Role Of Corin In Diabetic Nephropathy

ObjectiveDiabetes mellitus(DM)is a major disease endangering human health.In recent years,incidence of DM is increasing.Diabetic nephropathy(DN)is one of the most important diabetic microvascular complications,and it seriously affects the quality of life of patients with diabetes mellitus.Corin,a type II transmembrane serine protease,is mainly specifically expressed in heart,and also found in kidney,uterus,placenta,bone and other systems.Corin can cleave pro-atrial natriuretic peptide(pro-ANP)into biologically active atrial natriuretic peptide(ANP)with high specificity.Several studies have shown that Corin is involved in many kidney diseases.In patients with chronic kidney disease,urinary Corin level and renal Corin expression decreased significantly;In proteinuria nephropathy,decreased Corin levels caused water and sodium retention.Water and sodium retention occurred in Corin knockout mice and in nephrotic syndrome mice,decreased Corin levels caused water and sodium retention.However,there are no research on role of Corin in diabetic nephropathy.Therefore,this study induces HK-2 human renal tubular epithelial cell line in high ambient glucose,and to explore the role of Corin in DN by using si RNA,Real-time PCR,RT-PCR,Western blot,immunofluorescence and other related cell and molecular biological techniques.It is expected to elucidate the new molecular mechanism of DN and find new targets for prevention and treatment.Methods1.RT-PCR was used to detect Corin and ANP expression in m RNA level in high glucose-induced HK-2 human renal tubular epithelial cells.2.Western blot was used to detect Corin and ANP expression in protein level in high glucose-induced HK-2 human renal tubular epithelial cells.3.Immunofluorescence staining was used to detect Corin expression level in high glucose-induced HK-2 human renal tubular epithelial cells.4.Corin-si RNA HK-2 cells were constructed and real-time PCR was used to detect Corin silencing efficiency in Corin-si RNA HK-2 cells.5.Western blot further validated the Corin silencing efficiency of Corin-si RNA HK-2 cells.6.The proliferation of Corin-si RNA HK-2 cells was observed under optical microscope.7.MTT assay and trypan blue staining were used to detect the proliferation and viability of Corin-si RNA HK-2 cells.8.The endothelial cell scratching test detected the effect of supernatant of Corin-si RNA HK-2 cells on EA.hy926 endothelial cells.Results1.RT-PCR showed that in high glucose-induced HK-2 human renal tubular epithelial cells,Corin and ANP expression in m RNA level were decreased significantly compared with those in the Ctrl and OC groups.2.Western blot showed that in high glucose-induced HK-2 human renal tubular epithelial cells,Corin and ANP expression in protein level were decreased significantly compared with those in the Ctrl and OC groups.3.Immunofluorescence staining showed that in high glucose-induced HK-2 human renal tubular epithelial cells,Corin expression was reduced significantly compared with that in the Ctrl and OC groups(P < 0.05).4.Real-time PCR detected that the silencing efficiency of Corin-si RNA sequence 1 and sequence 2 in HK-2 cells was 45.45% and 44.18% respectively(P < 0.01).5.Western blot showed that in Corin-si RNA sequences 1 and sequence 2 HK-2 cells,Corin expression in protein level decreased significantly compared with NC-si RNA group.6.Optical microscope observed that Corin-siRNA HK-2 cells proliferation was obviously inhibited compared with NC-si RNA group.7.MTT assay showed that cell count of Corin-si RNA HK-2 cells decreased significantly compared with that of NC-si RNA group(P < 0.01).Trypan blue staining showed that cell viability decreased obviously in Corin-si RNA HK-2 cells in comparison to that in NC-si RNA group(P < 0.01).8.The endothelial cell scratching test detected that Corin-si RNA HK-2 cells inhibited repair after EA.hy926 endothelial cells damage.Conclusions1.Corin-siRNA HK-2 cells model was successfully constructed;2.Corin and ANP have protective effects on kidney;3.Corin deficiency in HK-2 cells affects the repair function of EA.hy926 endothelial cells after injury;4.Corin deficiency is involved in diabetic nephropathy endothelial dysfunction;5.Corin plays an important role in the pathogenesis of diabetic nephropathy.