转录因子Krüppel样因子15通过下调活化T细胞核因子胞质1型保护足细胞  

Transcription factor KLF15 protects podocytes by suppressing NFATc1 signaling pathway

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作  者:窦曹帅 张鸿[2] 柯贵宝 连智雯 陈雪芹 史伟[2] 梁馨苓[2] 章斌 刘双信[1,2] DOU Caoshuai;ZHANG Hong;KE Guibao;LIAN Zhiwen;CHEN Xueqin;SHI Wei;LIANG Xinling;ZHANG bin;LIU Shuangxin(School of Medicine,South China University of Technology,Guangzhou 510006,China;Department of Nephrology,Guangdong Provincial People's Hospital,Guangdong Academy of Medical Sciences,Guangzhou 510080,China)

机构地区:[1]华南理工大学医学院,广州510006 [2]广东省人民医院肾内科广东省医学科学院

出  处:《肾脏病与透析肾移植杂志》2019年第5期427-434,共8页Chinese Journal of Nephrology,Dialysis & Transplantation

基  金:国家自然科学基金面上项目(81670656,81870508);国家自然科学基金青年项目(81900658);广州市科技计划项目(201707010009)

摘  要:目的:探究转录因子Krüppel样因子15(Krüppel-Like Factor 15,KLF15)通过下调活化T细胞核因子胞质1型(Nuclear factor of activated T-cells cytoplasmic 1,NFATc1)保护足细胞的作用机制。方法:通过免疫荧光染色观察在正常人及不同类型肾小球疾病患者足细胞KLF15的表达情况;通过实时荧光定量PCR(RT-PCR)、Western blot检测体外培养的永生化小鼠足细胞在高糖(HG)、脂多糖(LPS)处理48h及阿霉素(ADR)处理24h后KLF15的表达情况,足细胞感染腺病毒瞬时过表达KLF15后KLF15过表达效率、促凋亡蛋白BAX、抗凋亡蛋白BCL2及NFATc1的表达情况,足细胞给予地塞米松处理后NFATc1表达情况;通过流式细胞术检测在过表达KLF15的足细胞中给予损伤刺激(ADR、LPS、HG)后,足细胞的凋亡情况;通过免疫染色质共沉淀(CHIP)检测足细胞在正常情况下与LPS处理后转录因子KLF15与NFATc1启动子的结合情况;通过RT-PCR检测足细胞过表达KLF15后,NFATc1下游基因的表达情况;结果:KLF15在不同类型的肾小球疾病患者足细胞表达降低;体外培养的足细胞在损伤刺激的情况下KLF15的mRNA及蛋白水平表达均降低;足细胞过表达KLF15后,促凋亡蛋白BAX表达降低,抗凋亡蛋白BCL2表达升高,在给予损伤刺激的情况下,过表达KLF15组的足细胞凋亡率较对照组明显减少;足细胞过表达KLF15后,NFATc1在mRNA及蛋白水平的表达降低;NFATc1下游目的基因转录减少;在正常的足细胞中,转录因子KLF15与NFATc1启动子区域有结合,且在LPS损伤刺激下结合减弱,过表达足细胞中的KLF15,NFATc1的下游基因(Fzd9、Rcan1、Plaur)在mRNA水平表达降低;在体外培养的足细胞给予地塞米松处理后,转录因子KLF15表达明显增高, NFATc1表达降低。结论:转录因子KLF15通过下调NFATc1保护足细胞,地塞米松可通过升高KLF15抑制NFATc1保护足细胞。Objective:To explore the molecular mechanism of Krüppel like Factor 15(KLF15)protecting podocytes by suppressing nuclear factor of activated T-cells cytoplasmic 1(NFATc1)signaling pathway. Methodology:The expressions of KLF15 in podocytes of normal people and patients with glomerular disease were observed by immunofluorescence staining;Real-time quantitative PCR(RT-PCR) and Western blot were used to detect expression of transcription factor KLF15 of the conditional immortalized mouse podocytes cultured in high glucose(HG) 30 mmol/L for 48 h,lipopolysaccharide(LPS) 100 μg/ml for 48 h and adriamycin(ADR) 0.25 μg/ml for 24 h;After podocyte transfection of adenovirus transiently overexpressed KLF15,the effect of KLF15 overexpression,apoptosis-promoting protein BAX and anti-apoptotic protein BCL2 were detected by real-time quantitative PCR(RT-PCR) and Western blot;Apoptosis of podocytes was detected by flow cytometry after injury stimulation(ADR,LPS,HG) was administered to podocytes after overexpression KLF15;After overexpression of KLF15 in podocytes,the expression of NFATc1 was detected by RT-PCR and Western blot;Binding of KLF15 to NFATc1 promoter was detected by chromatin-immunoprecipitation(CHIP);The expression of NFATc1 downstream gene was detected by RT-PCR.In vitro,podocytes were cultured and treated with dexamethasone,the expression of NFATc1 was observed by Western blot and RT-PCR. Results:In patients with glomerular disease,the expression of KLF15 in podocytes were decreased;The expression of KLF15 mRNA and protein were decreased in podocytes cultured in vitro;After overexpression of KLF15 in podocytes,the expression of pro-apoptotic protein BAX was decreased,and the expression of anti-apoptotic protein BCL2 was increased.Under the condition of injury stimulation,the apoptotic rate in overexpression of KLF15 in podocytes was significantly lower than that in the control group;The expression of NFATc1 at mRNA and protein levels was decreased after overexpression of KLF15 in podocytes;Downstream target gene

关 键 词:足细胞 Krüppel样因子15 活化T细胞核因子胞质1型 

分 类 号:R692[医药卫生—泌尿科学]

 

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