肾小管上皮细胞内质网应激时NGAL表达改变的调控机制研究

The Regulatory Mechanism of NGAL Overexpression in the Endoplasmic Reticulum Stress of Renal Tubular Epithelial Cells

目的:探讨人肾小管上皮细胞(HK-2细胞)发生内质网应激时,NGAL表达增加的上游调控机制。方法:将HK-2细胞分为对照组(正常HK-2细胞),TG(毒胡萝卜素,thapsigargin)组(5μmol/L TG处理8 h),单纯转染组(siRNAATF4试剂转染24 h),转染+TG组(siRNA-ATF4试剂转染24 h后,5μmol/L TG处理8 h),阴性对照组(siRNA-阴性对照物转染24 h),DMSO组(5μmol/L DMSO处理8 h)。采用Western blot检测各组细胞内质网源性转录因子(CHOP)、内质网分子伴侣葡萄糖调节蛋白78(GRP78)、中性粒细胞明胶酶相关性载脂蛋白(NGAL)、激活转录因子4(ATF4)的表达,采用Real-time PCR方法测得ATF4mRNA、NGALmRNA表达量。结果:与对照组相比,TG组细胞NGAL、ATF4、ATF4mRNA、NGALmRNA表达量显著提高(P <0. 05),而转染+TG组、单纯转染组、阴性对照组、DMSO组中ATF4及NGAL差异无统计学意义(P> 0. 05)。与TG组相比,转染+TG组ATF4、NGAL、ATF4mRNA及NGALmRNA表达量呈显著降低趋势(P <0. 05)。在TG组与转染+TG组细胞中,CHOP和GRP78呈过表达状态(P <0. 05),而转染+TG组细胞CHOP和GRP78提升趋势明显低于TG组细胞(P <0. 05)。结论:(1) TG可诱导人肾小管上皮HK-2细胞发生内质网应激反应。(2) HK-2细胞发生内质网应激反应时,抑制ATF4表达会引起NGAL降低,提示ATF4是NGAL表达的上游调控因子。(3) HK-2细胞发生内质网应激反应时,抑制ATF4不能阻止CHOP和GRP78发生过表达,但可降低其升高程度,提示ATF4及NGAL降低可能对内质网应激反应介导HK-2细胞损伤起到一定的缓解作用。

Objective:To investigate the upstream regulatory mechanism of NGAL overexpression in human renal tubular epithelial cells(HK-2 cells)of endoplasmic reticulum stress(ERS).Methods:The cells were divided:control group(normal HK-2 cells),TG group(HK-2 cells with TG by the concentration of 5μmol/L in 8 h),simple transfection group(HK-2 cells treated by siRNA-ATF4 transfection reagent in 24 h),TG+transfection group(HK-2 cells treated by siRNA-ATF4 transfection reagent in 24 h,then added TG by the concentration of 5μmol/L in 8 h),negative control group(HK-2 cells treated by siRNA-negative transfection reagent in 24 h),DMSO group(HK-2 cells with 5μmol/L DMSO in 8 h).Western blot was used to measure the expression of GRP78,CHOP,ATF4 and NGAL.Real-time PCR was used to detect ATF4mRNA and NGALmRNA.Results:Compared with control group,the expression of NGAL,ATF4,ATF4mRNA and NGALmRNA was increased significantly(P<0.05).Among control group,TG+transfection group,negative control group and DMSO group,NGAL and ATF4 was similar(P>0.05).Compared with TG group,ATF4,NGAL,ATF4mRNA and NGALmRNA was decreased significantly in TG+transfection group(P<0.05).Both TG and TG+transfection group,CHOP and GRP78 was overexpressed(P<0.05),but the uptrend of CHOP and GRP78 in TG+transfection group was lower than those in TG group(P<0.05).Conclusion:(1)TG induced ERS in HK-2 cells.(2)After inhibited ATF4 expression,NGAL was declining obviously in ERS to indicate that ATF4 is the upstream regulator of NGAL gene expression.(3)As ERS,the overexpression of CHOP and GRP78 was insusceptible to inhibition of ATF4 as the extent of increase was influenced to indicate that the decrease of ATF4 and NGAL may reduce the damage of HK-2 cells by ERS.