NDT Advance Access originally published online on May 19, 2009
Nephrology Dialysis Transplantation 2009 24(9):2665-2672; doi:10.1093/ndt/gfp215
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Endoplasmic reticulum stress induces autophagy in renal proximal tubular cells
1 Division of Nephrology and Endocrinology, University of Tokyo School of Medicine 2 Central Institute for Electron Microscopic Research, Nippon Medical School, Tokyo, Japan 3 Division of Pediatric Nephrology, Massachusetts General Hospital, Boston, MA, USA
Correspondence and offprint requests to: Masaomi Nangaku; E-mail: mnangaku-tky{at}umin.ac.jp
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Abstract |
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Background. Autophagy, an intracellular self-degradation system conserved throughout eukaryotes, plays an important role in a variety of biological processes, including cell death, development, cancer, defence against infection and neurodegeneration. However, little information about autophagy in renal tubular cells is available. We investigated the relationship of autophagy with endoplasmic reticulum (ER) stress in proximal tubular cells.
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.
Keywords: autophagy; ERK; ER stress; LC3
Received for publication: 27.10.08
Accepted in revised form: 20. 4.09