Role of GPR56 in glomerular endothelial cell injury in early diabetic kidney disease

GPR56在早期糖尿病肾病肾小球内皮细胞损伤中的作用

基本信息

批准号：
10214883
负责人：
Jia Fu
金额：
$ 14.05万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2026-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10214883
关键词：
ADGR1 gene Adhesions Advanced Glycosylation End Products Affect Albuminuria Attenuated Automobile Driving Cell Survival Cell physiology Cells Code Collagen Cyclic AMP Cyclic AMP-Dependent Protein Kinases Data Development Diabetes Mellitus Diabetic Nephropathy Disease Disease Progression End stage renal failure Endothelial Cells Endothelium Event Extracellular Matrix Proteins Fibronectins Functional disorder G-Protein Signaling Pathway G-Protein-Coupled Receptors Genes Glucose Heparin Human Immunity In Vitro Incidence Injury Kidney Kidney Diseases Knock-out Knockout Mice Learning Skill Ligands Mediating Mentors Methods Microalbuminuria Mus NOS3 gene Pathogenicity Pathway interactions Permeability Phosphorylation Play Postdoctoral Fellow Prevalence Principal Investigator Process Proteins Regimen Regulation Research Personnel Role Signal Pathway Signal Transduction Streptozocin Sulfate Techniques Testing Therapeutic Effect Therapeutic Intervention Tissues Transgenic Mice Tumor Biology Vascular Endothelial Growth Factors adhesion receptor angiogenesis base cell injury diabetic differential expression glomerular endothelium in vivo mRNA Expression melanoma neuron development non-diabetic novel novel therapeutics podocyte post-doctoral training promoter protein expression shear stress single-cell RNA sequencing transcriptome transcriptome sequencing transcriptomics

项目摘要

Glomerular endothelial cell (GEC) dysfunction is an early event in DKD which promotes the disease progression. However, the mechanisms of GEC injury in DKD remain unclear. Thus, a better understanding of the underlying processes of GEC injury is urgently required for the development of an early therapeutic intervention. Recently, we developed a novel method of effective isolation of GECs from transgenic mice expressing enhanced yellow fluorescent protein (EYFP) under the endothelium-specific Flk1 promoter (Fu J et al. KI, 2018). We were able to sort GECs from these mice with or without DKD for RNA-seq. We also performed single-cell RNA-seq of glomeruli isolated from these mice, which also allow us to compare the transcriptome of GECs at the single cell level between diabetic and non-diabetic mice (Fu J et al. JASN, 2019). From these studies, we found that many of the differentially expressed genes (DEGs) in diabetic GECs were involved in the regulation of endothelial injury in early DKD. Among these, G-protein coupled receptor-56 (GPR56) was found to be highly upregulated in diabetic GECs. GPR56 codes for an atypical G protein-coupled receptor and is also referred to as Adhesion G Protein-Coupled Receptor G1 (ADGRG1). Expression of Collagen III, a major ligand for GPR56, is also accumulated in diabetic kidney. GPR56 activates mainly G12/13-mediated RhoA-ROCK pathway, which is known to mediate endothelial cell dysfunction in DKD. Like other adhesion receptors, GPR56 also responds to shear stress, which is increased in GECs of diabetic kidneys due to glomerular hyperfiltration. Our key preliminary observations are: 1) Recent single-cell RNA-seq data confirm that GPR56 expresses predominantly in GECs in the glomeruli. 2) Both mRNA and protein expression of GPR56 increase in human DKD and correlate negatively with eGFR, suggesting an important role of GPR56 in human DKD. 3) GPR56 is upregulated in cultured GECs by high glucose and advanced glycation endproducts (AGE). 4) Collagen III treatment suppressed eNOS phosphorylation and expression through activation of GPR56. 5) GPR56 reduces eNOS phosphorylation likely through G12/13-mediated RhoA pathway and inhibits eNOS expression via Gi-mediated inhibition of cAMP/PKA/KLF4 pathway in cultured mGECs. 6) Knockout of GPR56 enhances eNOS and KLF4 expression in GECs and attenuated albuminuria and glomerular injury in mice with DKD. Based on these findings, we hypothesize that GPR56 mediates disease progression in DKD by increasing GEC injury. We propose to determine the role and mechanism of GPR56 signaling pathway in diabetes-induced GEC injury in vitro in GECs treated with diabetic condition and in vivo in mice with DKD. We believe that our studies will help us to determine whether GPR56 could be a potential new target to treat DKD by targeting GEC injury. The principal investigator will learn the skills and new techniques under the guidance of her mentoring team (Drs. John He, Ravi Iyenger, Weijia Zhang, Bi-sen Ding, and Evren Azeloglu) and her consultants (Drs. Xianhua Piao), who have a very strong track record of training postdoctoral fellows in transitioning into independent investigators.

