Pyk2在糖尿病小鼠的表达和肾小管上皮细胞的作用及ALM调控Pyk2延缓肾纤维化的研究  

Alpha-lipoamide attenuates renal fibrosis by regulating Pyk2 expression in renal tubular epithelial cells of diabetic mice

作　　者：蔡雪妮 杨雨星 张芮 曾永秦 文青 李智恒 周兴艳 严瑞 CAI Xueni;YANG Yuxing;ZHANG Rui;ZENG Yongqin;WEN Qing;LI Zhi‐heng;ZHOU Xingyan;YAN Rui(Department of Nephrology,Affiliated Hospital of Guizhou Medical University,Guiyang 550004,China;Department of Endocrinology and Metabolism,Affiliated Hospital of Guizhou Medical University,Guiyang 550004,China)

机构地区：[1]贵州医科大学附属医院肾内科,贵州贵阳550004 [2]贵州医科大学附属医院内分泌与代谢病科,贵州贵阳550004

出　　处：《中国病理生理杂志》2023年第4期623-631,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81960140);贵州省科学技术基金资助项目(黔科合基础-ZK〔2021〕一般379号);贵州省卫健委科技基金资助项目(No.gzwjkj2020-1-002);贵州省中医药管理局中医药、民族医药科学技术研究资助项目(No.QZYY‐2021-020)。

摘　　要：目的:探讨富含脯氨酸的酪氨酸激酶2(proline-rich tyrosine kinase 2,Pyk2)在糖尿病(diabetes mellitus,DM)小鼠中的表达和在肾小管上皮细胞的作用,以及α-硫辛酰胺(alpha-lipoamide,ALM)是否通过调控Pyk2延缓糖尿病肾病(diabetic kidney disease,DKD)肾纤维化。方法:选取8周龄野生型(wild-type,WT)小鼠和db/db小鼠各5只,正常饮食饲养至16周,Western blot检测肾组织纤维连接蛋白(fibronectin,FN)、III型胶原α1链(collagen type III alpha 1 chain,COL3A1)、Pyk2和p-Pyk2(Y402)的蛋白水平,免疫组织化学染色观察Pyk2和p-Pyk2(Y402)蛋白分布及表达情况,RT-qPCR检测Pyk2的mRNA水平。体外培养大鼠肾小管上皮细胞系NRK-52E,取对数生长期细胞予以高糖高脂(high glucose and high palmitic acid,HP)、Pyk2抑制剂PF-562271、ALM处理及转染Pyk2过表达质粒后,分为正常糖(normal glucose,NG)组、HP组、HP+PF-562271组、HP+vector组、HP+OE-Pyk2组、HP+ALM组和HP+ALM+OE-Pyk2组,每组重复3次。免疫荧光染色观察Pyk2蛋白定位及表达,Western blot检测FN、COL3A1、Pyk2和p-Pyk2(Y402)的蛋白水平,RT-qPCR检测Pyk2的mRNA水平。结果:与WT组相比,db/db组小鼠纤维化指标(FN和COL3A1)蛋白表达升高,Pyk2和p-Pyk2(Y402)主要在肾小管中表达且蛋白表达水平显著升高,Pyk2的mRNA水平显著升高(P<0.05)。与NG组比较,HP组中纤维化指标、Pyk2和p-Pyk2(Y402)蛋白水平显著升高,Pyk2的mRNA水平显著升高(P<0.05);予PF-562271处理后,与HP组比较,p-Pyk2(Y402)和纤维化指标蛋白水平显著降低,予转染Pyk2过表达质粒后,与HP+vector组比较,纤维化指标蛋白水平显著升高(P<0.05)。经ALM干预后,与HP组比较,Pyk2、p-Pyk2(Y402)及纤维化指标蛋白水平显著降低(P<0.05),与HP+ALM组比较,转染Pyk2过表达质粒后,纤维化指标的变化趋势被逆转(P<0.05)。结论:Pyk2在DM小鼠中表达升高,在肾小管上皮细胞表达可促进DKD肾纤维化进展;ALM可通过下调肾小管上皮细胞中Pyk2的表达及磷AIM:To investigate the expression of proline-rich tyrosine kinase 2(Pyk2)in diabetes mellitus(DM)mice and the role of Pyk2 in renal tubular epithelial cells,and to explore the inhibitory effect of alpha-lipoamide(ALM)on renal fibrosis in diabetic kidney disease(DKD)by regulating Pyk2.METHODS:Wild-type(WT)mice and db/db mice were selected and fed with a normal diet until 16 weeks.The protein levels of fibronectin(FN),collagen type III alpha 1 chain(COL3A1),Pyk2 and p-Pyk2(Y402)in the renal tissue were analysed by Western blot.Immunohistochemistry was used to observe the distribution and expression of Pyk2 and p-Pyk2(Y402)proteins.RT-qPCR was used to detect the Pyk2 mRNA level.Rat renal tubular epithelial NRK-52E cells were divided into normal glucose(NG)group,high glucose and high palmitic acid(HP)group,HP+PF-562271(Pyk2 inhibitor)group,HP+vector group,HP+Pyk2 overexpression(OE-Pyk2)group,HP+ALM group,and HP+ALM+OE-Pyk2 group.Immunofluorescence was used to observe the Pyk2 protein.Western blot was used to detect the protein levels of FN,COL3A1,Pyk2,and p-Pyk2(Y402),and RT-qPCR was used to detect Pyk2 mRNA level.RESULTS:The FN and COL3A1 were increased in db/db mice.The Pyk2 and p-Pyk2(Y402)were observed in renal tubules,and Pyk2 mRNA level was increased significantly.Compared with NG group,fibrosis markers,Pyk2 and p-Pyk2(Y402)protein levels and Pyk2 mRNA level in HP group were significantly increased(P<0.05).After treatment with PF-562271,the levels of p-Pyk2(Y402)and fibrosis-related proteins were significantly lower than those in HP group.After transfection with Pyk2 plasmid,the levels of fibrosis-related proteins were significantly higher than those in HP+vector group(P<0.05).After ALM intervention,the levels of Pyk2,p-Pyk2(Y402)and fibrosis-related proteins were significantly decreased compared with HP group(P<0.05).The trend of fibrosis was reversed in HP+ALM+OE-Pyk2 group(P<0.05).CONCLUSION:The expression of Pyk2 is increased in DM mice,and the expression of Pyk2 in renal tubular epithelial cells can promot

关 键 词：富含脯氨酸的酪氨酸激酶2 α-硫辛酰胺 糖尿病肾病 肾纤维化 

分 类 号：R587.1[医药卫生—内分泌] R363.2[医药卫生—内科学]