TNFα和TNFR1信号通路对小鼠急性肾损伤敏感性影响的研究

Tumor necrosis factor-alpha and tumor necrosis factor receptor 1 passway induced the high susceptibility to acute kidney injury in mice

目的本研究拟从衣霉素诱导的内质网应激反应着手,观察肿瘤坏死因子α(TNFα)及TNF受体1、受体2(TNFR1、TNFR2)在急性肾组织损伤过程中的作用。方法采用衣霉素腹腔注射TNFα、TNFR1、TNFR2基因敲除小鼠及TNFR1R2联合基因敲除小鼠,造成其肾组织急性内质网应激反应和急性肾损伤,对各种小鼠肾组织分别进行病理染色、mRNA和蛋白水平表达检测、凋亡细胞染色、免疫组化染色和油红O染色等。在体外应用免疫印迹方法,检测TNFR1基因敲除小鼠肾近曲小管细胞的真核翻译起始因子2α(eIF2α)的磷酸化蛋白表达水平,并检测磷酸化酶抑制剂(salubrinal)作用后eIF2α的磷酸化水平。结果与C57小鼠相比,TNFα基因敲除小鼠对衣霉素引起的肾损伤更严重,且其病变主要位于肾近曲小管,表现为广泛的空泡变性、脂质聚积和细胞凋亡。提前24h用TNFα腹腔注射TNFα基因敲除小鼠,能逆转衣霉素引起的肾损伤。用衣霉素作用于TNFR1、TNFR2基因敲除小鼠,发现只有TNFR1基因敲除小鼠出现严重的肾损伤,而TNFR2基因敲除小鼠未出现类似的反应,提示TNFα主要通过TNFR1通路发挥作用。衣霉素诱导的急性内质网应激反应,未引起TNFα和TNFR1基因敲除小鼠肾组织中eIF2α的活化(磷酸化)。在体外,衣霉素也未引起TNFR1基因敲除小鼠肾近曲小管细胞的eIF2α磷酸化,反而造成小管细胞凋亡增加;eIF2α磷酸化酶抑制剂作用于TNFR1基因敲除小鼠肾小管细胞后,能明显提高eIF2α的磷酸化,降低小管细胞凋亡率。结论阻断TNFα/TNFR1信号通路能导致肾组织eIF2α失调,提高了对急性内质网应激损伤的敏感性。

Objective To study the role of tumor necrosis factor-alpha （TNFct） and tumor necrosis factor recptor 1, 2 （TNFR1, R2） in acute kidney injury in mice from endoplasmic reticulum （ER） stress induced by tunicamycin. Methods TNFct, TNFR1, TNFR2 or TNFR1R2- knockout mice were treated with tunicamycin by intraperitoneal injection which induced acute ER stress in kidney and acute kidney injury. After harvesting above kidney tissues, PAS, TUNEL, immunohistochemistry or oil red O staining were preformed respectively for detection of cell death or apoptosis. And the measures of mRNA and protein levels were executed using RT-PCR, real-time PCR and western blot. In vitro, the level of phosphorylated eukaryotic translation initiation factor 2ct （elF2ct） of proximal tubular cells in TNFR1 knockout mice implemented with or without salubrinal, an inhibitor of elF2ct phosphatase, was determined. Results Much more severe renal damage induced by tunicamycin in TNFct knockout mice than in wild-type mice （C57）. And kidney injury in TNFct knockout mice was mostly distributed in proximal tubules, which showed extensive cell vacuolation, lipid accumulation, and apoptosis. TNFct knockout mice treated with TNFc~ 24 hours before tunicamycin injection reversed this renal damage. Severe kidney injury was found in TNFR1 but not TNFR2 knockout mice when TNF-receptor-deficient mice were managed with tunicamycin, suggesting the role of TNFa was through TNFR1. Kidney of TNFct or TNFRl-deficient mice did not show a significant increase in elF2a phosphorylation in response of tunicamycin-induced acute ER stress. In vitro, proximal tubular cells from TNFR1 knockout mice did not show high activity of elF2cc phosphorylation under tunicamycin and presented high ER stress-induced cell death. However, these proximal tubule cells treated with salubrinal increased the levels of phosphorylated elF2ct and substantially reduced tunicamycin-induced cell death. Conclusions Thus, disruption of TNFct/TNFR1 signaling pathway leads to dysregulation of elF2a and increased susceptibility to acute ER stress injury in the kidney.