Clearance of damaged mitochondria via mitophagy is important to the protective effect of ischemic preconditioning in kidneys

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). The protective effect of IPC was abolished by pharmacological inhibitors of autophagy and by the ablation of <i>Atg7</i> from kidney proximal tubules. Pretreatment with BECN1/Beclin1 peptide induced autophagy and protected against IRI. These results suggest the dependence of IPC protection on renal autophagy. During IPC, the mitophagy regulator PINK1 (PTEN induced putative kinase 1) was activated. Both IPC and BECN1 peptide enhanced mitolysosome formation during renal IRI in mitophagy reporter mice, suggesting that IPC may protect kidneys by activating mitophagy. We further established an in vitro model of IPC by inducing ‘chemical ischemia’ in kidney proximal tubular cells with carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Brief treatment with CCCP protected against subsequent injury in these cells and the protective effect was abrogated by autophagy inhibition. In vitro IPC increased mitophagosome formation, enhanced the delivery of mitophagosomes to lysosomes, and promoted the clearance of damaged mitochondria during subsequent CCCP treatment. IPC also suppressed mitochondrial depolarization, improved ATP production, and inhibited the generation of reactive oxygen species. Knockdown of <i>Pink1</i> 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. <b>Abbreviations</b>: 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: 4ʹ,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: 5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3,3ʹ-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

缺血预处理（ischemic preconditioning，IPC）可在包括肾脏在内的多种器官中发挥组织保护作用，但其潜在机制尚未阐明。本研究证实巨自噬/自噬（macroautophagy/autophagy）尤其是线粒体自噬（mitophagy）在IPC对肾脏的保护效应中扮演关键角色。IPC可诱导小鼠肾小管上皮细胞发生自噬，并抑制后续的肾缺血再灌注损伤（ischemia-reperfusion injury，IRI）。自噬的药理学抑制剂以及肾脏近端小管中<i>Atg7</i>的基因敲除均可消除IPC的保护作用。使用BECN1/Beclin1肽段进行预处理可诱导自噬，从而对抗IRI。上述结果表明，IPC的肾脏保护作用依赖于肾自噬。
在IPC过程中，线粒体自噬调节因子PTEN诱导推定激酶1（PTEN induced putative kinase 1，PINK1）被激活。在线粒体自噬报告基因小鼠中，IPC与BECN1肽段均可增强肾IRI过程中的线粒体溶酶体形成，提示IPC或可通过激活线粒体自噬发挥肾脏保护作用。本研究进一步利用羰基氰化物间氯苯腙（carbonyl cyanide 3-chlorophenylhydrazone，CCCP）诱导肾小管上皮细胞发生"化学性缺血"，构建了体外IPC模型。对细胞进行短暂CCCP处理可对抗后续的细胞损伤，该保护效应可被自噬抑制剂阻断。在体外IPC模型中，短暂CCCP处理可增加线粒体自噬体形成，促进线粒体自噬体向溶酶体的递送，并在后续CCCP处理过程中加速受损线粒体的清除。此外，IPC可抑制线粒体去极化，改善ATP生成，并减少活性氧（reactive oxygen species，ROS）的产生。敲低<i>Pink1</i>可抑制线粒体自噬，并削弱IPC的细胞保护作用。综上，上述结果表明，自噬尤其是线粒体自噬在IPC的保护效应中发挥重要功能。
<b>缩略语</b>: ACTB：肌动蛋白β（actin, beta）；ATG：自噬相关（autophagy related）；BNIP3：BCL2相互作用蛋白3（BCL2 interacting protein 3）；BNIP3L/NIX：BCL2相互作用蛋白3样（BCL2 interacting protein 3 like）；BUN：血尿素氮（blood urea nitrogen）；CASP3：半胱氨酸天冬氨酸蛋白酶3（caspase 3）；CCCP：羰基氰化物间氯苯腙（carbonyl cyanide 3-chlorophenylhydrazone）；COX4I1：细胞色素c氧化酶亚基4I1（cytochrome c oxidase subunit 4I1）；COX8：细胞色素c氧化酶亚基8（cytochrome c oxidase subunit 8）；DAPI：4',6-二脒基-2-苯基吲哚（4ʹ,6-diamidino-2-phenylindole）；DNM1L：动力蛋白1样（dynamin 1 like）；EGFP：增强型绿色荧光蛋白（enhanced green fluorescent protein）；EM：电子显微镜（electron microscopy）；ER：内质网（endoplasmic reticulum）；FC： floxed对照（floxed control）；FIS1：线粒体分裂1（fission, mitochondrial 1）；FUNDC1：FUN14结构域包含蛋白1（FUN14 domain containing 1）；H-E：苏木精-伊红（hematoxylin-eosin）；HIF1A：缺氧诱导因子1亚基α（hypoxia inducible factor 1 subunit alpha）；HSPD1：热休克蛋白家族D（Hsp60）成员1（heat shock protein family D (Hsp60) member 1）；IMMT/MIC60：线粒体内膜蛋白（inner membrane mitochondrial protein）；IPC：缺血预处理（ischemic preconditioning）；I-R：缺血再灌注（ischemia-reperfusion）；IRI：缺血再灌注损伤（ischemia-reperfusion injury）；JC-1：5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑羰花青碘化物（5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3,3ʹ-tetraethylbenzimidazolylcarbocyanine iodide）；KO：基因敲除（knockout）；MAP1LC3B/LC3B：微管相关蛋白1轻链3β（microtubule associated protein 1 light chain 3 beta）；mito-QC：线粒体质量控制（mito-quality control）；mRFP：单体红色荧光蛋白（monomeric red fluorescent protein）；NAC：N-乙酰半胱氨酸（N-acetylcysteine）；PINK1：PTEN诱导推定激酶1（PTEN induced putative kinase 1）；PPIB：肽基脯氨酰异构酶B（peptidylprolyl isomerase B）；PRKN：帕金RBR E3泛素连接酶（parkin RBR E3 ubiquitin protein ligase）；ROS：活性氧（reactive oxygen species）；RPTC：大鼠近端肾小管上皮细胞（rat proximal tubular cells）；SD：标准差（standard deviation）；sIPC：模拟缺血预处理（simulated IPC）；SQSTM1/p62：隔离小体1（sequestosome 1）；TOMM20：线粒体外膜转位酶20（translocase of outer mitochondrial membrane 20）；TUNEL：脱氧核糖核苷酸末端转移酶介导的dUTP缺口末端标记法（terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling）