生长抑制和DNA损伤诱导基因45β在足细胞损伤中的作用及机制

Growth arrest and DNA damage-inducible protein 45β plays a role in podocyte injury

目的:研究生长抑制和DNA损伤诱导基因45β(GADD45B)在足细胞损伤中的作用及机制。方法:微分离37例肾活检证实的局灶节段性肾小球硬化(FSGS)患者肾小球,qRT-PCR检测肾小球GADD45B基因表达,同时进行免疫组化检测GADD45B蛋白表达。进一步利用模式动物斑马鱼研究GADD45B基因功能,Podocin启动子驱动和UAS/GAL4系统构建足细胞上特异性表达GADD45B转基因斑马鱼,硝基还原酶/甲硝唑(NTR/MTZ)系统特异性诱导足细胞损伤,观察斑马鱼水肿发生率,定量检测蛋白尿及足细胞凋亡。结果:FSGS患者肾小球GADD45B基因和蛋白表达水平显著高于正常对照组,并与FSGS患者白尿、血白蛋白及血肌酐水平密切相关;斑马鱼研究结果显示,足细胞上高表达GADD45B能够显著加重MTZ诱导的足细胞损伤,GADD45Ba/b转基因鱼的水肿发生率和蛋白尿水平均显著高于野生型斑马鱼。活性caspase染色结果显示,GADD45B转基因鱼发生凋亡的足细胞数显著高于非转基因鱼。吗啉环寡聚核苷酸注射斑马鱼抑制GADD45B基因表达,则MTZ诱导的斑马鱼水肿发生率和蛋白尿水平显著降低。结论:高表达GADD45B显著加重足细胞受损,而抑制GADD45表达则能有效改善足细胞损伤,提示其可作为治疗足细胞损伤药物靶点。

Objective： To investigate the role of growth arrest and DNA damage-inducible protein 45β（GADD45B） in podocytes injury of zebrafish. Methodology：（1） Immunohistochemistry and QPCR were used to detect the expression of GADD45B in glomeruli of patients with focal segmental glomerulosclerosis（ FSGS）.（ 2） Correlation analysis was used to analyze the relationship between the expression level of GADD45B mRNA and the clinical related indexes of patients with FSGS.（ 3） RT-PCR detected the expression of GADD45Ba/bb in the glomeruli of zebrafish.（ 4）Transgenic zebrafish was screened by fluorescence microscopy,and in situ hybridization identification of transgenic zebrafish GADD45 Ba/bb was expressed on the glomeruli.（ 5） Metronidazole（ MTZ） induced podocytes damage in transgenic zebrafish.（ 6） ELISA was used to detect proteinuria,and apoptosis was detected by caspase staining. Results：GADD45B mRNA and protein levels were up-regulated in the glomeruli of patients with FSGS and the expression level was negatively correlated with renal function of the patients. In an in vivo zebrafish model of inducible podocyte injury that we previously established,we found that podocyte-specific overexpression of zebrafish orthologs of GADD45B exacerbated edema,proteinuria and foot effacement,while inhibition of GADD45B expression by morpholino oligo in zebrafish larvae ameliorated podocyte injury. Conclusion：Taken together,our findings demonstrated that GADD45B is an important mediator of podocyte apoptosis upon injury and may be a therapeutic target for the management of podocyte injury in glomerular diseases.