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

• 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
• 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

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，它促进了疾病的发生 进步。然而，DKD中GEC损伤的机制尚不清楚。因此，更好地理解 GEC损伤的潜在过程是开发早期治疗方法的迫切需要 干预。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。 内皮特异性Flk1启动子(Fu J Et)诱导表达增强型黄色荧光蛋白(EYFP) 艾尔Ki，2018)。我们能够从这些有或没有DKD的小鼠的GEC中分选出RNA-Seq。我们也 对从这些小鼠分离的肾小球进行单细胞rna-seq，这也使我们能够比较 糖尿病小鼠和非糖尿病小鼠之间在单细胞水平上的GEC转录组(Fu J等人)。JASN，2019年)。 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)是 参与DKD早期内皮损伤的调节。其中，G蛋白偶联受体-56 (GPR56)在糖尿病肾小球内皮细胞中高度上调。GPR56编码一种非典型的G蛋白偶联 受体，也称为黏附G蛋白偶联受体G1(ADGRG1)。的表达 III型胶原是GPR56的主要配体，也在糖尿病肾脏中积聚。GPR56主要激活 G12/13介导的RhoA-ROCK通路，已知在DKD中介导内皮细胞功能障碍。喜欢 其他黏附受体GPR56也对切应力有反应，切应力在糖尿病患者的肾小管上皮细胞中增加 肾小球高滤过所致的肾脏。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主要在肾小球内的GEC中表达。2)信使核糖核酸和蛋白质 GPR56在人DKD中的表达增加，并与EGFR呈负相关，提示 GPR56在人类DKD中的作用3)GPR56在高糖和晚期糖刺激下表达上调。 糖基化终末产物(年龄)。4)III型胶原处理抑制eNOS的磷酸化和表达 通过激活GPR56。5)GPR56可能通过G12/13介导的RhoA降低eNOS的磷酸化 Gi通过抑制cAMP/PKA/KLF4途径抑制eNOS的表达 MGEC。6)GPR56基因敲除可增强肾小管上皮细胞eNOS和KLF4的表达，减少蛋白尿 和DKD小鼠的肾小球损伤。基于这些发现，我们假设GPR56在 DKD的疾病进展通过增加GEC损伤来实现。我们建议确定其作用和机制。 GPR56信号通路在糖尿病大鼠血管内皮细胞损伤中的作用 在DKD小鼠体内。我们相信，我们的研究将帮助我们确定GPR56是否可能是一种 靶向GEC损伤治疗DKD的潜在新靶点。首席调查员将学习技能和 在她的指导团队(何约翰博士、拉维·艾扬格博士、张维佳博士、毕森博士)的指导下的新技术 Ding，Evren Azeloglu)和她的顾问(朴贤华博士)，他们在 培训博士后研究员过渡为独立调查人员。