高糖环境下IQGAP1在足细胞中的表达及其对足细胞骨架重排的调节作用

IQGAP1 expression in podocytes and its regulation to podocyte cytoskeleton rearrangement in high-glucose environment

目的观察高糖环境下IQ结构域GTP酶活化蛋白1(IQGAP1)在足细胞中的表达情况,并探讨IQGAP1在调节高糖诱导的足细胞骨架重排中的作用及其可能的机制。方法 (1)体外培养永生化的小鼠足细胞。取一部分足细胞加入不同浓度葡萄糖(5 mmol/L、10 mmol/L、15 mmol/L、20 mmol/L、25 mmol/L、30 mmol/L)刺激48 h,另取一部分细胞加入高浓度葡萄糖(30 mmol/L)刺激不同时间(0 h、3 h、6 h、12 h、24 h、48 h),检测各组足细胞IQGAP1蛋白的表达情况。(2)将足细胞分为正常糖浓度组(5 mmol/L葡萄糖,NG组)、高糖组(30 mmol/L葡萄糖,HG组)、高渗对照组(5 mmol/L葡萄糖+25 mmol/L甘露醇,HO组)、HG+IQGAP1质粒转染组、HG+空质粒转染组、NG+IQGAP1质粒转染组、NG+空质粒转染组。给予相应干预后,比较NG组、HG组、HO组足细胞的IQGAP1蛋白表达情况及纤维状肌动蛋白(F-actin)骨架分布,比较NG组、HG组、HG+IQGAP1质粒转染组、HG+空质粒转染组足细胞的F-actin骨架分布,比较6组足细胞的IQGAP1蛋白及细胞分裂周期蛋白42(Cdc42)表达情况、IQGAP1和Cdc42蛋白的结合情况。结果 (1) 30 mmol/L葡萄糖刺激后足细胞的IQGAP1蛋白表达水平低于5 mmol/L葡萄糖刺激后的水平(P<0.05),高糖刺激48 h后的足细胞IQGAP1蛋白表达水平低于高糖刺激0 h后的水平(P<0.05)。(2) HG组足细胞的F-actin排列紊乱,F-actin形成核外周环,F-actin外周环评分(CFS)高于NG组(P<0.05)。(3) HG+IQGAP1质粒转染组的足细胞骨架重排部分逆转,CSF低于HG组(P<0.05)。(4) HG+IQGAP1质粒转染组的IQGAP1蛋白及Cdc42表达水平、两者结合水平均高于HG组(均P<0.05)。结论高糖刺激可减少IQGAP1及Cdc42蛋白表达以及两者结合,并诱导足细胞骨架重排,而IQGAP1可能通过与Cdc42结合调节细胞骨架重排。

Objective To observe the expression of IQ domain GTPase-activating protein 1(IQGAP1) in podocytes in high-glucose environment,and to explore the role and its possible mechanism of IQGAP1 in regulating high glucose-induced podocyte cytoskeleton rearrangement. Methods (1)Immortalized mouse podocytes were cultured in vitro .The podocytes were stimulated by adding glucose of different concentrations(5 mmol/L,10 mmol/L,15 mmol/L, 20 mmol/L ,25 mmol/L and 30 mmol/L) for 48 h of stimulation,and another podocytes were stimulated by adding high-concentration glucose (30 mmol/L) for different durations(0 h,3 h,6 h,12 h,24 h and 48 h),then the expression of IQGAP1 protein was detected in podocytes of each group.(2)Podocytes were divided into normal-concentration glucose group(5 mmol/L glucose,NG group), high-glucose group(30 mmol/L glucose,HG group), hyperosmotic control group (5 mmol/L glucose+25 mmol/L mannitol,HO group),HG+IQGAP1 plasmid transfection group,HG+empty plasma transfection group,NG+IQGAP1 plasmid transfection group,and NG+empty plasma transfection group.After corresponding intervention,IQGAP1 protein expression and fibrous actin(F-actin) cytoskeleton distribution of podocytes were compared among the NG group,the HG group and the HO group,F-actin cytoskeleton distribution of podocytes was compared among the NG group,the HG group,the HG+IQGAP1 plasmid transfection group and the HG+empty plasma transfection group,expression of IQGAP1 protein and cell division cycle 42(Cdc42) in podocytes, as well as combination of IQGAP1 and Cdc42 proteins,was compared among the six groups. Results (1)The expression level of IQGAP1 protein in podocytes stimulated by 30 mmol/L glucose was higher than that in podocytes stimulated by 5 mmol/L glucose( P <0.05),and the expression level of IQGAP1 protein in podocytes stimulated by high-glucose for 48 h was higher than that in podocytes stimulated by high-glucose for 0 h ( P <0.05).(2)In the HG group,disorganized arrangement of F-actin and perinuclear ring formed by F-actin were observed ,and the cortical F-actin score(CFS) was higher than that in the NG group ( P <0.05).(3)The HG+IQGAP1 plasmid transfection group represented partial reversal of podocyte cytoskeleton rearrangement and a lower CFS compared with the HG group( P <0.05).(4)The expression levels of IQGAP1 protein and Cdc42 and their combination in the HG+IQGAP1 plasmid transfection group were higher than those in the HG group(all P <0.05). Conclusion High-glucose stimulation can decrease the expression of IQGAP1 and Cdc42 proteins and their combination,as well as induce cytoskeleton rearrangement of podocytes,whereas IQGAP1 might regulate cytoskeleton rearrangement by its combination with Cdc42.