内质网应激参与尿酸诱导的大鼠肾小球系膜细胞表型转化

Endoplasmic reticulum stress participates in uric acid-induced phenotypic transformation of rat glomerular mesangial cells

目的探讨内质网应激在尿酸(UA)诱导的大鼠肾小球系膜细胞(HBZY-1)表型转化过程中的作用。方法体外培养HBZY-1细胞,应用不同浓度UA(0.05,0.1,0.2,0.4 mmol/L)刺激48 h及应用0.2 mmol/L UA分别作用24,36,48 h,分别采用RT-PCR和Western blot印迹法检测细胞内GRP78、PDI mRNA及蛋白的表达。最后应用内质网应激反应抑制剂4-苯基丁酸(4-PBA,0.25μmol/L)预处理6 h后,再加入UA(0.2 mmol/L)处理后,应用倒置显微镜及透射电镜观察各组HBZY-1细胞的形态及超微结构变化,Western印迹法检测细胞内GRP78、PDI、α-SMA、TGF-β1、FN蛋白的表达。结果与0 mmol/L相比,0.2,0.4 mmol/L UA处理后HBZY-1细胞内质网应激指标GRP78、PDI mRNA及蛋白的表达显著上调(P<0.05),并呈时间依赖性。给予内质网应激反应抑制剂4-PBA后,UA诱导的HBZY-1细胞微绒毛密长,内质网轻度扩张,GRP78、PDI、α-SMA、TGF-β1、FN蛋白表达受到抑制(P<0.05)。结论UA可诱导HBZY-1细胞表型转化,上调内质网应激反应蛋白GRP78、PDI等指标,提示内质网应激反应可能参与UA诱导的HBZY-1细胞表型转化。

Objective To investigate the effect of endoplasmic reticulum stress on the phenotypic transformation of rat glomerular mesangial cells(HBZY-1)induced by uric acid(UA).Methods HBZY-1 cells were cultured in vitro and treated with different concentrations of UA(0,0.05,0.1,0.2,0.4 mmol/L)for 48 h and 0.2 mmol/L UA for 24,36,48 h,respectively.The mRNA and protein expression levels of GRP78 and PDI in cells were detected by RT-PCR and Western blot.HBZY-1 cells were pretreated with 4-phenylbutyric acid(4-PBA,0.25μmol/L)for 6 h and then given UA(0.2 mmol/L).The morphological and ultrastructural changes of HBZY-1 cells were observed by inverted microscope and transmission electron microscopy.The protein expression levels of GRP78,PDI,α-SMA,TGF-β1 and FN in HBZY-1 cells were detected by Western blot.Results Compared with 0 mmol/L UA,0.2,0.4 mmol/L UA significantly up-regulated the mRNA and protein expression levels of GRP78,PDI and ER stress index of HBZY-1 cells in a time dependent manner(P<0.05).After treatment with ER stress response inhibitor 4-PBA,the expression levels of GRP78,PDI,α-SMA,TGF-β1 and FN in HBZY-1 cells induced by UA were inhibited(P<0.05),and the microvilli of HBZY-1 cells were dense and ER was slightly dilated.Conclusion UA can induce the phenotypic transformation of HBZY-1 cells and up-regulate the indexes of ER stress response proteins GRP78 and PDI,suggesting that ER stress response may be involved in the phenotypic transformation of HBZY-1 cells induced by UA.