Renal Protection Effect And Underlying Mechanism Of Irisin Pretreatment On The Ischemia/reperfusion-induced Acute Kidney Injury

Acute kidney injury(AKI)is a global public health problem with a high morbidity and mortality,and current therapeutic options for AKI remain limited.Irisin is a muscle factor that plays a vital role in regulating glucose and lipid homeostasis.Recently,the anti-inflammatory,anti-apoptotic and anti-oxidative properties of irisin have received a great deal of attention.It is known that irisin can regulate the expression of uncoupling protein 1(UCP1),however UCP1 is mainly expressed in brown adipose tissue.And uncoupling protein 2(UCP2)is high expressred in the kidney,and can protect the kidney from ischemia/reperfusion injury by improving mitochondrial dynamics and inhibiting cell apoptosis.However,whether irisin can regulate the expression of UCP2 and play a role in the pathophysiology of ischemia/reperfusion-induced AKI is still unclear.In this study,we used an ischemia/reperfusion(I/R)-induced AKI C57BL/6 mouse model and a hypoxia/recovery(H/R)cell model to explore the role of irisin in the progression of AKIPART I Effect of Irisin on ischemia/reperfusion induced AKI Objective:To investigated the effect of irisin pretreatment on ischemia/reperfusion-induced AKI injury in vitro and in vivoMethods:(1)The male C57BL/6 mice were randomly divided into four groups(n=6 per group)a sham+saline group(Sham),a sham+irisin group(Sham+Irisin),an I/R+saline group(I/R),and an I/R+irisin group(I/R+Irisin).The Sham+Irisin and I/R+Irisin groups were given an intraperitoneal injection of irisin(100 ?g/kg body weight)once a day for 14 days.The same volume of sterile phosphate-buffered saline(PBS)was administered to mice in the control and I/R groups.All animals were accepted renal I/R surgery,after reperfusion 24 h,blood samples and kidney tissues were obtained for further experiments Including renal function,renal histology assays.Moreover,detected the levels of TNF-?,IL-6 and IL-1? by Western Blotting or Elisa.And detected the levels of MPO,MDA and SOD,which are the indicators of oxidative stress.Furthermore,Western Blotting and TUNEL assay were used to indicate cell apoptosis.(2)The cultured HK-2 human renal proximal tubular epithelial cells(PTECs)were randomly divided into 4 groups:CTL,CTL+Irisin,H/R and H/R+Irisin.When cells had grown to 80%confluence,a hypoxia/recovery(H/R)model was constructed.In brief,the cells were exposed to hypoxia for 3 h(5%C02,1%02,and 94%N2),followed by 3 h of reoxygenation(normal culture condition).Furthermore,before H/R,cells were starved for 24 h following treatment with recombinant irisin(1 ?g/ml).Then detected the mRNA levels of TNF-?,IL-6 and IL-1? by qRT-PCR.And the activation of caspase3 was detected by Western BlottingResults:(1)Compared with control group,H/R treatment induced HK-2 cells apoptosis and promoted cell inflammation in vitro.While,pretreatment of irisin decreased the mRNA levels of TNF-?,IL-6,IL-1? and activation of caspase 3 in cells subjected to 3 h of hypoxia and 3 h of recovery.(2)The inflammatory factors and cell apoptosis were notably increased in I/R mice compared with control mice,while the expression of TNF-?,IL-6,IL-1? and cell apoptosis were significantly lower in the I/R+Irisin group than in the I/R group.And after I/R injury,irisin decreased the level of MDA and MPO,at the same time,increased the level of SODConclusion:Pretreatment with irisin could alleviate I/R-induced kidney damage in mice and H/R-induced cell injury via decreasing cell apoptosis,inflammation and oxidative stressPart II The underlying mechanism of irisin in I/R-induced AKIObjective:Mouse model and cell experiment were used to declare the important role of uncoupling protein 2(UCP2)in the procession of Irisin alleviated renal tubular epithelial cells damage induced by I/R in mice or H/R in vivo.And to explore the potential signaling pathway of irisin in I/R-induced AKIMethods:(1)As described in the first part,the mice were randomly divided into four groups(n=6 per group):Sham,Sham+Irisin,I/R and I/R+Irisin group.After mice sacrificed,immunoblotting was used to explore the effect of irisin on pAMPK and UCP2 expression in I/R mouse model(2)The cultured HK-2 cells were randomly divided into 4 groups:CTL,CTL+Irisin,H/R and H/R+Irisin group.The level of UCP2 was examined by Western Blotting.Then,to determine the functional role of irisin in expression of UCP2 in an H/R model in vitro,HK-2 cells were treatment with UCP2 siRNA or nontarget siRNA with or without irisin.And the mRNA levels of IL-1? and IL-6 were detected by qRT-PCR,and after Annexin V-FITC and PI staining,apoptotic ratio of cells was detected by flow cytometric analysis(3)As described in the first part,the cultured HK-2 cells were randomly divided into 3 groups:CTL,Compound C pretreated(CC),and Irisin+CC group.Before treated with irisin or vehicle,cells were starved for 24-hour following treatment with Compound C (5?M)for 30 min.Then cells were harvested to detecting the level of UCP2 and phosphorylation of AMPKResults:(1)After the mice underwent 45 min of ischemia and 24 h of reperfusion,we obseversed that irisin significantly increased UCP2 and pAMPK expression in mice kidney after I/R treated.(2)In vitro,the expression of UCP2 in H/R group was substantially higher than that in the CTL group.Furthermore,supplementation with irisin increased the UCP2 level to those in H/R group.(3)In cultured HK-2 cells,to assess the cell apoptosis,we used flow cytometric analysis to determine the cell apoptotic ratio,and found that the H/R-induced cell apoptosis was not significantly relieved by irisin in the cells transfected with UCP2-siRNA compared to those transfected with nontarget siRNA.(4)In this study,we found that pretreatment of irisin significantly attenuated the phosphorylation of AMPK in a time dependent manner.(5)The HK-2 cells treated with CC exhibited significant suppression of AMPK phosphorylation and UCP2 expression compared with cells treated with vehicle.In addition,the regulation effects of irisin on UCP2 expression was suppressed by Compound CConclusion:The protective role of irisin in I/R mouse model and H/R cell model was mainly due to regulation of the expression of UCP2.And the effect of irisin on UCP2 expression is,at least partially,AMPK mediated.