糖尿病小鼠肾小管上皮细胞HNF4A和MUCDHL下调促进肾纤维化  

Down-regulation of HNF4A and MUCDHL in renal tubular epithelial cells promotes renal fibrosis of diabetic mice

作　　者：贾静 梁露群 谭万林 许晓晓 阮媛媛 李霜 陈荣誉 余雄 王方芳 陈钰婷 彭玉琳 郭兵 王圆圆 JIA Jing;LIANG Luqun;TAN Wanlin;XU Xiaoxiao;RUAN Yuanyuan;LI Shuang;CHEN Rongyu;YU Xiong;WANG Fangfang;CHEN Yuting;PENG Yulin;GUO Bing;WANG Yuanyuan(Department of Pathophysiology,School of Basic Medical Sciences,Guizhou Medical University,Guizhou Key Laboratory of Pathogenesis and Drug Research of Common Chronic Diseases,Guiyang 550025,China;Department of Pathology,Gui-zhou Provincial People's Hospital,Guiyang 550002,China)

机构地区：[1]贵州医科大学基础医学院病理生理教研室,贵州省常见慢性疾病发病机制及药物研究重点实验室,贵州贵阳550025 [2]贵州省人民医院病理科,贵州贵阳550002

出　　处：《中国病理生理杂志》2024年第6期1085-1096,共12页Chinese Journal of Pathophysiology

基　　金：贵州省基础研究计划重点项目(黔科合基础-ZK[2022]重点042号);贵州省科技拔尖人才项目(黔教合KY字[2021]032号);贵州省人民医院青年基金项目(GZSYQN[2018]12号)。

摘　　要：目的:探索肝细胞核因子4α(HNF4A)和μ-原钙黏蛋白(MUCDHL)在糖尿病小鼠肾脏中的作用和联系。方法:(1)选取12周龄的db/m小鼠和db/db小鼠各6只,正常饮食喂养至16周,Western blot检测肾组织纤维连接蛋白(FN)、Ⅲ型胶原(Col-Ⅲ)、上皮钙黏素(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)、HNF4A、Snail和MUCDHL的蛋白水平,免疫组织化学染色观察FN、HNF4A和MUCDHL蛋白的表达及分布情况。(2)体外培养小鼠肾小管上皮细胞(mRTEC),分为正常糖(NG)组、高糖(HG)组、过表达对照组(NG+vector组和HG+vector组)、过表达组(NG+OE-MUCDHL组、HG+OE-MUCDHL组、NG+OE-HNF4A组和HG+OE-HNF4A组)、敲减对照组(NG+control组和HG+control组)和敲减组(NG+si-MUCDHL组、HG+si-MUCDHL组、NG+si-HNF4A组和HG+si-HNF4A组),Western blot检测相关指标的蛋白水平。结果:(1)与db/m组相比,db/db组体重、血糖和尿白蛋白/肌酐比值均显著升高(P<0.05),提示db/db小鼠有明显肾损伤;与db/m组相比,db/db组FN、Col-Ⅲ、α-SMA和Snail蛋白表达升高,E-cadherin、HNF4A和MUCDHL蛋白表达降低(P<0.05);MUCDHL主要表达在肾小管上皮细胞顶端膜,FN主要表达在肾小管间质,HNF4A主要表达在肾小管细胞浆和细胞核。(2)与NG组相比,HG组FN、Col-Ⅲ、α-SMA和Snail蛋白表达升高,E-cadherin、HNF4A和MUCDHL蛋白表达降低(P<0.05);过表达MUCDHL后FN、Col-Ⅲ、α-SMA和Snail蛋白表达降低,E-cadherin和MUCDHL蛋白表达升高(P<0.05),HNF4A表达不变;敲减MUCDHL后相关指标表达与上述效应相反(P<0.05),HNF4A表达不变;过表达HNF4A可使MUCDHL表达增加,其余指标的表达变化与过表达MUCDHL一致;敲减HNF4A表达可使上述效应反转(P<0.05);MUCDHL可能是HNF4A的下游靶基因。结论:HNF4A和MUCDHL在糖尿病小鼠肾小管中表达降低。HNF4A可能通过上调MUCDHL的表达延缓糖尿病肾病肾纤维化进程。AIM:To explore the roles and associations of hepatocyte nuclear factor 4 alpha(HNF4A)and muprotocadherin(MUCDHL)in the kidney of diabetic mice.METHODS:(1)A cohort of six 12-week-old db/m mice and six db/db mice were selected and maintained on a standard diet until 16 weeks.The protein levels of fibronectin(FN),collagen type Ⅲ(Col-Ⅲ),E-cadherin,α-smooth muscle actin(α-SMA),HNF4A,Snail and MUCDHL in renal tissues were scrutinized using Western blot.Immunohistochemical staining was conducted to observe the distribution and expression of FN,HNF4A and MUCDHL.(2)Mouse renal tubular epithelial cells(mRTEC)were cultured in vitro and categorized into groups:normal glucose(NG)group,high glucose(HG)group,overexpression control groups(NG+vector and HG+vector),overexpression groups(NG+OE-MUCDHL,HG+OE-MUCDHL,NG+OE-HNF4A and HG+OE-HNF4A),knockdown control groups(NG+control and HG+control),and knockdown groups(NG+si-MUCDHL,HG+si-MUCDHL,NG+si-HNF4A and HG+si-HNF4A).The relevant protein levels were also detected by Western blot.RESULTS:(1)In db/db group,elevated body weight,blood glucose and urine albumin-to-creatinine ratio(UACR)indicated significant renal injury.Compared with db/m group,the mice in db/db group exhibited increased expression of FN,Col-Ⅲ,α-SMA and Snail,and decreased expression of E-cadherin,HNF4A and MUCDHL.MUCDHL was predominantly expressed in the apical membrane of renal tubular epithelial cells,FN in the tubular mesenchyme,and HNF4A in the plasma and nucleus of renal tubular cells.(2)In HG group,there was an up-regulation in the expression of fibrosis-related proteins and a down-regulation in the expression of E-cadherin,HNF4A and MUCDHL compared with NG group.Overexpression of MUCDHL led to a decrease in the expression of FN,Col-Ⅲ,α-SMA and Snail proteins,an increase in the expression of Ecadherin and MUCDHL proteins,and unaltered expression of HNF4A.Knockdown of MUCDHL resulted in a reversal of the aforementioned effects,with HNF4A expression remaining unaltered.Overexpression of HNF4A led to an

关 键 词：糖尿病肾病 纤维化 μ-原钙黏蛋白 肝细胞核因子4Α 

分 类 号：R587.2[医药卫生—内分泌] R692.6[医药卫生—内科学] R363[医药卫生—临床医学]