| Background Diabetic nephropathy(DN),one of the crucial microvascular complications of diabetes mellitus,is currently considered to be the major cause of ESRD.However,the pathogenesis of DN is complex and unclear and there is no effective therapeutic approach for DN.Many studies have shown that blocking vascular endothelial growth factor B(VEGF-B)could ameliorate DN via reducing renal lipotoxicity.However,there is no evidence to prove that antagonizing VEGF-B could exist beneficial effect on other pathogenic factors of DN,such as oxidative stress and inflammatory response.In addition,a large number of data demonstrated that protective effects of interleukin-22(IL-22)on DN might be due to reducing renal inflammatory response.Objective We aimed to develop the fusion protein with biological effect of anti-VEGF-B antibody and IL-22 and called it anti-VEGFB/IL22 fusion protein,and further exploring therapeutic effects of the fusion protein on DN and its underlying molecular mechanism in vivo.Methods1.The levels of VEGF-B and IL22 in the serum of non-diabetic subjects and DN patients were detected by the ELLSA kit.In addition,the level of VEGF-B in the renal tissue of DN patients was detected by immunohistochemistry.Moreover,the expression of NLRP3 and ADFP in the renal tissue of DN patients were detected by immunofluorescence.2.The gene sequence of anti-VEGFB antibody and IL22 were inserted into p TT5 plasmid vector and transfected into CHO-S cells.Finally,we collected cell supernatants and purified the fusion protein using a Protein A affinity chromatography column.The the molecular weight and the purity of anti-VEGFB/IL22 fusion protein were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and size exclusion chromatography.Affinity of the fusion protein and VEGF-B using the surface plasmon resonance.Furthermore,western blotting and confocal microscopy were used to verify whether the fusion protein could exist the biological effects of anti-VEGF-B monoclonal antibody and IL-22.3.DB/DB mice fed with high glucose and high fat diet were used to establish mouse model of DN.The levels of blood glucose,triglyceride,total cholesterol,serum creatinine,blood urea nitrogen,microalbuminuria,urine creatinine and urine protein were detected by assay kit and ELLSA kit.Moreover,HE staining was used to evaluate pathological injury of kidney tissues.Masson staining was used to evaluate fibrosis degree of kidney tissues.Electron microscope was used to observe pathological ultramicrostructure of the kidney tissues.Western blotting was used to analyse the levels of fibrosis-related proteins,such as collagen ?,vimentin and ?-SMA.4.Mitosox Red dye was used to detect the level of mitochondrial ROS and JC-1 kit was used to detect the level of mitochondrial membrane potential.Moreover,western blotting and immunohistochemistry were used to assess the levels of inflammation-related proteins,such as NLRP3,cleaved caspase-1,mature IL-1?,TGF-? and p-NF-?B p65.In addition,Oil Red O staining was used to evaluate neutral lipid aggregation.We detected the expression of FATP4 and ADFP,further assessing the changes of lipid transport and lipid deposition in the kidney tissues after anti-VEGFB/IL22 fusion protein treatment.Furthermore,western blotting was used to estimate the levels of insulin signaling-related proteins,such as p-IRS-1,IRS-1,p-ERK1/2,ERK1/2,p-Akt,Akt in the liver tissues and kidney tissues.And we evaluated the glycogen deposition by Glycogen staining(PAS staining).Results1.Our data suggested that the circulating VEGF-B level was notably higher and the circulating IL-22 level was substantially lower in DN patients than in nondiabetic subjects.Furthermore,we found a positive correlation of VEGF-B level with the levels of albumin/creatinine(ACR).We also observed a negative correlation of IL-22 level with the level of ACR.In addition,to further explore the effect of VEGF-B on the progression of DN,immunohistochemistry was conducted to detect the expression of VEGF-B in kidney tissues.Our data indicated that the expression of VEGF-B significantly increased in the kidney tissues of DN patients.Furthermore,we found that the expression of ADFP and NLRP3 inflammasome significantly increased in kidney tissues of DN patients,indicating that the development of DN was related to ectopic lipid accumulation and inflammatory responses.2.SDS-PAGE analysis and size-exclusion chromatography showed the molecular weight of the anti-VEGF-B antibody and anti-VEGFB/IL22 fusion protein,and showed that anti-VEGF-B antibody and anti-VEGFB/IL22 fusion protein had a high purty and and no polymer.The surface plasmon resonance results indicated that the anti-VEGF-B antibody and anti-VEGFB/IL22 fusion protein had a similar high-binding affinity to the VEGF-B antigen.In addition,we found that the anti-VEGFB/IL22 fusion protein could increase p-STAT3 and p-AKT expression and reduce the oxidative stress level and mitochondrial dysfunction in SV40 MES13 cells,indicating that the fusion protein activated IL22-related signaling.Furthermore,anti-VEGFB/IL22 fusion protein could block the high expression of FATP4 caused by the VEGF-B antigen in HUVECs,proving that the fusion protein and anti-VEGF-B antibody could repress the interaction of VEGF-B and VEGFR1/NRP-1.3.As expected,our data illustrated that there was a increase of ACR,24-hour urinary volume,24-hour urinary protein,Ccr,serum creatinine,BUN,blood glucose,triglyceride and total cholesterol in DN mouse model.Meanwhile,anti-VEGFB/IL22 fusion protein significantly downregulated the levels of ACR,24-hour urinary volume,24-hour urinary protein,Ccr,BUN,blood glucose,triglyceride and total cholesterol.In addition,the fusion protein improved the survival of db/db mice and effectively reduced diabetes-induced increase in the renal index.Renal pathological alterations were significantly improved in db/db mice as shown by H&E staining after administration of anti-VEGFB/IL22 fusion protein therapy for 8 weeks.Furthermore,transmission electron microscopy(TEM)results showed the protective effects of the fusion protein on the pathological ultramicrostructure of the kidney,including a reduction in glomerular basement membrane(GBM)thickness and inhibition of podocyte foot process effacement.Moreover,the anti-VEGFB/IL22 fusion protein markedly reduce the levels of fibrosis-related proteins,such as collagen ?,vimentin and ?-SMA.4.Our results indicated that anti-VEGFB/IL22 fusion protein significantly decreased the expression of NLRP3,Cleaved Caspase-1 and Mature IL-1 ? in kidney tissues of DN mice.Meanwhile,we found the fusion protein reduced the expression of TNF-? and p-NF-?B p65,indicating the fusion protein could effectively ameliorate inflammatory responses.Furthermore,this study demonstrated that the anti-VEGFB/IL22 fusion protein reduced oxidative stress level and mitochondrial dysfunction.In addition,Oil Red O staining result and immunofluorescence staining result showed that anti-VEGFB/IL22 fusion protein reduced renal lipid deposition.Moreover,the fusion protein could significantly alleviate renal glycogen deposition and increase the expression of p-IRS-1,p-ERK/2 and p-AKT,suggesting that the fusion protein existed beneficial effects on DN via improving glycometabolism.5.Conclusion Here,we found that serum level of VEGF-B was clearly upregulated and that IL-22 level was obviously downregulated in DN patients,which might be related to the progression of DN.To meet these challenges,we successfully constructed and purified a novel anti-VEGFB/IL22 fusion protein,simultaneously combining a VEGF-B antibody and IL-22.We further demonstrated the beneficial effects of the fusion protein on DN by ameliorating glomerular and tubular lipid accumulation while alleviating oxidative stress and inflammatory responses.Moreover,the fusion protein could increase insulin sensitivity.Overall,these findings indicated that anti-VEGFB/IL22 fusion protein might be expected to become a prospective therapeutic strategy for DN. |
PDF Full Text Request