Relaxin Abrogates Renal Interstitial Fibrosis By Regulating Macrophage Polarization Via Inhibition Of Toll-Like Receptor 4 Signaling

Purpose:1.To study the effects of Relaxin on renal macrophage polarization and renal fibrosis in obstructive nephropathy.2.To study on the effect of Relaxin on TLR4 signal pathway after obstructive nephropathy.3.To verify that RLX abrogates renal interstitial fibrosis by regulating macrophage polarization via inhibition of Toll-like receptor 4 signaling.Materials and method:1.Mice with unilateral ureteral obstruction in obstructive nephropathy model,observed renal fibrosis and renal tissue macrophage polarization after RLX treatment;RAW264.7 macrophage cell line induced into M1 macrophages and M2 macrophages,observed macrophage polarization changes after Relaxin treatment.2.Mice with unilateral ureteral obstruction in obstructive nephropathy model,observed the change of TLR4 signaling after RLX treatment;RAW264.7 macrophage cell line induced into M1 macrophages and M2 macrophages,observed the change of TLR4 signaling after Relaxin treatment.3.RAW264.7 macrophage cell line induced into M1 macrophages and M2 macrophages,observed macrophage polarization changes after Relaxin and TAK-242 treatment.Results:1.H&E and Sirius red staining showed that marked interstitial inflammation and fibrosis occurred 5 days after UUO and that relaxin can reduce cell infiltration and interstitial fibrosis.Compared with the kidneys of mice pretreated with relaxin,the kidneys of day 5 UUO-injured mice had higher fibronectin levels.Five days following UUO,gene expression analysis of macrophages from the kidneys revealed increased both M1 and M2 markers.Relaxin pretreatment caused significant increases in the gene expression levels of most renal macrophage M2 markers and decrease M1 markers.the expression levels of most of the M1 marker genes decreased while M2 markers increased when macrophages were treated with relaxin.2.TLR4,Myd88,p65 and pp65 expression levels were upregulated at day 5 following UUO,while relaxin significantly decreased their mRNA and protein expression levels.TLR4,Myd88,p65 and pp65 genes and proteins were expressed at low levels after relaxin treatment in the different macrophage phenotypes compared with those treated with vehicle3.The proteins and genes expression profiles of M1 markers and M2 markers in macrophage were not different between those treated with TAK-242 and those treated with TAK-242 and relaxin.Conclusions:Relaxin can downregulate the Toll-like receptor(TLR)4 signaling,shift macrophage polarization toward the M2 phenotype and ameliorate renal fibrosis at the early stages of UUO.In vitro experiments confirmed that relaxin can downregulate the TLR4 signaling and induce the M2 macrophage transition.Furthermore,the macrophage phenotype transition actions of relaxin were significantly blocked by TAK-242,a TLR4 antagonist,in vitro.