Supplementary Material for: Febuxostat Prevents Renal Interstitial Fibrosis by the Activation of BMP-7 Signaling and Inhibition of USAG-1 Expression in Rats

<b><i>Background:</i></b> Renal interstitial fibrosis (RIF) is a common pathology associated with end-stage renal diseases. The activation of bone morphogenetic protein-7 (BMP-7)-Smad1/5/8 pathway seems to alleviate RIF. Uterine sensitization-associated gene-1 (USAG-1), a kidney-specific BMPs antagonist, is associated with the development and prognosis of several renal diseases. Febuxostat is a xanthine oxidase inhibitor that can attenuate the renal dysfunction of patients. The purpose of this study was to investigate the effects of febuxostat on renal fibrosis and to clarify the mechanisms underlying these effects. <b><i>Methods:</i></b> Rats were randomly divided into 6 groups termed a sham-operated group, a unilateral ureteral obstruction (UUO) group, 3 doses of febuxostat groups (low, intermediate and high doses) and a sham group treated with high-dose febuxostat. After 14 days, renal function, relative kidney weight, accumulation of glycogen and collagens were examined by different methods. Expression of α-SMA, transforming growth factor-β1 (TGF-β1), BMP-7 and USAG-1 was detected by western blotting and RT-PCR, respectively. The phosphorylation level of Smad1/5/8 was also quantified by western blotting. <b><i>Results:</i></b> The renal function was declined, and large amounts of glycogen and collagens were deposited in the kidneys of UUO rats compared with the rats in the sham group. Besides, expression of α-SMA and USAG-1 in these kidneys was elevated, and the TGF-β1 was also activated, while the BMP-7-Smad1/5/8 pathway was inhibited. Febuxostat reversed the changes stated earlier, exhibiting protective effects on RIF induced by UUO. <b><i>Conclusion:</i></b> Febuxostat was able to attenuate RIF caused by UUO, which was associated with the activation of BMP-7-Smad1/5/8 pathway and the inhibition of USAG-1 expression in the kidneys of UUO rats.

**_背景：_** 肾间质纤维化（renal interstitial fibrosis, RIF）是与终末期肾病相关的常见病理状态。骨形态发生蛋白7（bone morphogenetic protein-7, BMP-7）-Smad1/5/8通路的激活似乎可缓解肾间质纤维化。子宫致敏相关基因1（uterine sensitization-associated gene-1, USAG-1）作为一种肾脏特异性骨形态发生蛋白拮抗剂，与多种肾脏疾病的发生发展及预后密切相关。非布司他是一种黄嘌呤氧化酶抑制剂，可减轻患者的肾功能异常。本研究旨在探讨非布司他对肾纤维化的作用，并阐明其潜在作用机制。
**_方法：_** 将大鼠随机分为6组：假手术组、单侧输尿管梗阻（unilateral ureteral obstruction, UUO）模型组、3个非布司他给药组（低、中、高剂量组）以及高剂量非布司他处理的假手术组。造模14天后，采用不同方法检测各组大鼠的肾功能、肾脏相对重量、糖原及胶原蛋白沉积情况。分别采用蛋白质印迹法（western blotting）和逆转录聚合酶链反应（RT-PCR）检测α-平滑肌肌动蛋白（α-SMA）、转化生长因子-β1（transforming growth factor-β1, TGF-β1）、BMP-7及USAG-1的表达水平。同时采用蛋白质印迹法检测Smad1/5/8的磷酸化水平。
**_结果：_** 与假手术组大鼠相比，UUO模型组大鼠肾功能下降，肾脏内出现大量糖原及胶原蛋白沉积。此外，该组大鼠肾脏内α-SMA与USAG-1的表达水平升高，TGF-β1被激活，而BMP-7-Smad1/5/8通路受到抑制。非布司他可逆转上述异常变化，对UUO诱导的肾间质纤维化发挥保护作用。
**_结论：_** 非布司他可减轻UUO诱导的肾间质纤维化，其作用机制与激活肾脏内BMP-7-Smad1/5/8通路并抑制USAG-1的表达密切相关。