The Role of LRG1 in Diabetic Kidney Disease

LRG1 在糖尿病肾病中的作用

• 批准号: 9752586
• 负责人: Kyung Lee
• 金额: $ 33.9万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9752586
• 关键词: Affect; Albuminuria; Architecture; Attenuated; Cell Line; Cells; Clinical Research; Clinical Trials; Complement; Cultured Cells; Development; Diabetes Mellitus; Diabetic Nephropathy; Diabetic mouse; Disease; Disease Progression; Early Intervention; End stage renal failure; Endothelial Cells; Endothelium; Event; Foundations; Functional disorder; Future; Genes; Genetic Models; Glucose; Glycoproteins; Human; In Vitro; Inflammatory Response; Injury; Kidney; Knock-out; Knockout Mice; Leucine; LoxP-flanked allele; Mediating; Methods; Microalbuminuria; Modeling; Molecular; Mus; NOS3 gene; Non-Insulin-Dependent Diabetes Mellitus; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Permeability; Process; Proteins; Public Health; Regulation; Role; Sampling; Serum; Signal Transduction; Streptozocin; TGF Beta Signaling Pathway; Testing; Therapeutic; Therapeutic Intervention; Tissues; Transforming Growth Factor beta; Transgenic Mice; Up-Regulation; Urine; angiogenesis; base; cancer cell; cell injury; db/db mouse; diabetes management; diabetic; differential expression; early detection biomarkers; effective therapy; endothelial dysfunction; glomerular endothelium; knock-down; mRNA Expression; malignant retina neoplasm; mouse model; non-diabetic; novel; overexpression; potential biomarker; promoter; single-cell RNA sequencing; transcriptome sequencing; transcriptomics

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.

项目概要 糖尿病肾病 (DKD) 是美国终末期肾病的最常见原因，但其影响有限 治疗方案。最近的几项临床试验尚未成功，可能是由于缺乏早期研究 干涉。肾小球内皮细胞 (GEC) 功能障碍促进 DKD 的进展，证据 表明 GEC 损伤是糖尿病肾脏的早期事件，可能先于糖尿病肾病的发展 微量白蛋白尿。然而，DKD 早期 GEC 损伤的机制仍不清楚。这样，更好 迫切需要了解 GEC 损伤的基本过程，以开发早期治疗方法。 治疗干预。迄今为止，DKD 中 GEC 损伤的研究依赖于全肾分析 组织或孤立的肾小球，提供间接和有限的信息。最近，我们开发并 优化了一种从表达增强黄色的转基因小鼠中有效分离 GEC 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 从链脲佐菌素 (STZ) 诱导的糖尿病内皮一氧化氮中分离出的 GEC 的转录组谱 合成酶（eNOS）缺失的小鼠，其 GEC 来自非糖尿病对照。许多差异表达基因 糖尿病 GEC 参与血管生成和内皮损伤的调节。其中，有一种分泌 富含亮氨酸的 α-2 糖蛋白 (LRG1) 在糖尿病 GEC 中高度上调。 LRG1 最近被证明是 TGF-β 信号传导的必要辅激活剂，对于 视网膜和癌细胞的新血管生成。因此，我们探讨LRG1是否具有调节功能 早期 DKD 中的血管生成。我们发现 1）LRG1 mRNA 表达在 DKD 的 GEC 中显着增加 糖尿病小鼠以及 DKD 患者的肾小球； 2）相应地，发现LRG1表达 主要存在于小鼠和人类肾脏的 GEC 中； 3）血清和尿液中LRG1蛋白增加 DKD 患者样本； 4)高糖诱导体外培养的肾小球和GECs中LRG1表达； 5） LRG1 敲除可减少糖尿病小鼠的蛋白尿和肾小球损伤，并减弱血管生成；和 6) LRG1 通过诱导 GEC 中的非经典 TGF-β 途径和敲低来促进血管生成 GEC 中 LRG1 的存在足以减少体外血管生成。基于这些显着的发现，我们 假设 LRG1 是早期 DKD 疾病进展的关键致病因素，通过增加 GEC 中的血管生成和损伤。因此，我们建议 1) 使用类型 1 确定 LRG1 在 DKD 中的作用 和2型DM小鼠模型； 2）确定LRG1在DKD早期和晚期的EC特异性作用；和 3)体外确定LRG1诱导糖尿病GEC损伤的分子机制。结果来自 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是否是一种 早期 DKD 的潜在生物标志物以及 LRG1 的全身阻断是否会带来治疗益处 干预 DKD 进展。