The Protective Role Of GPR124 In Diabetic-Induced Podocytes Injury

Background:Diabetic nephropathy is one of the main microangiopathies in diabetic patients. According the statistic data from America, Japan and many European countries, DN has been the first cause of the end-stage renal disease. Moreover, the morbidity of diabetic nephropathy shows a escalating trend in China. Because patients suffer a complicated metabolic disturbance, once it develops to ESRD, diabetic nephropathy will be more difficult to be treated than the other kidney diseases. Therefore, searching for further study has been a urgent mission to provide an effective measures. The pathogenesis of diabetic nephropathy is in a complexity, referring to a wild range of factors.G-protein coupled receptors are known as seven-transmembrane domain receptors, converting the extracelluar signals (hormones, neurotransmitter, peptides) into the intracelleluar signals, then acting upon effector molecules, causing the cellular reaction. These cellular response get involved in variety of biological functions, like five senses. According to the amino acid sequence homology and functional similarity, GPCRs can be grouped into 6 classes based on GRAFS system:Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2, Secretin. GPR124 is a member of the Adhesion GPCRs family of receptors which encodes a 1331 amino-acid open reading frame with a 760 amino acid N-terminal extracellular domain. The domain structure contains GPS (GPCR proteolysis site), HormR (hormone binding domain), Ig (immunoglobulin domain), LRR (leucine-rich repeat), LRRCT (C-terminal leucine-rich repeat), PDZB (PDZ binding domain), RGD (Argenine-Glycine-Aspartic acid motif),7TM (seven-transmembrane region). Currently, reports about GPR124 focused on the function in CNS (Central Nervous System). Research has reported that the deletion of GPR124 disrupted TGF-β signaling; in vivo, GPR124 functioned cell-autonomously in CNS endothelium to regulate sprouting, migration, and developmental expression of the GLUT1, whereas in vitro, GPR124 mediated Cdc42 dependent directional migration to forebrain-derived, VEGF-independent cues; silencing of GLUT1 expression in the abnormal glomeruloid-like bodies of Gpr124-/- brains was observed, as well as up-regulation of VEGF expression; GPR124 is a Wnt7-specific coactivator of β-catenin signaling in brain endothelium. All of the factors mentioned above play an essential role in the development of diabetic nephropathy, which indicates that GPR124 may involve in the development of DN, however, the function of GPR124 in diabetic nephropathy remains poorly understood.Objective:1. To explore GPR124 expression in diabetic nephropathy.2. To explore the actions and properties of GPR124 in diabetic mice.3. To explore the role of GPR124 in podocytopathies.Methods and Results:Expression of GPR124 in kidney was detected by WB, Real time RT-PCR and immunohistochemical staining. We found that expression of GPR124 was down-regulated in kidney from STZ-induced diabetic mice and ADR-induced nephropathy mice. In vitro, effects of different stimuli on the expression of GPR124 in podocytes, such as high glucose, advanced glycation end-product, tumor necrosis factor-a were carried out by Western blot. All of these factors decreased GPR124 expression in podocyte. Furtherly, by Cre-LoxP technology, we created podocyte conditional GPR124 knockout mice and found that podocyte-deletion of GPR124 exacerbated podocytes injury in diabetic mice. More severe nuclear accumulation, mesentery proliferation and thickeness of basement membrane was observed in Gpr124pod-/-mice by PAS staining and TEM. Furthermore, the recombinant adenovirus vector carrying GPR124 gene was constructed and successfully transfect podocytes. Overexpression of GPR124 promotes LC3B expression and inhibit the expression and phosphorylation of YAP, suggesting that GPR124 is an important component in the YAP-autophagy pathway.Conclusions:1. The expression of GPR124 was down-regulated in the kidney from diabetic mice and FSGS mice,as well as in the podocytes treated with common detrimental factors. Deficiency of GPR124 exacerbated podocyte Injury in diabetic nephropathy, indicating GPR124 play protective role in the development of podocytopathies.2. The overexpression of GPR124 ameliorated the podocytes injury through enhancing autophagy level by downregulating YAP expression,indicating that GPR124 is an one of critical components of a signal transduction pathway that links Hippo pathway to autophagy in podocytopathy.