KIM-1对AGEs诱导的肾小管上皮细胞EMT和凋亡的影响及其机制  

Effect of KIM-1 on EMT and apoptosis of renal tubular epithelial cells induced by AGEs and its mechanism

作　　者：张霄 张睿[2] 吴妍 李宁 曹雷 张长庚 ZHANG Xiao;ZHANG Rui;WU Yan;LI Ning;CAO Lei;ZHANG Chang-geng(Department of Clinical Laboratory,Hengshui People's Hospital,Hengshui 053000,China;Central Laboratory,The Frist Hospital of Hebei Medical University,Shijiazhuang 050031,China;Department of Nephrology,Hengshui People's Hospital,Hengshui 053000,China)

机构地区：[1]衡水市人民医院检验科,河北衡水053000 [2]河北医科大学第一医院中心实验室,河北石家庄050031 [3]衡水市人民医院肾内科,河北衡水053000

出　　处：《中国病理生理杂志》2022年第11期2005-2013,共9页Chinese Journal of Pathophysiology

基　　金：中央引导地方科技发展资金项目(No.206Z7701G);衡水市科技计划项目(No.2020014061Z)。

摘　　要：目的:探讨肾损伤分子1(KIM-1)对晚期糖基化终末产物(AGEs)诱导的人近端肾小管上皮HK-2细胞上皮-间充质转化(EMT)和凋亡的影响及其可能的机制。方法:将HK-2细胞分为对照组、AGEs组、AGEs+阴性对照小干扰RNA(NC siRNA)组和AGEs+KIM-1 siRNA组。免疫荧光检测KIM-1蛋白的表达;Western blot检测KIM-1、α-平滑肌肌动蛋白(α-SMA)、E-cadherin、Bcl-2、Bcl-2相关X蛋白(Bax)、caspase-3、细胞外信号调节激酶1/2(ERK1/2)和磷酸化ERK1/2(p-ERK1/2)蛋白水平;RT-qPCR检测KIM-1 mRNA的表达;MTS检测细胞活力;TUNEL染色和annexin V-FITC/PI双染流式细胞术检测细胞凋亡;流式细胞术检测细胞活性氧(ROS)水平。结果:与对照组比较,AGEs处理后的HK-2细胞中KIM-1蛋白、KIM-1 mRNA和α-SMA蛋白表达呈时间依赖性升高,E-cadherin蛋白表达和细胞活力呈时间依赖性降低,凋亡率呈时间依赖性升高,Bcl-2蛋白表达降低,Bax和caspase-3蛋白表达增加,ROS水平和ERK1/2磷酸化水平显著升高(P<0.05或P<0.01)。转染KIM-1 siRNA能够在AGEs条件下减少HK-2细胞中KIM-1的表达(P<0.01)。与AGEs+NC siRNA组相比,敲减KIM-1的HK-2细胞经AGEs诱导后,细胞α-SMA蛋白表达显著降低,E-cadherin蛋白表达和细胞活力显著升高,细胞凋亡率显著降低,Bcl-2蛋白表达增加,Bax和caspase-3蛋白表达降低,ROS水平和ERK1/2磷酸化水平显著降低(P<0.05或P<0.01)。结论:AGEs刺激能够呈时间依赖性地上调HK-2细胞中KIM-1的表达;敲减KIM-1可抑制AGEs诱导的HK-2细胞EMT和凋亡,其机制可能与降低ROS水平和抑制ERK通路的激活有关。AIM:To investigate the effect of kidney injury molecule-1(KIM-1)on epithelial-mesenchymal transition(EMT)and apoptosis induced by AGEs in human renal proximal tubular epithelial HK-2 cells and its possible mechanism.METHODS:The HK-2 cells were divided into the control group,AGEs group,AGEs+negative control small interfering RNA(NC siRNA)group,and AGEs+KIM-1 siRNA group.The KIM-1 protein expression was examined by immunofluorescence.The protein levels of KIM-1,α-smooth muscle actin(α-SMA),E-cadherin,Bcl-2,Bcl-2-associated X protein(Bax),caspase-3,extracellular signal-regulated kinase 1/2(ERK1/2)and phosphorylated ERK1/2(p-ERK1/2)were detected by Western blot.The KIM-1 mRNA expression was detected by RT-qPCR.Cell viability was detected by MTS assay,and the apoptosis was detected by TUNEL staining and flow cytometry with annexin V-FITC/PI double staining.Flow cytometry was used to detect reactive oxygen species(ROS).RESULTS:Compared with control group,the protein and mRNA levels of KIM-1,the cell apoptosis rate,the ROS generation,and the protein levels of α-SMA,Bax,caspase-3 and p-ERK1/2 were all increased(P<0.05 or P<0.01)in AGEs group,but the cell viability and the protein expression of E-cadherin and Bcl-2 were all decreased(P<0.05 or P<0.01).Transfection with KIM-1 siRNA decreased the expression of KIM-1 in HK-2 cells treated with AGEs.Compared with AGEs+NC siRNA group,the protein and mRNA levels of KIM-1,the cell apoptosis rate,the ROS generation,and the protein levels of α-SMA,Bax,caspase-3 and p-ERK1/2 were all decreased(P<0.05 or P<0.01)in AGEs+KIM-1 siRNA group,while the cell viability and the protein expression of E-cadherin and Bcl-2 were all increased(P<0.05 or P<0.01).CONCLUSION:The AGEs stimulate KIM-1 expression in HK-2 cells.Knockdown of KIM-1 inhibits AGEs-induced EMT and apoptosis of HK-2 cells,and the mechanism may be associated with reducing ROS levels and inhibiting the activation of ERK pathway.

关 键 词：肾损伤分子1 晚期糖基化终末产物 HK-2细胞 上皮-间充质转化 细胞凋亡 

分 类 号：R363.2[医药卫生—病理学] R692[医药卫生—基础医学]