The Protective Effects And Mechanisms Of Liraglutide On Acute Renal Ischemia-reperfusion Injury And Chronic Renal Fibrosis

Part ? Study on the protective effects and mechanisms of liraglutide on renal ischemia-reperfusion injury?Objective?To explore the protective effects and mechanisms of liraglutide(Lirag)on renal ischemia-reperfusion injury?Methods?A mouse model of fatal renal ischemia-reperfusion injury(IRI)was established,and five different Lirag administration regimens were used.Lirag(200?g/kg,q12h)was subcutaneously injected at 60 h,48 h,24 h,12 h and 1 h before surgery,respectively.Unilateral renal ischemia/reperfusion injury was performed with or without 200?g/kg Lirag injection 10 h after surgery.According to the experimental results,the drug regimen with the strongest protective effect was selected for subsequent mechanism study.Serum creatinine(SCr)and urea nitrogen(BUN)levels,pathological injury and inflammatory cells infiltration of renal tissue were detected 24 h after reperfusion.In addition,the expression of HMGB1 in renal tissues and plasma at different time points after ischemia and reperfusion was assessed by Western blot,immunohistochemistry and ELISA.The expression of HMGB1 downstream signaling pathway and inflammatory cytokines were detected by RT-PCR.In vitro,the inhibitory effect of Lirag on the nuclear-cytoplasmic translocation of HMGB1 in hypoxia-induced human renal tubular epithelial cell line(HK-2)was detected by immunofluorescence.The protective effect of Lirag was further confirmed by recombinant HMGB1(r HMGB1),GLP-1 receptor knockout mice(GLP-1R-/-),and GLP-1 receptor antagonist Exendin(9-39)to further explore the relationship between HMGB1 and GLP-1 receptors.?Results?By detecting SCr and BUN levels in mice 24 h after surgery,we found that the administration regimen starting from 60 h before surgery,6 doses before ischemia and 1 dose 10 h after reperfusion had the best protective effect on renal IRI.Under the treatment of this protocol,the HE,MPO,F4/80 and TUNEL staining of kidney tissue showed that kidney tissue damage,inflammatory cell infiltration and apoptosis were ignificantly reduced.Western blot,immunohistochemistry and ELISA results showed that Lirag significantly inhibited the nuclear-cytoplasmic translocation and release of HMGB1 in renal parenchymal cells after ischemia or at 1 h,3 h and 24 h after reperfusion.RT-PCR results showed that the increased expression of HMGB1 receptors(TLR2,TLR4 and RAGE)and inflammatory factors(IL-1?,IL-6,TNF-? and MCP-1)induced by IRI were significantly inhibited by Lirag.In vitro,Lirag significantly reduced hypoxia-induced the nuclear-cytoplasmic translocation of HMGB1 in HK-2 cells.Intraperitoneal injection of r HMGB1 immediately after reperfusion significantly weakened the protective effect of Lirag on renal IRI.The protective effect of Lirag on renal IRI was also partially reversed after the use of GLP-1R-/-mice or the additional use of GLP-1R antagonist Exendin(9-39).In vitro,Exendin(9-39)largely reversed the inhibitory effect of Lirag on the nuclear-cytoplasmic translocation of HMGB1 in hypoxia-induced HK-2 cells.?Conclusion? Lirag has a significant protective effect on fatal renal IRI in mice,which is related to the inhibition of the nuclear-cytoplasmic translocation and the release of HMGB1 in renal parenchymal cell,and the mechanism is partially dependent on the expression of GLP-1R in renal tissue.Part ? Liraglutide reduces the fibrosis caused by UUO and inhibits the nuclear-cytoplasmic translocation and release of HMGB1?Objective? To investigate whether the reduction of renal fibrosis by liraglutide(Lirag)is related to the inhibition of the release of HMGB1 in renal parenchymal cells.?Methods?Unilateral ureteral obstruction(UUO)model was established,and mice were injected with lirag subcutaneously for intervention.The experiments were divided into four groups: Normal group,Sham group,UUO group,UUO+Lirag group(300?g/kg,q12h).The kidney samples and peripheral blood serum samples of mice were collected on the 7th day after operation.HE staining and Masson staining were erformed on renal tissue to determine the degree of renal injury and fibrosis.RT-PCR,Western blot and immunohistochemistry were used to detect the expression of fibrosis related indicators(?-SMA,TGF-? and Collagen I).TUNEL staining and immunohistochemical staining were used to detect apoptosis of renal tubular epithelial cells and infiltration of inflammatory cells(macrophages and neutrophils)in the kidney.The production of inflammatory factors in renal tissues was detected by RT-PCR.Western blot,immunohistochemistry,and ELISA were used to determine the nuclear-cytoplasmic translocation of HMGB1 in renal tubular epithelial cells and the extent of HMGB1 release into peripheral blood.The expression of TLR4,the main receptor of HMGB1,and the entry of phosphorylated NF-?B into the nucleus were detected by Western blot.Exogenous recombinant HMGB1(r HMGB1)was used to further verify whether the protective effect of Lirag on renal fibrosis was related to the inhibition of HMGB1 release.?Results?HE staining showed that compared with the Normal and Sham groups,the kidney tissue in the UUO group was significantly damaged,and MASSON staining indicated more collagen deposition in the kidney tissue.The detection of renal fibrosis related indicators(?-SMA,Collagen I,TGF-?)indicated that the renal tissue had obvious fibrosis.However,Lirag treatment significantly improved renal tissue damage,inhibited collagen deposition in the interstitium,and reduced the expression of fibrosis related indicators in the kidney.Immunohistochemistry and RT-PCR indicated that inflammatory cells infiltration and inflammatory cytokines production were significantly increased in renal tissue of UUO group.After the intervention of Lirag,the inflammatory cell infiltration and the production of inflammatory factors were significantly reduced.On the 7th day after operation,the nuclear-cytoplasmic translocation and release of HMGB1 were observed in the UUO group.After Lirag intervention,the nuclear-cytoplasmic translocation and release of HMGB1 were significantly reduced.At the same time,treatment with Lirag also significantly inhibited the increase of the downstream receptor TLR4 of HMGB1 and the nuclear entry of phosphorylated NF-?B.Exogenous r HMGB1 injection largely offset the bove protective effects of Lirag.?Conclusion? Lirag can significantly inhibit renal injury and fibrosis induced by UUO model,and this protective effect may be related to the inhibition of the nuclear-cytoplasmic translocation and release of HMGB1 from renal tubular epithelial cells,the expression of TLR4 and the activation of NF-?B signaling pathway in renal tissues after UUO operation.