Endoplasmic reticulum stress induces autophagy in renal proximal tubular cells
Endoplasmic reticulum stress induces autophagy in renal proximal tubular cells
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DOI：
10.1093/ndt/gfp215
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发表时间：
2009-09-01
影响因子：
6.1
通讯作者：
Nangaku, Masaomi
中科院分区：
文献类型：
--
作者：
Kawakami, Takahisa;Inagi, Reiko;Nangaku, Masaomi
方法。将永生化大鼠近端小管细胞暴露于经典内质网应激诱导剂tunicamycin或brefeldin a中，检测自噬主要采用免疫印迹分析LC3（一种广泛使用的自噬标记物）、免疫荧光细胞化学和电镜检测LC3。使用自噬体降解抑制剂巴菲霉素A1研究了这一现象的生物学意义。我们还利用MAPK通路抑制剂研究了内质网应激后的信号转导通路。在免疫印迹分析中，两种内质网应激诱导剂均显著增加LC3-II作为自噬的标志物。LC3免疫细胞化学和电镜也显示内质网应激激活自噬。巴菲霉素A1的抑制表明内质网应激后的自噬完成了其固有功能，即细胞质成分的降解。此外，使用MEK 1/2抑制剂U0126，抑制内质网应激诱导的自噬诱导和ERK激活，表明ERK， MAPK家族成员，是诱导自噬所必需的。我们首次证明了内质网应激诱导肾小管自噬，并阐明了其机制。这些发现为进一步研究肾脏疾病中的自噬奠定了基础。
Methods. Immortalized rat proximal tubular cells were exposed to the classic ER stress inducers tunicamycin or brefeldin A. Autophagy was detected mainly by immunoblot analysis of LC3, a widely used marker of autophagy, and also by immunofluorescent cytochemistry of LC3 and electron microscopy. Biological significance of the phenomenon was studied using bafilomycin A1, an inhibitor of autophagosome degradation. Signal transduction pathways following ER stress were also investigated using inhibitors of the MAPK pathway.Results. Both ER stress inducers significantly increased LC3-II as a marker of autophagy in immunoblot analysis. Immunocytochemistry of LC3 and electron microscopy also showed activation of autophagy by ER stress. Inhibition by bafilomycin A1 showed that autophagy following ER stress fulfilled its intrinsic function, namely degradation of cytoplasmic components. Further, use of the MEK 1/2 inhibitor U0126, which inhibits ER stress-induced autophagy induction and ERK activation, showed that ERK, a MAPK family member, was necessary to the induction of autophagy.Conclusions. For the first time, we demonstrate the induction of autophagy following ER stress in renal tubules, and clarify its mechanism. These findings serve as the foundation for further investigation into autophagy in renal diseases.