肾小球内皮细胞 (GEC) 功能障碍是 DKD 的早期事件，可促进疾病的发展进展。然而，DKD 中 GEC 损伤的机制仍不清楚。从而更好地理解迫切需要了解 GEC 损伤的基本过程，以开发早期治疗方法干涉。最近，我们开发了一种从转基因小鼠中有效分离 GEC 的新方法在内皮特异性 Flk1 启动子下表达增强型黄色荧光蛋白 (EYFP)（Fu J 等等人。 KI，2018）。我们能够对这些患有或不患有 DKD 的小鼠的 GEC 进行分类，以进行 RNA 测序。我们也对从这些小鼠中分离出的肾小球进行了单细胞 RNA 测序，这也使我们能够比较糖尿病小鼠和非糖尿病小鼠之间单细胞水平的 GEC 转录组（Fu J et al. JASN, 2019）。从这些研究中，我们发现糖尿病 GEC 中的许多差异表达基因 (DEG) 参与早期 DKD 内皮损伤的调节。其中，G蛋白偶联受体56 (GPR56)被发现在糖尿病GEC中高度上调。 GPR56 编码非典型 G 蛋白偶联受体，也称为粘附 G 蛋白偶联受体 G1 (ADGRG1)。表达 III 型胶原蛋白是 GPR56 的主要配体，也在糖尿病患者的肾脏中积累。 GPR56主要激活 G12/13 介导的 RhoA-ROCK 通路，已知可介导 DKD 中的内皮细胞功能障碍。喜欢与其他粘附受体一样，GPR56 也对剪切应力做出反应，糖尿病患者的 GEC 中剪切应力增加肾小球过度滤过所致的肾脏损害。我们的主要初步观察结果是：1) 最近的单细胞 RNA-seq 数据证实 GPR56 主要在肾小球的 GEC 中表达。 2) mRNA和蛋白质人类 DKD 中 GPR56 的表达增加，并与 eGFR 呈负相关，这表明 GPR56 的表达具有重要意义 GPR56 在人类 DKD 中的作用。 3) GPR56 在培养的 GEC 中因高葡萄糖和高级药物而上调糖基化终产物（AGE）。 4) III 型胶原蛋白治疗抑制 eNOS 磷酸化和表达通过激活 GPR56。 5) GPR56 可能通过 G12/13 介导的 RhoA 降低 eNOS 磷酸化途径并通过 Gi 介导的抑制培养物中的 cAMP/PKA/KLF4 途径来抑制 eNOS 表达 mGEC。 6) GPR56 的敲除增强了 GEC 中 eNOS 和 KLF4 的表达并减轻了蛋白尿以及 DKD 小鼠的肾小球损伤。基于这些发现，我们假设 GPR56 介导 DKD 的疾病进展是通过增加 GEC 损伤来实现的。我们建议确定 GPR56 信号通路在糖尿病诱导的体外 GEC 损伤中的作用患有 DKD 的小鼠体内。我们相信，我们的研究将帮助我们确定 GPR56 是否可以成为通过针对 GEC 损伤来治疗 DKD 的潜在新靶标。首席研究员将学习技能和在她的导师团队（John He博士、Ravi Iyenger博士、Weijia 张博士、Bi-sen博士）指导下的新技术 Ding 和 Evren Azeloglu）及其顾问（Xianhua Piao 博士）在培训博士后研究员转变为独立研究者。