Intermedin Attenuates Renal Fibrosis By Induction Of Heme Oxygenase-1 In Rats With Unilateral Ureteral Obstruction

Objective:Intermedin [IMD,adrenomedulin-2(ADM-2)] attenuates renal fibrosis by inhibition of oxidative stress.However,the precise mechanisms remain unknown.Heme oxygenase-1(HO-1),an antioxidant agent,is associated with antifibrogenic effects.ADM is known to induce HO-1.Whether IMD has any effect on HO-1 is unclear.Herein,we determined whether the antifibrotic properties of IMD are mediated by induction of HO-1.Methods:Renal fibrosis was induced by unilateral ureteral obstruction(UUO)performed on male Wistar rats.Rat proximal tubular epithelial cell line(NRK-52E)was exposed to rhTGF-?1(10 ng/ml)to establish an in vitro model of epithelial-mesenchymal transition(EMT).IMD was over-expressed in vivo and in vitro using the vector pcDNA3.1-IMD.Zinc protoporphyrin(ZnPP)was used to block HO-1 enzymatic activity.IMD effects on HO-1 expression in the obstructed kidney of UUO rat and in TGF-?1-stimulated NRK-52 E were analyzed by real-time RT-PCR,Western blotting or immunohistochemistry.HO activity in the obstructed kidney,contralateral kidney of UUO rat and NRK-52 E was examined by measuring bilirubin production.Renal fibrosis was determined by Masson trichrome staining and collagen I expression.Macrophage infiltration and IL-6 expression were evaluated using immunohistochemical analysis.In vivo and in vitro EMT was assessed by measuring ?-smooth muscle actin(?-SMA)and E-cadherin expression using Western blotting or immunofluorescence,respectively.Results:HO-1 expression and HO activity were increased in IMD-treated UUO kidneys or NRK-52 E.The obstructed kidneys of UUO rats demonstrated significant interstitial fibrosis on day 7 after operation.In contrast,kidneys that were treated with IMD gene transfer exhibited minimal interstitial fibrosis.The obstructed kidneys of UUO rats also had greater macrophage infiltration and IL-6 expression.IMD restrained infiltration of macrophages and expression of IL-6 in UUO kidneys.The degree of EMT was extensive in obstructed kidneys of UUO rats as indicated by decreased expression of E-cadherin and increased expression of ?-SMA.In vitro studies using NRK-52 E confirmed these observations.EMT was suppressed by IMD gene delivery.However,all of the above beneficial effects of IMD were eliminated by ZnPP,an inhibitor of HO enzyme activity.Conclusion:IMD attenuates renal fibrosis by induction of HO-1.