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，可促进该疾病进展。但是，DKD中GEC损伤的机制尚不清楚。因此，更好地理解 GEC损伤的基本过程迫切需要早期治疗的发展干涉。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启动子下表达增强的黄色荧光蛋白（EYFP）（Fu J et al。 Ki，2018年）。我们能够从或不带有DKD的RNA-seq中对这些小鼠的GEC进行分类。我们也是从这些小鼠中分离出的肾小球的单细胞RNA-seq，这也使我们能够比较 GEC在糖尿病和非糖尿病小鼠之间的单细胞水平上的转录组（Fu J等人Jasn，2019年）。从这些研究中，我们发现糖尿病性GEC中的许多差异表达基因（DEG）是参与DKD早期内皮损伤的调节。其中，G蛋白耦合受体-56 （GPR56）在糖尿病GEC中被高度上调。 GPR56代码非典型G蛋白偶联受体，也称为粘附G蛋白偶联受体G1（ADGRG1）。表达胶原蛋白III是GPR56的主要配体，也积累在糖尿病肾脏中。 GPR56主要激活 G12/13介导的Rhoa-Rock途径，已知可以介导DKD中的内皮细胞功能障碍。喜欢其他粘附受体，GPR56也会响应剪切应力，在糖尿病的GEC中增加了剪切应力由于肾小球过滤而引起的肾脏。我们的关键初步观察是：1）最近的单细胞RNA-seq 数据证实GPR56主要在肾小球中的GEC中表达。 2）mRNA和蛋白质 GPR56的表达人类DKD增加并与EGFR负相关，这表明一个重要的 GPR56在人类DKD中的作用。 3）GPR56在培养的GEC中被高葡萄糖和晚期上调糖基化最终产物（年龄）。 4）胶原蛋白III处理抑制了eNOS磷酸化和表达通过激活GPR56。 5）GPR56可通过G12/13介导的RhoA降低ENOS磷酸化途径并通过gi介导的CAMP/PKA/KLF4途径抑制eNOS表达 MGEC。 6）GPR56的敲除可以增强GEC中的eNOS和KLF4表达并减弱蛋白尿 DKD小鼠的肾小球损伤。基于这些发现，我们假设GPR56介导通过增加GEC损伤，DKD的疾病进展。我们建议确定糖尿病诱导的GEC损伤的GPR56信号通路在经过糖尿病状况和治疗的GEC中带有DKD的小鼠体内。我们认为，我们的研究将帮助我们确定GPR56是否可以成为通过靶向GEC损伤来治疗DKD的潜在新目标。首席调查员将学习技能和在她的指导团队的指导下（John He，Ravi Iyenger博士，Weijia Zhang，Bi-Sen）的新技术丁和Evren Azeloglu）和她的顾问（Xianhua piao博士），他们的记录很强培训博士后研究员过渡到独立调查员。