ISCHEMIC PRECONDITIONING ATTENUATES RENAL ISCHEMIA-REPERFUSION INJURY THROUGH ACTIVATING THE NRF2/HO-1 PATHWAY ENHANCED AUTOPHAGY

Abstract Background and Aims Ischemic preconditioning (IPC), which is a brief and nonlethal episode of ischemia, confers protection against subsequent ischemia-reperfusion (I/R) through the up-regulation of endogenous protective mechanisms. Recent studies have reported that the activation of autophagy is associated with IPC. However, the underlying mechanisms still need to be determined. In this study, we investigated the role of IPC in renal I/R injury and demonstrated that IPC could ameliorate renal I/R injury by activating the Nrf2/HO-1 pathway and induction of autophagy. Method In order to gain insights into IPC–induced alterations at the cellular level, an in vitro model for IPC was designed using the human proximal tubule epithelial cell line HK-2. In vitro I/R was induced by layering mineral oil (for 60 min) over a thinfilm of media covering the cells followed by 120 min of reperfusion in normal media. IPC was performed by a 10-min period of incubation of cells under mineral oil followed by a 30-min recovery period prior to the 120 min reperfusion. To further determine the role of autophagy in IPC-induced renoprotection, the cells were treated with bafilomycin A1 (200 nM), prior to I/R injury. In a renal I/R injury model, mice were subjected to 30 min of renal ischemia followed by 24 h of reperfusion. IPC was produced by 5 min of ischemia followed by 10 min of reperfusion prior to sustained ischemia. Results We found that IPC significantly attenuated I/R-induced kidney injury and suppressed oxidative stress and inflammatory responses. IPC also increased LC3-II and decreased SQSTM/p62 and cleaved caspase-3 during renal I/R injury, as well as increased the number of intracellular double-membrane vesicles in injured renal cells. IPC-induced renal protection was efficiently attenuated by pretreatment with Bafilomycin A1 (0.3 mg/kg, IP). Pretreatment with IPC also dynamically affected the expression of Nrf2/HO-1 signaling components. The beneficial effects of IPC were abolished by Brusatol (2 mg/kg, IP), potent inhibitors of Nrf2. Moreover, the Nrf2 agonist t-BHQ (50 mg/kg, IP) showed similar activity as IPC. Conclusion In summary, the present study shows that Nrf2 plays a critical role in regulating the HO-1 production induced by I/R. Also, upregulation of Nrf2 expression might represent one of the major mechanisms whereby IPC confers protection against I/R-induced kidney injury. The beneficial effects of IPC could be attributed to its antioxidative stress and anti-inflammatory properties