糖基化终产物诱导小鼠足细胞内质网应激和细胞凋亡的研究

Advanced glycation end products induce endoplasmic reticulum stress and apoptosis of murine podocytes

目的内质网应激是独立于线粒体途径诱导细胞凋亡的新途径。文中将观察糖基化终产物(advanced glycation end products,AGEs)对肾小球足细胞凋亡和内质网应激标记蛋白-葡萄糖调解蛋白78(glucose-regulated protein 78,GRP78)表达以及足细胞内游离钙离子浓度([Ca2+]i)的影响,探讨内质网应激在糖尿病肾病(diabetic nephropathy,DN)发病中的作用。方法体外培养小鼠肾小球足细胞,不同浓度的AGEs、牛血清清蛋白(bovine serum albumin,BSA)刺激小鼠足细胞6、12、24和48 h后收集细胞。采用Western blot方法检测GRP78蛋白水平,通过激光共聚焦显微镜检测足细胞内游离钙离子浓度([Ca2+]i),Annexin V/PI双染色流式细胞术检测足细胞凋亡率。结果 AGEs呈时间和剂量依赖性地诱导足细胞凋亡和GRP78蛋白表达。同时,AGEs可诱导小鼠足细胞[Ca2+]i快速上升。结论 AGEs诱导内质网应激标记蛋白表达,并激发肾小球足细胞[Ca2+]i升高。AGEs可通过内质网应激诱导足细胞凋亡。

Objective Cell apoptosis could be induced by an endoplasmic retieulum （ER） stress pathway independent from mitoehondria. However, the mechanism of advanced glycation end products （AGEs） triggering the apoptosis of podoeytes re- mains to be elucidated. This study was to investigate the role of ER stress in the pathogenesis of diabetic nephropathy by observing the effects of AGEs on the apoptosis of podocytes, the expression of the ER stress marker glucose-regulated protein 78 （ GRP78 ） and the concentration of Ca2＋ in podocytes. Methods We incubated murine podoeytes in vitro with different concentrations of AGEs and bovine serum albumin for 6, 12, 24 and 48 hours. We measured the expression level of GRP78 by Western blot, detected the intraeellular calcium concentration （ [ Ca2 ＋ ] i） by laser confoeal microscopy, and determined the AGEs -induced apoptosis of podo- eytes by Annexin V/PI double staining flow cytometry. Results AGEs induced the apoptosis of podoeytes, increased the expres- sion of GRP78 in a dose-and time-dependent manner, and meanwhile mediated a rapid rise of [ Ca2＋ ] i in the murine podocytes. Conclusion AGEs increase the expression of the ER stress marker GRP78, elevate the level of [ Ca2 ＋ ] i in podocytes, and induce the apoptosis of podocytes via ER stress.