Involvement of phosphatase and tensin homolog-induced putative kinase 1-Parkin-mediated mitophagy in septic acute kidney injury
Involvement of phosphatase and tensin homolog-induced putative kinase 1-Parkin-mediated mitophagy in septic acute kidney injury
复制标题
DOI：
10.1097/cm9.0000000000000448
复制
发表时间：
2019-10-05
影响因子：
6.1
通讯作者：
Ai, Yu-Hang
中科院分区：
文献类型：
--
作者：
Dai, Xin-Gui;Xu, Wei;Ai, Yu-Hang
背景：研究报道了急性肾损伤（AKI）中肾小管上皮细胞（RTECs）的自噬激活。磷酸酶和紧张素同源诱导的推定激酶1 （PINK1）和E3泛素蛋白连接酶Parkin参与线粒体自噬调节；然而，关于PINK1-Parkin有丝分裂在脓毒性AKI中的作用知之甚少。在这里，我们研究了PINK1-Parkin有丝分裂通路是否参与脓毒性AKI及其对体外细胞凋亡和体内肾功能的影响。方法：采用Western blot法检测细菌脂多糖（LPS）刺激的人RTEC细胞株HK-2和盲肠结扎穿孔（CLP）诱导的脓毒性AKI大鼠RTEC细胞中自噬相关基因的表达。电镜观察大鼠rtec细胞自噬相关超微结构特征。采用功能增益和功能丧失方法来研究PINK1-Parkin通路在HK-2细胞有丝分裂中的作用。采用自噬激活剂和自噬抑制剂研究自噬调节对体外细胞凋亡和体内肾功能的影响。结果：LPS刺激可显著诱导体外培养4 h LC3-II和BECN-1蛋白表达（LC3-II: 1.72±0.05比1.00±0.05,P < 0.05; BECN-1: 5.33±0.57比1.00±0.14,P < 0.05）。LC3-II、BECN-1蛋白水平也明显升高，并在术后2 h达到峰值（LC3-II: 3.33±0.12∶1.03±0.15,P < 0.05; BECN-1: 1.57±0.26∶1.02±0.11,P < 0.05）。在脓毒症大鼠rtec中观察到线粒体变形和有丝分裂酶介导的线粒体清除。与lps刺激的HK-2细胞相比，PINK1敲低显著降低LC3-II蛋白表达（1.35±0.21比2.38±0.22,P < 0.05），而PINK1过表达显著提高LC3-II蛋白表达（2.07±0.21比1.29±0.19,P < 0.05）。lps诱导的促凋亡蛋白表达在自噬激活剂处理的HK-2细胞中保持不变，在pink1过表达的细胞中显著减弱，但在自噬抑制剂处理和pink1缺失的细胞中显著上调。流式细胞术细胞凋亡实验和大鼠肾功能指标结果一致。结论：pink1 - parkin介导的线粒体自噬可能在脓毒性AKI中发挥保护作用，可能是脓毒性AKI的潜在治疗靶点。
Abstract Background: Studies have reported mitophagy activation in renal tubular epithelial cells (RTECs) in acute kidney injury (AKI). Phosphatase and tensin homolog-induced putative kinase 1 (PINK1) and E3 ubiquitin-protein ligase Parkin are involved in mitophagy regulation; however, little is known about the role of PINK1-Parkin mitophagy in septic AKI. Here we investigated whether the PINK1-Parkin mitophagy pathway is involved in septic AKI and its effects on cell apoptosis in vitro and on renal functions in vivo. Methods: Mitophagy-related gene expression was determined using Western blot assay in human RTEC cell line HK-2 stimulated with bacterial lipopolysaccharide (LPS) and in RTECs from septic AKI rats induced by cecal ligation and perforation (CLP). Autophagy-related ultrastructural features in rat RTECs were observed using electron microscopy. Gain- and loss-of-function approaches were performed to investigate the role of the PINK1-Parkin pathway in HK-2 cell mitophagy. Autophagy activators and inhibitors were used to assess the effects of mitophagy modulation on cell apoptosis in vitro and on renal functions in vivo. Results: LPS stimulation could significantly induce LC3-II and BECN-1 protein expression (LC3-II: 1.72 ± 0.05 vs. 1.00 ± 0.05, P < 0.05; BECN-1: 5.33 ± 0.57 vs. 1.00 ± 0.14, P < 0.05) at 4 h in vitro. Similarly, LC3-II, and BECN-1 protein levels were significantly increased and peaked at 2 h after CLP (LC3-II: 3.33 ± 0.12 vs. 1.03 ± 0.15, P < 0.05; BECN-1: 1.57 ± 0.26 vs. 1.02 ± 0.11, P < 0.05) in vivo compared with those after sham operation. Mitochondrial deformation and mitolysosome-mediated mitochondria clearance were observed in RTECs from septic rats. PINK1 knockdown significantly attenuated LC3-II protein expression (1.35 ± 0.21 vs. 2.38 ± 0.22, P < 0.05), whereas PINK1 overexpression markedly enhanced LC3-II protein expression (2.07 ± 0.21 vs. 1.29 ± 0.19, P < 0.05) compared with LPS-stimulated HK-2 cells. LPS-induced proapoptotic protein expression remained unchanged in autophagy activator-treated HK-2 cells and was significantly attenuated in PINK1-overexpressing cells, but was remarkably upregulated in autophagy inhibitor-treated and in PINK1-depleted cells. Consistent results were observed in flow cytometric apoptosis assay and in renal function indicators in rats. Conclusion: PINK1-Parkin-mediated mitophagy might play a protective role in septic AKI, serving as a potential therapeutic target for septic AKI.