Activation Of Farnesoid X Receptor Downregulates Visfatin And Attenuates Diabetic Nephropathy

Background and objective:Diabetic nephropathy(DN)is the leading cause of end-stage renal diseases(ESRD)worldwide and results in higher risk of morbidity and mortality.Clinically,the patients in DN will develop proteinuria,decreased glomerular filtration rate(GFR),decline of renal function and finally renal failure.Pathologically,DN is characterized by fibrosis,oxidative stress,inflammation,and metabolic disorder.The pathology of DN is complicated and multiple pathways are involved,including advanced glycation end products,oxidative stress,inflammatory cytokines and profibrotic factors.The farnesoid X receptor(FXR)belongs to the nuclear receptor superfamily and is mainly expressed in kidney,liver,adrenal glands,aorta and intestines.FXR plays a pivotal role in glucose homeostasis,as well as in the regulation of bile acid and lipid metabolism and also in a wide range of disease of the liver,biliary tract,intestine and cardiovascular systems.FXR has been highlighted and well established in the development of DN.Activation of FXR chemically and genetically improves the hyperglycemia and hyperlipidemia in diabetic mice.Deficiency of FXR exacerbates DN in type I diabetes model.The mechanistic explanations for these effects have been attributed to many targets genes regulated by FXR.However,the underlying mechanism remains not fully understood and needs more investigation.Visfatin,also termed as pre-B cell-enhancing factor(PBEF),is first discovered as an adipocytokine that preferentially secreted by visceral adipocytes and later reported to be expressed in macrophage in atherosclerotic lesion,endothelial dysfunction in diabetic patients and synovial fibroblasts in rheumatoid patients.Visfatin expression is modulated by a broad spectrum of cytokine that induce insulin resistance,such as interleukin 1(IL-1),interleukin 6(IL-6),and tumor necrosis factor alpha(TNF-α).Interestingly,all these inflammation factors play important roles in the development of DN.Furthermore,we have demonstrated that visfatin contributes to lipid dysregulation and endothelial dysfunction in patients suffering from chronic kidney diseases.However,whether visfatin mediates FXR signaling in diabetic nephropathy is poorly reported.In this study,we aimed to elucidate the role and regulation mechanism between FXR and visfatin in the pathophysiology of diabetic nephropathy,and hopfully provide a possible target for the treatment in diabetic nephropathy.Methods:1.Clinical study:Patients with type 2 diabetic nephropathy diagnosed by renal biopsy diagnosis were enrolled from June 2014 to December 2015 in the Department of Nephrology,Xinqiao Hospital,and divided into incipient DN group(13 cases),manifest DN group(16 cases)and advanced DN group(18 cases)according to the pathological stage;The normal tissue was collected from patients subjected to renal tumor surgery.The specimens were excised far from the tumor tissue and considered as the control group.The collected renal tissue specimens were subjected to PAS staining and visfatin immunohistochemical staining.The clinical data,including age,gender,24 h urinary albumin,serum creatinine(Scr),blood urea nitrogen(BUN),and the estimated glomerular filtration rate(e GFR)were collected.The 24 h urinary albumin,Scr,BUN,e GFR and visfatin were compared between each group.Meanwhile the correlation betweteen expression of visfatin and 24 h urinary albumin,Scr,BUN,e GFR were also analyzed.2.In vitro study:(1).FXR agonist GW4064 and FXR antagonist Guggulsterone were used to treat the human mesangial cells(HMC).The m RNA and protein levels of visfatin were detected by Real-time PCR and Western blot.(2).High glucose(HG)induced HMC were treated with either visfatin si RNA transfection,GW4064,or exogenous visfatin.The expression levels of visfatin,NF-κB,IκBα were determined by using Real-time PCR and Western blot.MCP-1 concentration were detected with ELISA kits.The m RNA and protein levels of TGF-β1,smad2/3,p-smad2/3,α-SMA,Collagen IV and FN were determined by using Real-time PCR and Western blot.To detect HMC proliferation,CCK-8 kits were used.The proliferation related PCNA expression were determined by Real-time PCR and Western blot.