Supplementary Material for: Indoxyl sulfate aggravates podocyte damage through the TGF-β1/Smad/ROS signaling pathway

Introduction: Hyperglycaemia induces the production of a large quantity of reactive oxygen species (ROS) and activates the transforming growth factor β1(TGF-β1)/Smad signalling pathway, which is the main initiating factor in the formation of diabetic nephropathy. Indoxyl sulfate (IS) is a protein-binding gut-derived uraemic toxin that localizes to podocytes, induces oxidative stress and inflames podocytes. The involvement of podocyte damage in diabetic nephropathy through the TGF-β1/) signalling pathway is still unclear. Methods: In this study, we cultured differentiated rat podocytes in vitro and measured the expression levels of nephrin, synaptopodin, CD2AP, SRGAP2a and α-SMA by quantitative real-time PCR (qRT‒PCR) and western blotting after siRNA-mediated TGF-β1 silencing, TGF-β1 overexpression and the presence of the ROS inhibitor acetylcysteine. We detected the expression levels of nephrin, synaptopodin, CD2AP, SRGAP2a, SRGAP2a in the Smad2/3, phosphorylated-Smad2/3 (p-Smad2/3), Smad7, NADPH oxidase 4 (NOX4), and ROS levels under high glucose (HG) and IS conditions. Results: The results indicated that nephrin, synaptopodin, CD2AP and SRGAP2a expressions were significantly upregulated and α-SMA expression was significantly downregulated in the presence of HG under siRNA-mediated TGF-β1 silencing or after the addition of acetylcysteine. However, in the presence of HG, the expressions of nephrin, synaptopodin, CD2AP and SRGAP2a were significantly downregulated, and the expression of α-SMA was significantly upregulated with the overexpression of TGF-β1. IS supplementation under HG conditions further significantly reduced the expressions of nephrin, synaptopodin, CD2AP and SRGAP2a; altered the expressions of Smad2/3, p-Smad2/3, Smad7 and NOX4; and increased ROS production in podocytes. Conclusions: This study suggests that IS may modulate the expression of nephrin, synaptopodin, CD2AP and SRGAP2a by regulating the ROS and TGF-β1/Smad signalling pathways, providing new theoretical support for the treatment of diabetic nephropathy.引言：高血糖可诱导大量活性氧簇（reactive oxygen species, ROS）生成，并激活转化生长因子β1（transforming growth factor β1, TGF-β1）/Smad信号通路，该通路是糖尿病肾病（diabetic nephropathy）形成的核心起始因素。吲哚硫酸酯（indoxyl sulfate, IS）是一类蛋白结合型肠道源性尿毒症毒素，可靶向定位于足细胞，诱导氧化应激并介导足细胞炎症反应。目前，通过TGF-β1/Smad信号通路介导足细胞损伤参与糖尿病肾病发生的具体分子机制仍有待阐明。 方法：本研究体外培养分化成熟的大鼠足细胞，分别在小干扰RNA（siRNA）介导的TGF-β1沉默、TGF-β1过表达以及活性氧抑制剂乙酰半胱氨酸干预的条件下，采用实时荧光定量聚合酶链反应（quantitative real-time PCR, qRT-PCR）与蛋白质印迹法（western blotting）检测nephrin、synaptopodin、CD2AP、SRGAP2a及α-SMA的表达水平；此外，在高糖（high glucose, HG）与吲哚硫酸酯干预条件下，检测Smad2/3、磷酸化Smad2/3（phosphorylated-Smad2/3, p-Smad2/3）、Smad7、烟酰胺腺嘌呤二核苷酸磷酸氧化酶4（NADPH oxidase 4, NOX4）的表达水平以及细胞内活性氧水平。 结果：实验结果表明，在siRNA介导的TGF-β1沉默或加入乙酰半胱氨酸的高糖环境中，nephrin、synaptopodin、CD2AP及SRGAP2a的表达水平显著上调，而α-SMA的表达水平显著下调。反之，在高糖环境下过表达TGF-β1时，nephrin、synaptopodin、CD2AP及SRGAP2a的表达水平显著下调，α-SMA的表达水平则显著升高。在高糖环境中补充IS可进一步显著降低nephrin、synaptopodin、CD2AP及SRGAP2a的表达水平，异常调控Smad2/3、p-Smad2/3、Smad7及NOX4的表达，并增加足细胞内活性氧的生成。 结论：本研究证实，IS可通过调控活性氧与TGF-β1/Smad信号通路，调节nephrin、synaptopodin、CD2AP及SRGAP2a的表达水平，为糖尿病肾病的临床治疗提供了全新的理论支撑。