The Role of LRG1 in Diabetic Kidney Disease

LRG1 在糖尿病肾病中的作用

• 批准号: 10415161
• 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 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10415161
• 关键词: Affect; Albuminuria; Architecture; Attenuated; Cell Line; Cells; Clinical Research; Clinical Trials; Complement; 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; Injury to Kidney; 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.

项目摘要 糖尿病肾病（DKD）是美国终末期肾病的最常见原因， 治疗方案。最近的几项临床试验没有成功，可能是由于缺乏早期治疗。 干预肾小球内皮细胞（GEC）功能障碍促进了DKD的进展， 提示GEC损伤是糖尿病肾脏的早期事件，可能先于 微量白蛋白尿然而，DKD早期GEC损伤的机制尚不清楚。因此， 了解GEC损伤的潜在过程是迫切需要的， 治疗干预迄今为止，DKD中GEC损伤的研究依赖于全肾分析 组织或分离的肾小球，其提供间接和有限的信息。最近，我们开发和 优化了一种从表达增强型黄色荧光素的转基因小鼠中有效分离GECs的新方法， 荧光蛋白（EYFP）在内皮特异性Flk 1启动子。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的GEC的转录组学谱 合成酶（eNOS）-无效小鼠与来自非糖尿病对照的GEC。许多差异表达的基因， 糖尿病GECs参与血管生成和内皮损伤的调节。其中， 糖蛋白，富含亮氨酸的α-2糖蛋白（LRG 1）被发现在糖尿病GEC中高度上调。 LRG 1最近被证明是TGF-β信号传导的必要共激活剂，TGF-β信号传导在 视网膜和癌细胞的新血管生成。因此，我们探讨了LRG 1是否具有调节功能， 我们发现：1）LRG 1的mRNA表达在DKD早期的GECs中显著增加， 糖尿病小鼠肾小球及DKD患者肾小球中LRG 1的表达 主要在小鼠和人肾脏的GEC中; 3）LRG 1蛋白在血清和尿液中增加 高糖诱导体外培养的肾小球和GECs表达LRG 1; LRG 1敲除减少糖尿病小鼠的白蛋白尿和肾小球损伤，并减弱血管生成;和 6)LRG 1通过诱导GECs中的非经典TGF-β途径促进血管生成， GEC中LRG 1的含量足以减少体外血管生成。基于这些重要的发现，我们 假设LRG 1是早期DKD疾病进展关键致病因素， GECs中的血管生成和损伤。因此，我们建议：1）使用1型胶原酶确定LRG 1在DKD中的作用。 2）确定LRG 1在DKD早期和晚期的EC特异性作用;和 3)探讨LRG 1诱导体外糖尿病GEC损伤的分子机制。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LRG 1是否是一种 早期DKD的潜在生物标志物以及LRG 1的全身阻断是否会提供治疗获益 干预DKD进展。