The Role And Mechanism Of NNMT And Its Metabolite MNAM In Renal Tubulointerstitial Fibrosis

Background:As the prevalence of diabetes continues to climb,diabetic kidney disease(DKD)has become the leading cause of chronic kidney disease(CKD),surpassing primary glomerulonephritis.Pathogenesis of DKD is complicated and new therapeutic options are urgently required.The damage of tubular epithelial cells(TECs)is closely related to the deterioration of DKD.The continuous aggravation of tubulointerstitial fibrosis(TIF)plays an important role in CKD progression to end-stage renal disease.Therefore,protecting renal tubules from repeated damage and restoring the function of renal tubules may be a priority and effective treatment for kidney disease.Nicotinamide N-methyltransferase(NNMT)is the main nicotinamide metabolizing enzyme that catalyzes the transfer of reactive methyl groups from S-adenosylmethionine(SAM)to nicotinamide(NAM),which generates S-adenosylhomocysteine(SAH)and 1-methylnicotinamide(MNAM).MNAM is not only the main metabolite of NAM but also owns pharmacological effects such as anti-inflammatory and vascular protection.However,there is no research to clarify the role and specific mechanism of NNMT and its metabolite MNAM in TIF.(I)The role of NNMT in TIF.Objective:Explore the role of NNMT in TIF.Methods:(1)In vitro:① TECs were exposed to TGF-β1.Western blot and qRT-PCR were used to detect the expression of NNMT.② After inhibiting or overexpressing NNMT in TECs,TGF-β1 was applied to induce profibrotic changes.Western blot and qRT-PCR were used to detect the expression of Fibronectin,Collagen I and α-SMA.(2)In vivo:① We established a successfully unilateral ureteral obstruction(UUO)model and randomly divided mouse into several groups:Sham operation(sham)mice,UUO(day 4,day 7,day 14)mice.Immunofluorescence staining,Western blot and qRT-PCR were used to detect the expression of NNMT.②We randomly divided mouse into several groups:Vector(pGV362)mice,UUO(pGV362)mice and UUO+pGV362-NNMT mice.NNMT was overexpressed in the kidneys of mice by injecting NNMT plasmid via tail vein.Masson’s staining was performed to detect the fibrosis in kidneys;Immunohistochemical technology(IHC),Western blot and qRT-PCR were used to detect the expression of Fibronectin,Collagen I,α-SMA and NNMT.Results:(1)In vitro:TGF-β1 induced the expression of NNMT in TECs.Overexpression of NNMT in TECs downregulated the extracellular matrix(ECM)deposition induced by TGF-β1 while inhibiting NNMT promoted ECM deposition.(2)In vivo:The expression of NNMT is elevated in UUO mice.Overexpression of NNMT can alleviate TIF induced by the UUO.Conclusion:The expression of NNMT increased in renal tubular interstitial fibrosis.Overexpression of NNMT reduced TIF.(Ⅱ)The role of MNAM metabolized by NNMT in TIF.Objective:To explore whether NNMT reduced TIF by increasing the concentration of MNAM.Methods:(1)In vitro:① Inhibited or overexpressed NNMT in TECs,and detected the changes in MNMA concentration by LC/MS/MS.② TECs were pre-treated with different concentrations of MNAM and then stimulated with TGF-β1.Western blot and qRT-PCR were used to detect the expression of Fibronectin,Collagen I and α-SMA.③We inhibited the expression of NNMT in TECs and then stimulated with MNAM and TGF-β1.Western blot was used to detect the expression of Fibronectin,Collagen I and α-SMA.(2)In Vivo:① We overexpressed the expression of NNMT in UUO mice and the concentration of MNMA in mouse serum was detected by LC/MS/MS.②We randomly divided mouse into several groups:Sham mice,Sham+MNAM mice,UUO mice,UUO+MNAM mice.Masson’s staining was performed to detect the fibrosis in kidneys;IHC,Western blot and qRT-PCR were used to detect the expression of Fibronectin,Collagen I,α-SMA and NNMT.qRT-PCR was used to detect the mRNA expression of TNF-α、MCP-1、IL-1β、IL-6.Results:(1)In vitro:① The concentration of MNAM downregulated,after inhibiting the expression of NNMT in TECs.Overexpression of NNMT increased the concentration of MNAM.②The protein and mRNA expression levels of Fibronectin,Collagen I and NNMT decreased when stimulated with MNAM and TGF-β1.③The protein expression of NNMT,Fibronectin,Collagen I and α-SMA downregulated when MNAM and TGF-β1 stimulating TECs which low expression of NNMT.(2)In vivo:① The serum MNAM concentration increased in UUO mice which overexpression of NNMT.② The expression levels of Fibronectin,Collagen I andα-SMA in MNAM mice were significantly downregulated and the mRNA expression levels of TNF-α,MCP-1,IL-1β and IL-6 were downregulated at the same time.Conclusion:NNMT overexpression reduced TIF by increasing the concentration of MNAM.(Ⅲ)The mechanism of MNAM’s inhibitory effect against TIF.Objective:To explore the mechanism of MNAM’s inhibitory effect against TIF.Methods:(1)In vitro:①TECs were pre-treated with different concentrations of MNAM and then stimulated with TGF-β1.Western blot was used to detect the protein expression of p-Smad2,p-Smad3,Smad4,Smad7.②The binding of Smad2,Smad3 and TβR Ⅰ and TβR Ⅱ was analyzed by Co-immunoprecipitation experiments.(2)In vivo:We randomly divided mouse into several groups:sham mice,sham+MNAM mice,UUO mice,and UUOMNAM mice.The expression levels of p-Smad2,p-Smad3,Smad4,Smad7 were detected by Western blot.Results:(1)In vitro:①When TECs were pre-treated with different concentrations of MNAM and then stimulated with TGF-β1,the expression of phosphorylation of Smad3 was downregulated.However,there is no effect on the expression of Smad2,Smad4 and Smad7.②The results of co-immunoprecipitation experiment showed that MNAM inhibited the binding of Smad3 to TβR I and TβR Ⅱ,but had no effect on the binding of Smad2 to TβR I and TβR Ⅱ.(2)In vivo:Compared with the UUO mice,the MNAM mice inhibited the phosphorylation of Smad3 and had no effect on the expression of Smad2,Smad4 and Smad7.Conclusion:MNAM specifically inhibited the phosphorylation of Smad3 to reduce TIF.