AKAP1在高糖诱导的足细胞线粒体分裂中的作用

Roles of AKAP1 in high- glucose induced mitochondrial fission in podocytes

目的研究A激酶锚定蛋白（AKAP1）在高糖诱导足细胞线粒体分裂中的作用及可能的信号转导通路。方法体外培养条件永生性人足细胞，完全分化并同步化处理后进行后续实验。实验分组：（1）葡萄糖浓度分为5、15、25、35mmol／L组刺激足细胞48h；（2）高糖（35mmol／L）培养基分别刺激足细胞12、24、36、48h；（3）正常对照组（5mmol／L葡萄糖）、高渗对照组（30mmol／L甘露醇＋5mmol／L葡萄糖）、高糖组（35mmol／L葡萄糖）、高糖（35mmol／L葡萄糖）＋AKAP1siRNA组。免疫荧光染色检测足细胞中AKAP1的表达和分布，Western印迹检测足细胞中AKAP1、线粒体动力相关蛋白1（dynamin related protein1，Drp1）、P-Drp1蛋白水平。Mitotracker Red染色观察各组足细胞线粒体形态，对长度、纵横比、形状系数定量分析。流式细胞术评价足细胞凋亡水平。结果与正常组比较，高糖刺激导致足细胞凋亡增加（P〈0．05），线粒体分裂增加、纵横比及形状系数降低（均P〈0．05），AKAP1蛋白表达水平、P-Drp1／Drp1以时间、浓度依赖方式增加（P〈0．05）；与高糖组比较，AKAP1siRNA转染后线粒体分裂减少，线粒体纵横比及形状系数升高（均P〈0．05），足细胞凋亡减少；AKAP1蛋白水平及P-Drp1／Drp1下降（P〈0．05）；上述各项高渗对照组与正常对照组差异均无统计学意义（均P〉0．05）。结论高糖刺激下AKAP1可能通过线粒体Drp1调控足细胞线粒体分裂，导致足细胞损伤。

Objective To investigate the roles of A kinase anchoring proteinl（AKAP1）in high- glucose induced mitochondrial fission in podocytes. Methods Conditionally immortalized human podocytes were cultured in serum-free medium for 24 hours, and then exposed to different glucose concentration conditions in different time periods. The protein expressions of AKAP1 were observed by immunofluorescence, and AKAP1, dynamin related proteinl （Drp1） and phospho Ser 637-Drp1 （p-Drp1） were analyzed by Western blotting. AKAP1 siRNA was transfected to block AKAP1 expression. Podocytes were then divided into normal control group （5 mmol/L glucose）, hypertonic group （30 mmol/L mannitol ＋ 5 mmol/L glucose）, high glucose group （35 mmol/L glucose）, and high glucose ＋ AKAP1 siRNA group. Mitochondria] morphological changes were assessed by mitotracker red staining. Podocyte apoptosis was assessed by flow cytometry. Results Compared with normal group, high- glucose induced more podocytes apoptosis （P 〈 0.05）, more mitochondrial fission with decreased aspect ratio and form factor （all P 〈 0.05）. Upregulated AKAP1 protein level, and increased ratio of p-Drp1/Drp1（all P 〈 0.05） in time and concentration dependent manners were also observed. Compared with high glucose group, transfection of AKAP1 siRNA showed less apoptosis （P 〈 0.05）, less mitochondrial fission with increased aspect ratio and form factor （all P 〈 0.05）, and down-regulated AKAP1 protein level as well as p-Drp1/Drp1 ratio （all P 〈 0.05）. Conclusion High glucose induced mitochondrial fission might be induced through AKAP1-Drp1 pathway.