Supplementary Material for: LRG1 Mitigates Renal Interstitial Fibrosis through Alleviating Capillary Rarefaction and Inhibiting Inflammatory and Pro-Fibrotic Cytokines

<b><i>Introduction:</i></b> Increasing evidence has demonstrated that loss of peritubular capillaries plays a critical role in renal interstitial fibrosis. Leucine-rich α2-glycoprotein-1 (LRG1) has been observed promoting angiogenesis in the ocular disease mouse model and myocardial infarction model. We aimed to explore the role of LRG1 in renal interstitial fibrosis. <b><i>Methods:</i></b> We analyzed the expression of LRG1 in the plasma and kidney of CKD patients by ELISA and immunohistochemistry. Relationships between the expression of LRG1 in plasma and kidney and renal fibrosis and inflammation were analyzed. Tube formation assay was used to detect the angiogenesis in the human umbilical vein endothelial cell lines (HUVECs). And real-time PCR was used to detect the mRNA expression of LRG1, inflammatory factors, renal tubular injury indicators, pro-fibrotic cytokines, and CD31. We examined the effects of genetic ablation of LRG1 on renal fibrosis induced by unilateral ureteral obstruction (UUO) mice model at day 7. <b><i>Results:</i></b> We demonstrated that the expression of LRG1 in renal tissues and plasma samples was upregulated in CKD patients. And the expression of LRG1 was elevated in human renal tubular epithelial cell line (HK-2) cells in response to the stimulation of TNF-α in vitro, and in kidney after UUO in vivo. The deficiency of the LRG1 gene aggravated renal fibrosis, inflammatory cells infiltration, and capillary rarefaction after UUO. In vitro, LRG1 promoted the tube formation of HUVEC cells. LRG1 inhibits fibronectin secretion induced by TGF-β1 in HK-2 and overexpression of LRG1 in HK-2 cells decreased fibronectin secretion. <b><i>Conclusion:</i></b> LRG1 may prevent renal fibrosis by inhibiting the secretion of inflammatory and pro-fibrotic cytokines and promoting angiogenesis.

**引言**：越来越多的证据表明，肾小管周毛细血管丢失在肾间质纤维化中发挥关键作用。富亮氨酸α2糖蛋白1（Leucine-rich α2-glycoprotein-1，LRG1）已被证实可在眼部疾病小鼠模型与心肌梗死模型中促进血管生成。本研究旨在探讨LRG1在肾间质纤维化中的作用。**材料与方法**：本研究通过酶联免疫吸附试验（Enzyme-Linked Immunosorbent Assay，ELISA）与免疫组织化学法，分析了慢性肾脏病（Chronic Kidney Disease，CKD）患者血浆与肾脏组织中LRG1的表达水平；并分析了血浆及肾脏组织中LRG1的表达与肾纤维化、炎症反应的相关性。采用管形成实验检测人脐静脉内皮细胞（Human Umbilical Vein Endothelial Cells，HUVECs）的血管生成能力；通过实时荧光定量PCR（real-time PCR）检测LRG1、炎症因子、肾小管损伤指标、促纤维化细胞因子及CD31的mRNA表达水平。此外，本研究观察了LRG1基因敲除对造模第7天单侧输尿管梗阻（Unilateral Ureteral Obstruction，UUO）小鼠模型诱导的肾纤维化的影响。**结果**：本研究证实，慢性肾脏病患者的肾脏组织与血浆样本中LRG1的表达水平均显著上调。体外实验中，肿瘤坏死因子α（Tumor Necrosis Factor-α，TNF-α）刺激后人肾小管上皮细胞系（HK-2）的LRG1表达水平升高；体内实验中，UUO模型小鼠肾脏组织的LRG1表达同样上调。LRG1基因敲除可加重UUO模型小鼠术后的肾纤维化、炎症细胞浸润与毛细血管稀疏化。体外实验显示，LRG1可促进HUVECs的管形成能力。在HK-2细胞中，LRG1可抑制转化生长因子β1（Transforming Growth Factor-β1，TGF-β1）诱导的纤维连接蛋白分泌；而HK-2细胞过表达LRG1则可降低纤维连接蛋白的分泌水平。**结论**：LRG1或可通过抑制炎症因子与促纤维化细胞因子的分泌，并促进血管生成，从而发挥预防肾纤维化的作用。