72 h. Conclusion Active leflunomide metabolite A771726 may protect podoeytes through blocking the high glucose-induced signaling pathway of NF-κBp65.

关 键 词：足细胞 细胞骨架 转录因子RelA 来氟米特 高糖 

分 类 号：R692[医药卫生—泌尿科学]