The Role Of LRG1 In Chronic Kidney Disease Through Angiogenesis

Background:Glomerular endothelial cell(GEC)injury and increased angiogenesis are early key events in pathogenesis of diabetic kidney disease(DKD).DKD is becoming the most common cause of chronic kidney disease(CKD)and end-stage renal disease(ESRD)in the world.However,the current treatments provide only partial therapeutic effects and several recent clinical trials in DKD treatments were unsuccessful.One major cause of these failures is due to the lateness of intervention.Glomerular endothelial cell injury is prior to podocyte injury and plays an irreplaceable role in kidney disease,in the early stage of DKD endothelial cell stimulated by high glucose and cytokines also with glomerular hyper-perfusion and hypertension all lead to microvascular disease,which can perform as abnormal neovascularity and lead to DKD.The neovascularity is well researched in the field of cancer and atherosclerosis but the study in kidney disease is not fully elucidated.Through the research on the transcriptome of podocytes and endothelial cells in diabetic nephropathy mice,we have found that LRG1(leucine-rich α-2 glycoprotein1)is enriched in glomerular endothelial cells rather than podocytes,and the expression of transcripts is increased in diabetic nephropathy.it is known that LRG1 can promote angiogenesis by activating non-classical ALK1/TGF-β signaling pathway in endothelial cells.However,LRG1 antibody can block this process and relieve angiogenesis,so whether LRG1,as an important regulatory protein of angiogenesis in endothelial cells,plays an important role in the progression and outcome of diabetic nephropathy through TGF-β signaling pathway is unclear.Objective:To clarify the role of LRG1 in the progression and outcome of diabetic nephropathy mouse models through angiogenesis and TGF-β signaling pathway;To verify that TGF-β receptor subtypes play important physiological and pathological roles in regulating different TGF-β signaling pathways;To explore that secreted LRG1 can act as cross-talk on the endothelial cell and other cells to regulate different TGF-β receptor subtypes and function differently.Methods:In vitro by using the primary isolated glomerular endothelial cell stimulated by high glucose to validate the transcriptomic results,by in situ hybridization to make sure the location and expression of LRG1 under pathologic condition.LRG1 knock down by shRNA and lenti-virus to make sure the loss function of LRG1 can influent the angiogenesis factors and pathway in vitro,pull down the overexpressed LRG1 by V5-tag to explore the co-operated TGF-β receptor and accessory receptors.In vivo LRG1 gene knockout mice were compared with wild-type mice after STZ induced type Ⅰ diabetes.The renal injury degree of the knockout mice included proteinuria,renal function and the difference of angiogenesis in vivo.According to the time difference of angiogenesis phenomenon,two time points of 12 weeks(early)and 20 weeks(late)were respectively selected to observe and clarify the role of LRG1 in early angiogenesis and the protective effect of late renal function in the diabetic nephropathy model of mice.Furthermore,vascular endothelial restriction eNOS-/-gene knockout mice and LRG1-/-double knockout mice were used to observe the degree of proteinuria and diabetic nephropathy injury,to further clarify the role and mechanism of LRG1 and important vascular regulatory factors in DKD endothelial injury.Results:1)LRG1 expression is localized predominantly in GECs,and mRNA level is elevated in mouse and human kidney disease,the expression has a correlation with human renal eGFR function.2)We found that high glucose exposure stimulated LRG1 expression in freshly isolated primary mouse GECs.Suppression of LRG1 expression specifically inhibited the ALK1-Smad1/5/8 pathway in GECs under high glucose conditions and decreased angiogenesis in vitro.3)LRG1 can bind to TGF-β RII and accessory co-receptor endoglin(ENG)in endothelial cells and activating the phosphorylation of downstream angiogenesis promoting S mad 1/5/8 pathway.4)Loss of LRG1 in vivo weakens diabetic nephropathy angiogenesis 12 weeks after streptozotocin(STZ)-induced+unilateral nephrectomy,and alleviates albuminuria and diabetic glomerulopathy,which is related to decreased activation of glomerular Smadl/5/8.5)20 weeks after STZ induction+unilateral nephrectomy showed that the absence of LRG1 provided similar renal protection in renal function,indicating that long-term blocking of LRG1 in the late DKD also had protective effect on diabetic renal injury.6)In addition,compared with diabetic mice with eNOS-/-knockout,double knock endothelial cell eNOS-/-and LRG1-/-mice showed that LRG1 still had renal protective effect on the premise of endothelial cell damage even after eNOS knockout.Conclusion:LRG1 is enriched in glomerular endothelial cells and promotes angiogenesis by activating nonclassical ALK1/TGF-β RII signal and recruitment accessory co-receptor endoglin(ENG)in endothelial cells,phosphorylated downstream Smad1/5/8 pathway.LRG1 gene knockout mice can relieve the proteinuria、pathological renal injury、angiogenesis in vivo and protect the renal function by compared with wild-type mice.LRG1 is not only a risk factor but also contributes to the progress of DKD.