草酸钙晶体诱导自噬加重肾小管上皮细胞间质转分化的进程  

Calcium Oxalate Crystals Trigger Autophagy and Aggravate Renal Tubular Epithelial Cell Mesenchymal Transition

作　　者：李璐 张陵艳 谌卫 丁家荣 赵婷婷 郭志勇 LI Lu;ZHANG Lingyan;CHEN Wei(Department of Nephrology,Changhai Hospital,Navy Medical University,Shanghai 200433)

机构地区：[1]海军军医大学长海医院肾内科,上海200433

出　　处：《中国中西医结合肾病杂志》2022年第6期482-487,I0002,共7页Chinese Journal of Integrated Traditional and Western Nephrology

基　　金：上海市卫生和计划生育委员会基金资助项目(No.20164Y0082)。

摘　　要：目的:在体内外模型中研究草酸钙晶体诱导的肾脏自噬和上皮间质转分化(epithelial mesenchymal transition,EMT)以及自噬在EMT中的作用。方法:(1)将20只C57BL/6小鼠随机分为对照组和模型组。检测小鼠血肌酐及尿素氮水平;取肾组织做免疫组化及westernblot检测。(2)应用一水草酸钠刺激HK2细胞株构建细胞模型,予雷帕霉素、3MA干预后检测细胞活性、自噬及EMT蛋白表达情况。结果:(1)造模组小鼠肾脏皮髓质交界处可见大量草酸钙晶体,伴随肾小管上皮细胞损伤;肾组织westernblot检测提示:LC3B、Beclin-1、α-SMA蛋白表达显著升高,E-cadherin表达显著下调(P<0.05)。(2)一水草酸钠处理HK2细胞后由鹅卵石样变成梭形,伴随细胞损伤,成时间和剂量依赖性;(3)一水草酸钠作用12 h后自噬及EMT蛋白表达升高,24 h到达高峰,随着时间延长逐渐下降,且细胞损伤加重;加予雷帕霉素处理后,α-SMA及Vimentin的表达进一步增加,细胞死亡程度加重。结论:自噬参与并加重早期草酸钙结晶肾损伤EMT的进程;通过抑制自噬可以改善草酸钙结晶肾损伤程度。Objective:To investigate calcium oxalate crystal-induced kidney autophagy and epithelial mesenchymal transition(EMT)and the role of autophagy on EMT in epithelial cells,in both in vivo and in vitro models.Methods:(1)Twenty male C57BL/6 mice(7-weeks-old,22~25 g)were randomly divided into two groups:a blank control group(Con)and a model group(Crystal).After the experiment,blood was collected for renal function evaluation.Kidneys were harvested for H&E staining,Von Kossa staining,immunohistochemistry and western blot.(2)Sodium oxalate monohydrate-treated Human renal tubular epithelial cells-HK2,were subjected to mimic calcium oxalate crystal model in vitro,and used to explore autophagy and EMT expression,with or without 3-MA and rapamycin administration.We used CCK8 kit to detect cell activity,western-blot and immunofluorescence to detect the protein expression of LC3B,Beclin-1,ATG5,ATG7,vimentin,α-SMA,E-cad.Results:(1)A large number of calcium oxalate crystals were detected at the junction of the renal cortex and medulla,accompanied with surrounding renal tubular epithelial cell injury.The LC3B,Beclin-1,α-SMA protein level was significantly increased,and the expression of E-cadherin was significantly down-regulated(P<0.05).(2)In vitro,the morphology of HK2 cells changed from cobblestone-like to spindle-shaped after OX treatment,accompanied by cell damage and death,in a time and dose-dependent manner;(3)We found that after 12 h the expression of autophagy-related proteins and EMT began to increase,reached a peak at 24 h,and then gradually decreased.After treatment with rapamycin,the expression of interstitial markerα-SMA and Vimentin further up-regulated,and the degree of cell death enhanced.Conclusion:Autophagy activated by calcium oxalate crystal can aggravates renal tubular epithelial cell EMT,inhibiting autophagy can improve renal injury triggered by calcium oxalate.

关 键 词：草酸钙结晶 自噬 上皮间质转分化 肾小管上皮细胞 

分 类 号：R692.4[医药卫生—泌尿科学]