TRPC6对糖尿病肾病小鼠肾小管间质炎症的影响及其机制  

Influence of transient receptor potential cation channel 6 on renal tubulointerstitial inflammation in mice with diabetic kidney disease and its mechanism

作　　者：刘丛聪 张行健 丁琳 张瑶[1] 张栋杰 马瑞霞[1] LIU Congcong;ZHANG Xingjian;DING Lin;ZHANG Yao;ZHANG Dongjie;MA Ruixia(Department of Nephrology,The Affiliated Hospital of Qingdao University,Qingdao 266003,China)

机构地区：[1]青岛大学附属医院肾病科,山东青岛266003 [2]湖北民族大学附属民大医院肾病内科

出　　处：《精准医学杂志》2024年第5期377-382,388,共7页Journal of Precision Medicine

基　　金：山东省自然科学基金面上项目(ZR2022MH-161);泰山学者工程专项项目(tstp20230665);青岛市医药卫生科研计划项目(2021-WJZD189);青岛市医疗卫生重点学科建设项目;青岛市临床重点专科。

摘　　要：目的探讨瞬时受体电位阳离子通道6(TRPC6)对糖尿病肾病(diabetickidneydisease,DKD)小鼠肾小管间质炎症的影响及其机制。方法将6周龄雄性C57BL/6J小鼠36只随机分为6组,每组6只。对照组(A组)和DKD组(B组)分别腹腔注射0.1mmol/L柠檬酸盐缓冲液和10g/L链脲佐菌素(后简称给药);DKD+生理盐水干预组(C组)和DKD+线粒体自噬激活剂干预组(D组)在B组基础上分别灌胃生理盐水和10mmol/L尿石素A;DKD+阴性对照慢病毒转染组(E组)和DKD+TRPC6敲降慢病毒转染组(F组)在B组基础上分别尾静脉注射阴性对照慢病毒和TRPC6敲降慢病毒。测定各组小鼠给药后第12周时的全血空腹血糖(FBG)水平、尿微量白蛋白肌酐比(ACR)和血尿素氮(BUN)水平,PAS染色观察小鼠肾小管损伤情况并进行评分,实时荧光定量PCR(RT-qPCR)技术检测小鼠肾组织中炎性因子(IL-1β、MCP-1、TNF-α)mRNA水平,免疫组织化学染色观察小鼠肾组织中TRPC6蛋白表达水平,蛋白免疫印迹检测小鼠肾组织中TRPC6、LC3B、P62、PINK1、Parkin相对表达量,透射电镜观察小鼠肾小管细胞中线粒体自噬体数量变化。将HK-2细胞分为高糖+TRPC6siRNA+DMSO干预组(G组,TRPC6siRNA转染+35.0mmol/L葡萄糖+0.06%DMSO)和高糖+TRPC6siRNA+线粒体自噬抑制剂干预组(H组,TRPC6siRNA转染+35.0mmol/L葡萄糖+12μmol/L千层纸素A),RT-qPCR技术检测细胞中炎性因子(IL-1β、MCP-1、TNF-α)mRNA的水平。结果给药后第12周时,B组小鼠全血FBG水平、ACR、BUN水平、肾小管损伤评分、肾组织炎性因子mRNA水平及TRPC6、P62蛋白表达水平均显著高于A组(t=2.77~13.61,P<0.05),肾组织LC3B-Ⅱ/LC3B-Ⅰ及PINK1、Parkin蛋白表达水平显著低于A组(t=3.33~14.63,P<0.05),肾小管细胞中线粒体自噬体数量减少;D组小鼠ACR、BUN水平、肾小管损伤评分、肾组织炎性因子mRNA水平及P62蛋白表达水平显著低于C组(t=2.40~23.50,P<0.05),肾组织LC3B-Ⅱ/LC3B-Ⅰ及PINK1、Parkin蛋白表达水平显著Objective To investigate the influence of transient receptor potential cation channel 6(TRPC6)on renal tubulointerstitial inflammation in mice with diabetic kidney disease(DKD)and its mechanism.Methods A total of 36 male C57/BL6J mice,aged 6 weeks,were randomly divided into control group(group A),DKD model group(group B),DKD+normal saline intervention group(group C),DKD+mitophagy activator intervention group(group D),DKD+negative control lentivirus transfection group(group E),and DKD+TRPC6 knockdown lentivirus transfection group(group F),with 6 mice in each group.The mice in groups A and B were respectively given intraperitoneal injection of 0.1 mmol/L citrate buffer and 10 g/L streptozotocin(hereinafter referred to as administration);the mice in groups C and D were respectively given normal saline and 10 mmol/L urolithin A by gavage in addition to the treatment in group B;the mice in groups E and F were respectively injected with negative control lentivirus and TRPC6 knockdown lentivirus via the tail vein in addition to the treatment in group B.The levels of fasting blood glucose(FBG),urinary albumin-to-creatinine ratio(ACR),and blood urea nitrogen(BUN)were measured at week 12 after administration;PAS staining was used for the observation and scoring of renal tubular injury;RT-qPCR was used to measure the mRNA expression levels of inflammatory factors[interleukin-1β(IL-1β),monocyte chemoattractant protein-1(MCP-1),and tumor necrosis factor-α(TNF-α)]in renal tissue;immunohistochemical staining was used to measure the protein expression level of TRPC6 in renal tissue,and Western blotting was used to measure the relative expression levels of TRPC6,LC3B,P62,PINK1,and Parkin in renal tissue;transmission electron microscopy was used to observe the change in the number of mitophagosomes in renal tubular cells of mice.HK-2 cells were divided into high glucose+TRPC6 siRNA transfection+DMSO intervention group(group G,treated with TRPC6 siRNA+35.0 mmol/L glucose+0.06%DMSO)and high glucose+TRPC6 siRNA transfection+mitop

关 键 词：糖尿病肾病 TRPC阳离子通道 线粒体自噬 肾炎 间质性 疾病模型 动物 小鼠 近交C57BL 

分 类 号：R587.24[医药卫生—内分泌] R692.33[医药卫生—内科学]