High Level Of Insulin Affect The Reabsorption Of Glucose And Albumin By Renal Tubular Epithelial Cells

Objective:Diabetic nephropathy(DN) is one of the characteristic of diabetic microvascular complications. The pathogenesis is still not clear and the pathology of renal lesions in type 2 diabetes is widely heterogeneity. Traditionally, DN has been thought of as a primarily glomerular disease, and microalbuminuria(MAU) is an early risk marker of nephropathy. Howerer early tubular injury has been reported in patients with diabetes mellitus whose glomerular function is intact, and the major clinical manifestation is renal tubular proteinuira and enzyme urine. It is now widely accepted and histological studies have confirmed that the deterioration of renal function in chronic kidney disease(CKD) and the decline of glomerular filtration rate(GFR) better correlates with tubular and interstitial changes than with glomerular changes. Therefore, the structural and functional changes of renal tubular, the mechanism and causes of renal tubular damage in diabetic nephropathy should be taken seriously. Our previous studies found that there was a different degree of renal tubular dysfunction in 78.2%of the patients with DM before the urine albumin increased and there was significant damage of renal tubular epithelial cells and interstitial microvascular endothelial cells in impaired glucose tolerance(IGT) in spontaneous type 2 diabetes OLETF rat, and the rate of urinary excretion of NAG, RBP, and NGAL is elevated, but there is no abnormal of the structure and biochemical indicators of glomerulus. Therefore, what causes the structural and functional damage of renal tubular in pre-diabietes, and what is the exact mechanism?Insulin resistence(IR)/hyperinsulinemia is an independent risk factor for patients with chronic kidney disease. There is hyperinsulinemia on the prophase and even after a long period of time of type 2 diabetes. Insulin resistance and the compensatory hyperinsulinemia are important pathophysiological changes of IGT. Therefore,whether hyperinsulinemia can affect the structure and function of renal tubule before hyperglycemia? Our study was based on renal tubule epithelial cells(NRK-52E)cultured in vitro, observed the changes of reabsorption of albumin and glucose by cells, and the changes of megalin, cubilin, SGLT2, GLUT2, SGLT1 and GLUT1 after being stimulated by insulin at different concentrations and different intervention time,and further observed the changes of insulin metabolism pathway, in order to explore how hyperinsulin effect on renal tubular epithelial cell and further influence its function.Methods:1. After being stimulated by insulin at different concentrations, the viability of NRK-52 E cells were determined by MTT methods, in order to determine the intervention concentration of insulin.2. Immunofluorescence method,western blot and fluorescence quantitative RT-PCR were used to assess the expression of megalin, cubilin, SGLT2, GLUT2, SGLT1 and GLUT1 in cells after being stimulated by insulin at different concentrations.3. Immunofluorescence method,western blot and fluorescence quantitative RT-PCR were used to assess the expression of the receptor proteins in cells after being stimulated by insulin at different intervention time.4. Laser scanning confocal microscope was used to observe the NRK-52 E cells intake TRITC-BSA and 2-NDBG.5. Fluorescence quantitative RT-PCR and western blot were used to detect the expression of IRS-1, PI3-K and Akt.6. We set up the control group, PI3-K inhibitor group and high intervention concentration of insulin group, to observe the NRK-52 E cells intake TRITC-BSA and 2-NDBG, and further to detect the receptor proteins in the three groups.Results:1. The intervention concentrations of insulin were 0ng/ml(the control group),5ng/ml(low concentration group), 10ng/ml(moderate concentration, physiological level), 50ng/ml(high concentration group).2. The maximal expression of megalin and cubilin appeared at the low concentration group, whereas decreases after that, and the minimal expression present to the high concentration group.The maximal expression of megalin and cubilin appeared at 6h after stimulated by insulin, whereas decreases after that,and the minimal expression appeared at 48 h.3. The maximal amount of NRK-52 E cells intake TRITC-BSA appeared at the low concentration group, whereas decreases after that.4. The expression of SGLT1 had no significant difference in different intervention concentrations of insulin; The expression of GLUT1 increased in the high concentration group; The expression of SGLT2 and GLUT2 increased with the increase of intervention concentrations of insulin.5. The amount of NRK-52 E cells intake 2-NDBG increased with the increase of intervention concentrations of insulin.6. The expression of IRS-1 and PI3-K decreased in high concentration group.Conclusion:1. In high concentration of insulin condition, the insulin metabolism pathway was restrained in NRK-52 E cell.2. High concentration of insulin inhibited the expression of pathway IRS-1/PI3-K/Akt, and decreased the expression of megalin and cubilin, and further inhibited the uptake of albumin by NRK-52 E cell.3. Insulin stimulated the expression of SGLT2 and GLUT2 and further promoted the uptake of glucose.4. High level of insulin affected the reabsorption of glucose by renal tubular epithelial cells is not through the insulin signaling pathway IRS-1/PI3-K/Akt, its mechanism remains to be further research.