The Protective Effect And Mechanism Of RTA-408 On Renal Ischemia-reperfusion Injury

Background: Acute kidney injury(AKI)is a common critical conundrum characterized by acute progressive renal dysfunction,sharp decline in glomerular filtration rate and azotemia accompanied by electrolyte,acid-base balance disorders and oliguria or even no urine.Renal ischemia-reperfusion injury is an important cause of acute kidney injury.Heart surgery,kidney transplantation and shock,can induce renal ischemia / reperfusion injury during which a large amount of reactive oxygen species are produced,resulting in acute kidney injury characterized by acute tubular necrosis.Studies have shown that Nrf2 is an important nuclear transcription factor in cellular defense against oxidative stress,which regulates the expression of a variety of downstream antioxidant genes in the nucleus when combined with antioxidant elements(AREs).RTA-408 is a potent Nrf2 inducer which is recently successfully synthesized,and in vitro experiments confirmed its antioxidant,anti-apoptotic biological function,suggesting that it has a protective effect of acute kidney injury caused by ischemia-reperfusion.However,despite significant need and historical trials,there are no effective drugs in use for the prevention or treatment of acute kidney injury(AKI).Therefore,it is of great significance to study the molecular mechanism of ischemia-reperfusion injury and actively explore effective drug and molecular therapy targets to reduce and avoid the loss of nephron caused by ischemia-reperfusion.Aim: Here,this study was aimed to determine whether and how RTA-408,a novel oleanane triterpenoid,could confer protection against renal ischemia-reperfusion injury(IRI)in male mice.To evaluate the effect of RTA-408 on renal function,oxidative stress and apoptosis during renal ischemia-reperfusion injury,and to reveal the pharmacological effects of RTA-408 on renal ischemia-reperfusion injury and its key drug target.Methods and materials: Renal ischemia-reperfusion injury model was established in the C57 mice with left renal pedicle clamping for 30 min,accompanied by right nephrectomy.The effects of RTA-408 on C57 mice were evaluated by renal function(serum creatinine,urea nitrogen)and pathology(PAS staining),meanwhile,TUNEL(marker of apoptosis),Ki-67(marker of proliferation)and MPO(marker of neutrophil infiltration)were also evaluated.ROS production(ROS immunofluorescence),total antioxidant capacity(T-AOC,t-GSH,GSH / GSSG ratio),changes in oxidative end products(MDA and carbonylation protein)were also measured to show RTA-408's role in oxidative stress.Western Blot was used to evaluate the effect of RTA-408 on the nuclear translocation and expression of Nrf2 and downstream genes(HO-1,NQO-1,GSR,GCLc)GSH synthesis / metabolizing enzymes(GCL,GST,Gpx)activity in renal homogenates were also measured by enzymes activity assay.To further clarify the mechanism by which RTA-408 regulates GSH synthesis / metabolism enzymes,HK-2 cell hypoxia / reoxygenation model was used to assess the transcriptional regulation of Nrf2,HO-1 and GCLc by RTA-408,and GCLc subcellular localization and expression were assessed by immunofluorescence.Meanwhile the GCL inhibitor BSO were used to observe that whether the protective effect of RTA-408 was blunted by GCL inhibitor.In order to clarify the molecular target of RTA-408,a model of renal ischemia-reperfusion injury was established in Nrf2 knockout mice.The levels of serum creatinine,urea nitrogen,morphological(PAS staining of kidney sections)and oxidative stress ROS immunofluorescence)to evaluate the effect of RTA-408 on renal ischemia-reperfusion injury in Nrf2 Knockout mice.Results: Mice treated with RTA-408 undergoing unilateral ischemia followed by contralateral nephrectomy had improved renal function and histological outcome,as well as decreased apoptosis,ROS production,and oxidative injury marker compared with vehicle-treated mice.Also,we had found that RTA-408 could strengthen the total antioxidant capacity by increasing Nrf2 nuclear translocation and subsequently increased Nrf2 downstream GSH-related antioxidant gene expression and activity.In vitro study demonstrated that GSH biosynthesis enzyme GCLc could be an important target of RTA-408.Furthermore,Nrf2-deficient mice treated with RTA-408 had no significant improvement in renal function,histology,ROS production,and GSH-related gene expression.Conclusions: Thus,by upregulating Nrf2 and its downstream antioxidant genes,RTA-408 presents a novel and potential approach to renal IRI prevention and therapy.