Involvement of phosphatase and tensin homolog-induced putative kinase 1–Parkin-mediated mitophagy in septic acute kidney injury

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 apoptosisin vitro and on renal functionsin 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 apoptosisin vitro and on renal functionsin vivo.Results: LPS stimulation could significantly induce LC3-II and BECN-1 protein expression (LC3-II: 1.72 ± 0.05vs. 1.00 ± 0.05,P < 0.05; BECN-1: 5.33 ± 0.57vs. 1.00 ± 0.14,P < 0.05) at 4 hin vitro. Similarly, LC3-II, and BECN-1 protein levels were significantly increased and peaked at 2 h after CLP (LC3-II: 3.33 ± 0.12vs. 1.03 ± 0.15,P < 0.05; BECN-1: 1.57 ± 0.26vs. 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 ± 0.21vs. 2.38 ± 0.22,P < 0.05), whereas PINK1 overexpression markedly enhanced LC3-II protein expression (2.07 ± 0.21vs. 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.