(3).The full-length and truncated visfatin promoter luciferase vectors were constructed,according to the bioinformatics prediction results.The full-length luciferase vector were transfected into HG induced HMC treated with different concentrations of GW4064(0.5μM,1μM,5μM)or DMSO for 24 h,and the visfatin promoter activity was assayed using a dual-luciferase reporter assay system.FXR expression plasmid and truncated visfatin promoter luciferase vector was co-transfected into 293 cells incubated with DMSO or GW4064(5μM)for 24 h,and the visfatin promoter activity was analyzed.3.In vivo study:The db/db mice were employed as animal model of diabetic nephropathy,and db/m mice were used as controls.The db/db mice were divided into four groups: db/db group(12w),db/db(16w)group,db/db(20w)group and db/db(20w)+GW4064 treatment group.The physiological,biochemical,and renal functional parameters were detected,including body weight,kidney weight,blood glucose,24 h urinary albumin,Scr and BUN.Histology staining and immunohistochemistry staining of visfatin,TGF-β1,α-SMA and FN were performed in the kidney tissues of db/db mice and db/m mice.The expression of visfatin,NF-κB,IκBα,TGF-β1,smad2/3,p-smad2/3,α-SMA,Collagen IV and FNt were detected by Western blot.Results:1.Clinical study:(1).Immunohistochemical staining showed that visfatin expression was mainly located in the glomerular tissue.The visfatin expression gradually increased with the progression of diabetic nephropathy.Compared with the control group,the expression of visfatin was significantly increased in DN group(P<0.01).(2).The correlation analysis showed that the expression of visfatin in renal tissue was positively correlated with 24 h urinary albumin(R=0.868,P<0.01),Scr(R=0.913,P<0.01),and BUN(R=0.938,P<0.01),negatively correlated with e GFR(R=-0.979,P<0.01)in the incipient and manifest group.However,in advanced DN group,the expression of visfatin was negatively correlated 24 h urinary albumin(R=-0.499,P<0.05),but no correlation with Scr(R=-0.099,P >0.05),BUN(R=0.281,P>0.05)and e GFR(R=0.026,P>0.05).2.In vitro study:(1).Compared with the control group,cell proliferation and expression of PCNA protein in HG group significantly increased(P<0.01).Cell proliferation and expression of PCNA protein in GW4064 group was significantly lower than that in HG group(P<0.01).Compared with GW4064 group,the cell proliferation and PCNA expression in GW4064 and visfatin co-treated group were significantly reduced(P<0.01).(2).Phospho-P65 and MCP-1 were significantly upregulated in HG group(P<0.01),while the expression of IκBα was significantly downregulated compared with the control group(P<0.01).Compared with the HG group,expression of p-P65 and MCP-1 were reduced significantly in GW4064 group(P<0.01),while Ik Bα was increased significantly(P<0.01).However,visfatin treatment could reverse GW4064 induced downregulation of p-P65 and MCP-1(P<0.01).Likewise,visfatin treatment significantly blunted Ik Bα increase induced by GW4064(P<0.01).(3).Compared with the control group,the expression of TGF-β1,p-smad2/3,α-SMA,Collagen IV and FN in HG group were significantly upregulated(P<0.01).However,the expression of TGF-β1,p-smad2/3,α-SMA,Collagen IV and FN were significantly downregulated in GW4064 group(P<0.01).Visfatin treatment significantly inhibited the expression of TGF-β1,p-smad2/3,α-SMA,Collagen IV and FN compared with GW4064 group(P<0.01).(4).The luciferase reporter assay showed that the visfatin promoter activity in HG group was significantly enhanced compared with the control group,and the FXR agonist GW4064 could inhibit the visfatin promoter activity in a concentration-dependent manner(P<0.01).In the truncated luciferase reporter assay,the activity of visfatin promoter was significantly increased in the plasmid containing the-1192 bp to the initiation site(P<0.01),demonstrating that the binding site might locate between-1607 bp and-1192 bp.3.In vivo study:(1).Compared with db/m group,24 h urinary albumin,Scr and BUN were significantly increased in the db/db group(P<0.01),and gradually increased with the increase of age(P<0.01).The 24 h urinary albumin,Scr and BUN in GW4064 treatment group were significantly lower than those in the db/db group(P<0.01,compared with 16 w and 20w).(2).PAS and Masson staining showed that the db/db mice progressed with glomerular hypertrophy,glycogen deposition and mesangial ECM accumulation compared with db/m mice.GW4064 maintained the glomerular structure and reduced the ECM accumulation compared to the db/db mice.Immunohistochemistry staining showed that visfatin was stained positive in the glomeruli.The signal was much higher during the diabetic nephropathy progression,and could be reduced by FXR agonist GW4064.Similarly observations were seen on TGF-β1,a-SMA and FN staining.(3).Western blot showed that the expression of visfatin in db/db group was significantly higher than that in db/m group(P<0.01).The expression of p-P65,TGF-β1,p-Smad2/3,α-SMA,Collagen IV and FN were significantly correlated with visfatin(P<0.01,compared with 16 w and 20w).The expression of visfatin in the GW4064 group was significantly downregulated compared with the db/db group(P<0.01,compared with 16 w and 20w).Likewise,the expression of p-P65,TGF-β1,p-Smad2/3,α-SMA,Collagen IV and FN were significantly downregulated(P<0.01,compared with 16 w and 20w).Conclusion:In the present study,we determined that activation of FXR inhibited visfatin expression,inflammatory response,TGF-β/smad signaling and cell proliferation and thus ameliorated the progression of the DN in vivo and vitro.Activation of FXR reduced visfatin transcription through directly binding to the visfatin promoter.The binding site might be between-1607 bp and-1192 bp of visfatin promoter.