The Role of LRG1 in Diabetic Kidney Disease

项目总结 糖尿病肾病(DKD)是美国终末期肾脏疾病最常见的原因，有限 治疗方案。最近的几项临床试验都没有成功，可能是因为缺乏早期的 干预。肾小球内皮细胞(GEC)功能障碍促进DKD进展 提示GEC损伤是糖尿病肾脏的早期事件，可能先于糖尿病的发生。 微量白蛋白尿。然而，DKD早期GEC损伤的机制尚不清楚。因此，更好地 了解GEC损伤的潜在过程是发展早期GEC的迫切需要 治疗性干预。到目前为止，对DKD中GEC损伤的研究主要依赖于全肾的分析。 组织或孤立的肾小球，提供间接和有限的信息。最近，我们开发了和 一种高效分离表达增强型黄色素转基因小鼠肾小管上皮细胞的新方法 内皮特异性Flk1启动子下的荧光蛋白(EYFP)。使用这种方法，我们比较了 链脲佐菌素诱导的糖尿病内皮细胞一氧化氮诱导的GECs转录图谱 合酶(ENOS)缺失的小鼠，其GEC来自非糖尿病对照组。许多差异表达的基因在 糖尿病血管内皮细胞参与了血管生成和内皮损伤的调节。其中，一个秘密的 糖蛋白，亮氨酸丰富的α-2糖蛋白(LRG1)在糖尿病肾小管上皮细胞中高度上调。 最近研究表明，lrg1是转化生长因子-β信号转导系统中必不可少的辅助激活剂。 视网膜和癌细胞的新生血管生成。因此，我们探索了LRG1是否在 DKD早期血管生成的实验研究发现：(1)DKD大鼠肾小管上皮细胞LRG1mRNA表达显著增加。 2)相应地发现LRG1在糖尿病小鼠和DKD患者的肾小球中表达 主要在小鼠和人肾小管上皮细胞中表达；3)血清和尿液中LRG1蛋白表达增加 4)高糖诱导体外培养的肾小球和肾小球内皮细胞LRG1的表达； LRG1基因敲除可减少糖尿病小鼠的蛋白尿和肾小球损伤，并抑制血管生成； 6)LRG1通过诱导肾小管上皮细胞非规范的转化生长因子-β途径促进血管生成，并下调 LRG1在血管内皮细胞中的表达足以抑制体外血管生成。基于这些突出的发现，我们 假设LRG1是早期DKD疾病进展的关键致病因素 血管生成与血管内皮细胞损伤。因此，我们建议1)使用类型1确定LRG1在DKD中的作用 和2型糖尿病小鼠模型；2)确定LRG1在DKD早期和晚期的EC特异性作用；以及 3)探讨LRG1诱导的糖尿病肾小管上皮细胞损伤的分子机制。调查结果： 拟议的研究将为未来的临床研究奠定坚实的基础，以确定LRG1是否为一种 早期DKD的潜在生物标志物及全身阻断LRG1是否提供治疗益处 以干预DKD的进展。

PROJECT SUMMARY Diabetic kidney disease (DKD) is the most common cause of end-stage renal disease in the US, with limited treatment options. Several recent clinical trials have not succeeded, likely owing to the lack of early intervention. Glomerular endothelial cell (GEC) dysfunction promotes the progression of DKD, and evidence suggests that GEC injury is an early event in diabetic kidneys that may precede the development of microalbuminuria. However, the mechanisms of early GEC injury in DKD remain unclear. Thus, better understanding of the underlying processes of GEC injury is urgently required for development of early therapeutic intervention. To date, the study of GEC injury in DKD has relied on the analysis of whole kidney tissues or isolated glomeruli, which provides indirect and limited information. Recently, we developed and optimized a novel method of effective isolation of GECs from transgenic mice expressing enhanced yellow fluorescent protein (EYFP) under the endothelium-specific Flk1 promoter. Using this approach, we compared the transcriptomic profiles of GECs isolated from streptozotocin (STZ)-induced diabetic endothelial nitric oxide synthase (eNOS)-null mice with GECs from nondiabetic controls. Many of the differentially expressed genes in diabetic GECs were involved in the regulation of angiogenesis and endothelial injury. Among these, a secreted glycoprotein, leucine-rich alpha-2 glycoprotein (LRG1) was found to be highly upregulated in diabetic GECs. LRG1 was recently shown to be the necessary co-activator of TGF-β signaling that is essential in neoangiogenesis of retinal and cancer cells. Thus, we explored whether LRG1 has a regulatory function in angiogenesis in early DKD.We found that 1) mRNA expression of LRG1 is significantly increased in GECs of diabetic mice, as well as in glomeruli of DKD patients; 2) correspondingly, LRG1 expression was found predominantly in GECs of mouse and human kidneys; 3) LRG1 protein is increased in serum and urine samples of DKD patients; 4) high glucose induces LRG1 expression in glomeruli and GECs cultured in vitro; 5) LRG1 knockout reduces albuminuria and glomerular injury, and attenuates angiogenesis in diabetic mice; and 6) LRG1 promotes angiogenesis via induction of the non-canonical TGF-β pathway in GECs, and knockdown of LRG1 in GECs is sufficient to diminish angiogenesis in vitro. Based on these salient findings, we hypothesize that LRG1 is a key pathogenic contributor of disease progression in early DKD by increasing angiogenesis and injury in GECs. Therefore, we propose to 1) determine the role of LRG1 in DKD using type 1 and type 2 DM mouse models; 2) determine the EC-specific role of LRG1 in early and late stages of DKD; and 3) determine the molecular mechanism of LRG1-induced diabetic GEC injury in vitro. Results from the proposed study will build the strong foundation for the future clinical studies to determine whether LRG1 is a potential biomarker of early DKD and whether the systemic blockade of LRG1 would offer therapeutic benefit to intervene against DKD progression.