Abstract
BackgroundIntermedin [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.MethodsRenal 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-β1 (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-β1-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 α-smooth muscle actin (α-SMA) and E-cadherin expression using Western blotting or immunofluorescence, respectively.ResultsHO-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 α-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.ConclusionThis study demonstrates that IMD attenuates renal fibrosis by induction of HO-1.
Highlights
Intermedin [IMD, adrenomedullin-2 (ADM-2)] attenuates renal fibrosis by inhibition of oxidative stress
We showed that after 7 days of transfection, obstructed kidneys from rats treated with pcDNA3.1-IMD plasmid exhibited significant increase in IMD expression compared with that from rats treated with control empty vector (Fig. 1a–c)
Heme oxygenase (HO)-1 is upregulated and HO activity is increased in the obstructed kidney of ureteral obstruction (UUO) rats, and is further induced by IMD gene transfer Heme oxygenase-1 (HO-1) mRNA and protein expression was very low in the sham-operated kidneys, whereas there was a significant increase in HO-1 mRNA and protein abundance in the obstructed kidney after UUO
Summary
Intermedin [IMD, adrenomedullin-2 (ADM-2)] attenuates renal fibrosis by inhibition of oxidative stress. Regardless of disease etiology, fibrosis is a final common pathogenic process for CKD leading to end stage renal diseases (ESRD) [2]. Reactive oxygen species (ROS) play an important pathogenic role in the development of renal fibrosis by inducing epithelial-mesenchymal transition (EMT) of tubular epithelial cells and by inducing macrophage infiltration [3,4,5]. Intermedin [IMD, known as adrenomedullin-2 (ADM-2)] is a novel member of the calcitonin/calcitonin gene-related peptide family [6]. Our previous study demonstrated that kidney-specific IMD gene transfer significantly ameliorated unilateral ureteral obstruction (UUO)-mediated fibronectin upregulation and the fibrotic changes, and the antifibrotic effect is achieved by inhibition of oxidative stress [7].
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