Role Of Corin-mediated Pro-ANP Activation Pathway In Endothelial Dysfunction In Diabetic Nephropathy

Objective Diabetes(diabetes mellitus,DM)is a common endocrine and metabolic disease characterized by hyperglycemia.With the change of people’s lifestyle,its incidence is increasing,which seriously affects human health.Diabetic nephropathy(DN)is one of the major vascular complications of diabetes and one of the leading causes of death in patients with diabetes.Corin is a type Ⅱ transmembrane serine protease that can cleave pro-atrial natriuretic peptide(pro-ANP)into active atrial natriuretic peptide(ANP),and then regulates water and salt metabolism balance.Corin is a heart-specific protease and is also expressed in kidney,cerebellum,skin and bone.At present,most of Corin’s researches are focusing on cardiovascular diseases such as hypertension,myocardial infarction,and heart failure.Corin is also associated with certain kidney diseases,such as nephrotic syndrome and glomerulonephritis.Our previous research found that Corin plays an important role in the pathogenesis of diabetic cardiomyopathy and diabetic nephropathy,the main vascular complication of diabetes,and the lack of Corin can also cause endothelial dysfunction.However,the molecular mechanism of Corin deficiency in endothelial dysfunction is not quite clear yet.Therefore,in this study we used the SD rat animal model of diabetic nephropathy and si RNA to interfere with the CORIN gene to treat HK-2 renal tubular epithelial cells to study the mechanism of endothelial dysfunction caused by Corin deficiency with cell biology and molecular biology techniques,and hope to provide a theoretical basis for the study of new molecular targets for the diagnosis and treatment of diabetic nephropathy.Methods1.Using the animal model of diabetic nephropathy SD rats successfully constructed by this research group,the expression of Corin and ANP in the kidney tissues of diabetic nephropathy rats and normal control rats was detected by immunohistochemistry.2.Si RNA technology was used to treat HK-2 renal tubular epithelial cells to interfere the Corin gene expression.At the same time,control group and NC-si RNA group was set.The HK-2 cell supernatants were collected after 48 hours of culture,and endothelial cells were cultured with the collected supernatants 24 hours.3.Cell scratch test was used to observe the changes of proliferation and repair function of EA.hy926 endothelial cells in control group,NC-si RNA group and Corin-si RNA group.4.Western blot was used to detect changes in phosphorylation expression levels of mitogen-activated protein kinase(MAPK)including extracellular regulated protein kinases(ERK),p38 protein kinase(p38),c-jun N-terminal kinase(JNK)protein in EA.hy926 endothelial cells of control group,NC-si RNA group and Corin-si RNA group.5.Western blot was used to detect changes in phosphorylation expression levels of endothelial nitric oxide synthase(e NOS)protein in EA.hy926 endothelial cells of control group,NC-si RNA group and Corin-si RNA group.6.In the supernatant of HK-2 cells interfered with CORIN,exogenous ANP protein was added to culture EA.hy926 endothelial cells.Cell scratch test was used to observe the changes of proliferation and repair function of EA.hy926 endothelial cells in control group,NC-si RNA group,Corin-si RNA group and Corin-si RNA+ANP group.7.Western blot was used to detect changes in phosphorylation expression levels of MAPK including protein in EA.hy926 endothelial cells of control group,NC-si RNA group,Corin-si RNA group and Corin-si RNA+ANP group.8.Western blot was used to detect changes in phosphorylation expression levels of e NOS protein in EA.hy926 endothelial cells of control group,NC-si RNA group,Corin-si RNA group and Corin-si RNA+ANP group.Results1.Compared with the normal control group,the expression levels of Corin and ANP protein in the kidney tissue of diabetic nephropathy SD rats were decreased.2.Compared with the control group and the NC-si RNA group,the proliferation and repair function of endothelial cells in Corin-si RNA group was decreased.3.Compared with the blank control group and the NC-si RNA group,the MAPK signaling pathway ERK,p38,and JNK protein phosphorylation levels of EA.hy926 endothelial cells were significantly increased and the e NOS protein phosphorylation levels were significantly decreased in the Corin-si RNA group.4.Compared with the Corin-si RNA group,the proliferation and repair capacity of EA.hy926 endothelial cells was consistent with normal levels after administration of exogenous ANP protein.5.Compared with the Corin-si RNA group,the degree of phosphorylation of ERK,p38,and JNK proteins in EA.hy926 endothelial cells decreased to normal levels and the degree of phosphorylation of e NOS protein increased to normal levels after administration of exogenous ANP protein.Conclusions In summary,in our study we found that the expression of Corin in diabetic nephropathy decreased,indicating that Corin is involved in the pathogenesis of diabetic nephropathy,and the lack of Corin will lead to endothelial dysfunction,which is accomplished by up-regulating MAPK signals and down-regulating e NOS.After administration of exogenous ANP,endothelial cell function was improved,and the phosphorylation levels of ERK,p38,JNK and e NOS proteins in endothelial cells are restored.The above results indicate that Corin plays an important role through ANP.Therefore,we conclude that Corin-mediated pro-ANP activation pathway plays an important role in endothelial dysfunction of diabetic nephropathy through MAPK /e NOS signaling pathway,and Corin is expected to become a new molecular target for the diagnosis and treatment of diabetic nephropathy.