Desflurane Preconditioning Ameliorates Renal Ischemia/Reperfusion Injury Through Suppression Of Inflammation And Pyroptosis Via NF-κB/NLRP3 Signaling

Background:Both in vivo and in vitro studies confirmed the organ protective effects of inhaled anesthetics against the ischemia-reperfusion injury which showed great potential for clinical application.However,the underlying molecular mechanisms are still not fully elucidated.Desflurane is a fluorinated methyl ethyl ethe.Current research on the organ protective roles of desflurane is very limited.It has been demonstrated the protective role against cardiac ischemiareperfusion injury,but few studies conducted on renal ischemia-reperfusion injury.Acute kidney injury(AKI)is a serious pathological condition defined as an abrupt decline in renal function and tissue injury.Due to the special structure and function of kidney,it is very sensitive to ischemia–reperfusion(I/R)injury which therefore become the primary cause of AKI.As such,it is very essential to study the protective roles of inhaled anesthetics in renal ischemia-reperfusion injury.In the aspect of underlying mechanisms,AKI is associated with the production and release of inflammatory cytokines,and inflammation related pyroptosis is a major type of cell death in proximal renal tubular cells.Base on the evidence that inhaled anesthetics could reduce inflammation and inflammatory cytokines production and release in multiple organs,we hypothesized that desflurane exert a protective role on renal ischemia-reperfusion injury through regulating inflammation related signaling pathways.In all,this study investigated the regulated roles of desflurane on inflammation related pyroptosis and focused on NLRP3 inflammasome axis.Objectives:In this study,we aimed to investigate the protective role of desflurane preconditioning on a mouse model of renal ischemia-reperfusion injury and on a cellular model of hypoxia/reoxygenation injury,as well as the function of desflurane preconditioning on reducing cytokines production and the mechanisms underlying pyroptosis inhibition during renal IRI.Methods:A renal ischemia-reperfusion injury mouse model with right-sided nephrectomy and unilateral renal ischemia followed by reperfusion was employed in this study.Desflurane was given prior to the surgery.The concentration of desflurane for preconditioning remains constant at 8%(desflurane in oxygen/nitrogen gas mixture).The preconditioning duration is 1hour,followed by a recovery time of 20 minutes.In the control group of mice(sham surgery control group and renal ischemia-reperfusion surgery control group),they are exposed to normal airflow at the same duration.The total urine volume,osmolarity,blood urea nitrogen and creatinine were evaluated and the endogenous creatinine clearance was calculated.The kidney injury was evaluated by HE staining and kidney injury score.Both immunohistochemistry and western blot was used to analyze the expression of AQP1 and AQP3.The expression of IL-1β,IL-6,TNF-α and MCP-1 in the kidney was confirmed by enzyme-linked immunosorbent assay and Western blot.To analyze the expression of pyroptosis related proteins,Western blot was used to detect the level of caspase-1,kim-1 and GSDMD.To induct hypoxia/reoxygenation,HK-2 cells were cultured in the medium without nutrients and subjected to hypoxia condition for 12 hours followed by reoxygenation for 8 hours.The cell viability was confirmed by CCK-8 assay,the NF-κ B nuclear translocation was confirmed by immunocytochemistry staining,and the expression of pyroptosis related proteins,NLRP3,kim-1,IL-1β and caspase-1 were assessed by Western blot.Results:1.Desflurane preconditioning alleviates the decline in renal function in ischemia-reperfusion mice,manifested by a reduction in the decrease of postoperative urine osmolality,the extent of increase in creatinine and blood urea nitrogen levels,and an improvement in the decreased creatinine clearance caused by renal ischemia-reperfusion.2.Desflurane preconditioning reduces the expression and release of inflammatory cytokines IL-1β,IL-6,TNF-α,and chemokine MCP-1 in the kidneys of ischemia-reperfusion mice.3.Desflurane preconditioning mitigates renal pathological changes and attenuates pyroptosis in ischemia-reperfusion mice,manifested by inhibiting the activation of key pyroptosis protease Caspase-1,suppressing the expression of pyroptosis execution protein GSDMD,and kidney tubular injury marker protein Kim-1.4.Desflurane preconditioning partially restores the downregulation of aquaporin 1 and 3,water channel proteins,in the kidneys of ischemiareperfusion mice.5.In cell experiments,pre-treatment with desflurane slows down the death of HK-2 cells induced by hypoxia/reoxygenation.6.Pre-treatment with desflurane regulates hypoxia/reoxygenation-induced necrosis in HK-2 cells through the NF-KB/NLRP3 signaling pathway,manifested by inhibiting the nuclear translocation of NF-KB,reducing the upregulation of NLRP3 expression after hypoxia/reoxygenation,decreasing the upregulation of cytokine IL-1β expression,and inhibiting the activation of key necrosis protease Caspase-1.Conclusions:Our results suggested that desflurane preconditioning protected mice from renal ischemia-reperfusion injury and this effect was through the inhibition of NF-κB/NLRP3 inflammasome induced pyroptosis.