Autophagy
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Ischemic preconditioning (IPC) affords tissue protection in organs including kidneys; however, the underlying mechanism remains unclear. Here we demonstrate an important role of macroautophagy/autophagy (especially mitophagy) in the protective effect of IPC in kidneys. IPC induced autophagy in renal tubular cells in mice and suppressed subsequent renal ischemia-reperfusion injury (IRI). ⋯ Knockdown of Pink1 suppressed mitophagy and reduced the cytoprotective effect of IPC. Together, these results suggest that autophagy, especially mitophagy, plays an important role in the protective effect of IPC. Abbreviations: ACTB: actin, beta; ATG: autophagy related; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; BUN: blood urea nitrogen; CASP3: caspase 3; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; COX4I1: cytochrome c oxidase subunit 4I1; COX8: cytochrome c oxidase subunit 8; DAPI: 4',6-diamidino-2-phenylindole; DNM1L: dynamin 1 like; EGFP: enhanced green fluorescent protein; EM: electron microscopy; ER: endoplasmic reticulum; FC: floxed control; FIS1: fission, mitochondrial 1; FUNDC1: FUN14 domain containing 1; H-E: hematoxylin-eosin; HIF1A: hypoxia inducible factor 1 subunit alpha; HSPD1: heat shock protein family D (Hsp60) member 1; IMMT/MIC60: inner membrane mitochondrial protein; IPC: ischemic preconditioning; I-R: ischemia-reperfusion; IRI: ischemia-reperfusion injury; JC-1: 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide; KO: knockout; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; mito-QC: mito-quality control; mRFP: monomeric red fluorescent protein; NAC: N-acetylcysteine; PINK1: PTEN induced putative kinase 1; PPIB: peptidylprolyl isomerase B; PRKN: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; RPTC: rat proximal tubular cells; SD: standard deviation; sIPC: simulated IPC; SQSTM1/p62: sequestosome 1; TOMM20: translocase of outer mitochondrial membrane 20; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.
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Mitochondrial dynamics is highly implicated in a plethora of cellular processes including apoptosis and mitophagy. However, little is known about the scope and precise functions of mitochondrial dynamics proteins for mitochondrial quality control and cellular homeostasis. Whether mitochondrial dynamics proteins serve in cellular processes reliant on mitochondrial fission-fusion is still not fully explored. ⋯ The recruitment of PRKN to depolarized mitochondria modulated the UPS-dependent degradation of MFN2 (mitofusin 2) and FIS1 (fission, mitochondrial 1) specifically, to further promote mitophagy. Our findings uncover a bridging role of MIEF1 integrating cell death and mitophagy, unlikely dependent on mitochondrial dynamics, implying new insights to mechanisms determining cellular fate. Abbreviations: ActD: actinomycin D; BAX: BCL2 associated X, apoptosis regulator; BAK1: BCL2 antagonist/killer 1; BCL2L1: BCL2 like 1; BMH: 1,6-bismaleimidohexane; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CHX: cycloheximide; CQ: chloroquine; CYCS: cytochrome c, somatic; DIABLO: diablo IAP-binding mitochondrial protein; DKO: double knockout; DNM1L/DRP1: dynamin 1 like; FIS1: fission, mitochondrial 1; GFP: green fluorescent protein; IP: immunoprecipitation; MFN1: mitofusin 1; MFN2: mitofusin 2; MG132: carbobenzoxy-Leu-Leu-leucinal; MIEF1/MiD51: mitochondrial elongation factor 1; MIEF2/MiD49: mitochondrial elongation factor 2; MOMP: mitochondrial outer membrane permeabilization; MTR: MitoTracker Red; OA: oligomycin plus antimycin A; OCR: oxygen consumption rate; OMM: outer mitochondrial membrane; PARP: poly(ADP-ribose) polymerase; PI: propidium iodide; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; SD: standard deviation; STS: staurosporine; TNF: tumor necrosis factor; UPS: ubiquitin-proteasome system; VDAC1: voltage dependent anion channel 1.