Intermedin attenuates renal fibrosis by induction of heme oxygenase-1 in rats with unilateral ureteral obstruction

Background: Intermedin [IMD, adrenomedullin-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-beta 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-beta 1-stimulated NRK-52E were analyzed by real-time RT-PCR, Western blotting or immunohistochemistry. HO activity in the obstructed kidney, contralateral kidney of UUO rat and NRK-52E 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 alpha-smooth muscle actin (alpha-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-52E. 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 alpha-SMA. In vitro studies using NRK-52E 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: This study demonstrates that IMD attenuates renal fibrosis by induction of HO-1.