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, ...