Qiao Xi, Wang Lihua, Wang Yanhong, Su Xiaole, Qiao Yufeng, Fan Yun, Peng Zhiqiang
Department of Nephrology, Second Hospital of Shanxi Medical University, Shanxi Kidney Disease Institute, 382, WuYi Road, Taiyuan, 030001, Shanxi, People's Republic of China.
Department of Microbiology and Immunology, Shanxi Medical University, 56, Xinjian Road, Taiyuan, 030001, Shanxi, People's Republic of China.
BMC Nephrol. 2017 Jul 11;18(1):232. doi: 10.1186/s12882-017-0659-6.
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.
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-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.
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 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.
This study demonstrates that IMD attenuates renal fibrosis by induction of HO-1.
中间介质[IMD，肾上腺髓质素2（ADM - 2）]通过抑制氧化应激减轻肾纤维化。然而，确切机制尚不清楚。血红素加氧酶-1（HO-1）作为一种抗氧化剂，与抗纤维化作用相关。已知ADM可诱导HO-1。IMD对HO-1是否有任何影响尚不清楚。在此，我们确定IMD的抗纤维化特性是否由HO-1的诱导介导。
通过对雄性Wistar大鼠进行单侧输尿管梗阻（UUO）诱导肾纤维化。将大鼠近端肾小管上皮细胞系（NRK-52E）暴露于重组人转化生长因子-β1（rhTGF-β1，10 ng/ml）以建立上皮-间质转化（EMT）的体外模型。使用载体pcDNA3.1-IMD在体内和体外过表达IMD。使用锌原卟啉（ZnPP）阻断HO-1的酶活性。通过实时逆转录-聚合酶链反应（RT-PCR）、蛋白质印迹法或免疫组织化学分析IMD对UUO大鼠梗阻肾脏和TGF-β1刺激的NRK-52E中HO-1表达的影响。通过测量胆红素生成来检测UUO大鼠梗阻肾脏、对侧肾脏和NRK-52E中的HO活性。通过Masson三色染色和I型胶原蛋白表达确定肾纤维化。使用免疫组织化学分析评估巨噬细胞浸润和白细胞介素-6（IL-6）表达。分别通过蛋白质印迹法或免疫荧光法测量α-平滑肌肌动蛋白（α-SMA）和E-钙黏蛋白表达来评估体内和体外的EMT。
在接受IMD治疗的UUO肾脏或NRK-52E中，HO-1表达和HO活性增加。UUO大鼠的梗阻肾脏在术后第7天表现出明显的间质纤维化。相比之下，接受IMD基因转移治疗的肾脏间质纤维化程度最小。UUO大鼠的梗阻肾脏中巨噬细胞浸润和IL-6表达也更高。IMD抑制UUO肾脏中巨噬细胞的浸润和IL-6的表达。如E-钙黏蛋白表达降低和α-SMA表达增加所示，UUO大鼠梗阻肾脏中的EMT程度广泛。使用NRK-52E进行的体外研究证实了这些观察结果。IMD基因传递抑制了EMT。然而，HO酶活性抑制剂ZnPP消除了IMD的所有上述有益作用。
本研究表明，IMD通过诱导HO-1减轻肾纤维化。
