MSS4在缺血再灌注损伤大鼠肾脏中的表达及作用

Expression and effect of MSS4 in rat kidney after ischemia-reperfusion injury

目的探索MSS4在缺血再灌注损伤(IRI)大鼠肾脏中的表达情况,以及在缺氧/复氧大鼠肾小管上皮细胞中的作用。方法雄性成年SD大鼠分为假手术组和IRI组,分别予以假手术和肾脏缺血再灌注处理(双侧肾蒂夹闭45 min后再灌注),分别于术后0、12、24、48、72 h收集血液及肾组织标本。自动分析法检测各组大鼠血尿素氮、血肌酐水平,肾组织PAS染色评估肾小管损伤情况,Western blot检测肾脏组织中MSS4的表达水平,免疫荧光染色检测MSS4、Vimentin在肾组织中的定位及表达情况。大鼠肾小管上皮细胞(NKR-52E)分为对照组和复氧组,对照组正常培养,复氧组缺氧6 h后分别复氧0、12、24、48、72 h。免疫荧光检测各时间点MSS4和α-SMA的表达,大鼠肾小管上皮细胞转染siRNA MSS4后检测缺氧/复氧后α-SMA的表达变化。结果与假手术比较,IRI组血尿素氮、血肌酐及肾小管损伤评分术后12 h明显升高(P<0.05),72 h肾功恢复正常,肾小管损伤未完全恢复。MSS4的表达水平在IRI后48 h开始升高,72 h更明显(P<0.05)。肾组织免疫荧光染色显示MSS4与Vimentin均定位于肾小管上皮细胞,72 h表达水平明显高于假手术组(P<0.01)。细胞免疫荧光染色显示,与对照组比较,复氧组复氧72 h后MSS4及α-SMA的表达水平明显增加(P<0.05),转染siRNA MSS4后α-SMA的表达明显降低(P<0.01)。结论MSS4在IRI大鼠肾脏中表达增加,参与AKI后肾小管上皮细胞上皮间质转化的调控。

Objective To investigate the expression of MSS4 in rat kidneys after ischemia-reperfusion injury(IRI),and to explore its role in rat renal tubular epithelial cells after hypoxia/reoxygenation(H/R).Methods Sprague dawley(SD)male adult rats were divided into the sham operation group and the IRI group,which were treated with the sham operation and renal ischemia reperfusion respectively(bilateral renal pedicles were clamped for 45 minutes and then reperfused).Blood and kidney tissue samples were collected at 0,12,24,48,and 72 hours after the surgery.Then,the levels of serum urea nitrogen and serum creatinine in rats were measured by an automatic analyzer,and the renal injury was evaluated by PAS staining.Western blot was used to detect the expression of MSS4 in kidney tissues,and immunofluorescence staining was used to detect the localization and expression of MSS4 and Vimentin in kidney tissues.Rat renal tubular epithelial cells(NKR-52E)were divided into the control group and the reoxygenation group.The control group was cultured normally.The reoxygenation group was hypoxic for 6 hours and reoxygenated for 0,12,24,48,72 hours.The expression of MSS4 andα-SMA at each time point was detected by immunofluorescence,and the changes in the expression ofα-SMA after H/R were detected after the NKR-52E cells were transfected with siRNA MSS4.Results Compared with the sham operation group,the serum creatinine,serum urea nitrogen,and renal tubular injury scores in the IRI group were significantly increased at 12 hours after the operation(P<0.05),the renal function returned to normal at 72 hours,but the tubular injury was not fully recovered.The expression level of MSS4 began to increase at 48 hours after IRI,and was more obvious at 72 hours(P<0.05).Immunofluorescence staining showed that both MSS4 and Vimentin were located in renal tubular epithelial cells,and their expression was more significant 72 hours after IRI compared with the sham operation group(P<0.01).Compared with the control group,the expression of MSS4 andα-SMA in the reoxygenation group increased after 6 hours of hypoxia and reoxygenation for 72 hours(P<0.05),and the expression ofα-SMA decreased significantly after transfection of siRNA MSS4(P<0.01).Conclusion The expression of MSS4 has been increased in the kidney of IRI rats,and MSS4 regulates the epithelial-mesenchymal transition(EMT)of renal tubular epithelial cells after AKI.