The Effects Of High Glucose And Valsartan On The Cytokines Expression In The Rat Isolated Perfused Kidney And Mesangial Cells

Background:Diabetic nephropathy (DN) is one of serious complications of diabetes and the main cause of leading the end stage renal disease and characterized by an expansion of the glomerular mesangium, proteinuria and progressive renal insufficiency, caused by mesangial cell proliferation and an excessive accumulation of extracellar matrix (ECM) proteins and tubular basement membranes, which ultimately progress to glomerulosclerosis and tubulo-interstitial fibrosis. And high blood glucose has deleterious effects on kidney cells. High glucose (HG) activates various pathways via similar mechanisms in different types of the kidney cells and stimulates some cytokines production. Simultaneously, impressive series of investigations, both in vitro and in vivo, have suggested that three particular growth factors, that are transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), contribute to the pathophysiological process leading to the development of DN and glomerulosclerosis. AngⅡis the main effect molecule of the renin-angiotensin system (RAS), and plays an important role in the high glucose-induced renal damage. As AT1 receptor antagonist, valsartan has become the first-line drug in the treatment of DN. Therefore, in this study, we investigated whether high glucose and valsartan would influence the expression of TGF-β1, VEGF and PDGF-BB using IPK and GMCs, and sought possible relationships between TGF-β1, VEGF and PDGF-BB protein expression in the early period within a hyperglycemic environment, in order to provide a theoretical basis for the occurrence and development of the prevention and treatment of DN. Experimental Methods:The isolated perfused kidney and mesangial cells were used in this study as models, in order to observe how high glucose (30.0mmol/L) and valsartan (10-6mol/L) influenced the expression of TGF-β1, VEGF and PDGF-BB at 0.5h, 1h,2h,4h and 6h.Part one The effects of high glucose and valsartan on the cytokines expression in the rat isolated perfused kidney:The isolated rat kidney was perfused continuously in vitro at 37℃, where perfusate was divided into control group (glucose 10.0mmol/L), high glucose group (glucose 30.0mmol/L) and valsartan group (glucose 30.0mmol/L+valsartan 10-6mol/L). Perfusion pressure was stabilized at 80mmHg throughout the process, when irrigation flow rate was about 10ml/min. The perfused kidney was taken, of which urine flow was greater than 0.04 ml/min. The urine was collected and recorded at 0.5h, 1h,2h,4h and 6h after 20min perfusion.1. The effects of high glucose and valsartan on the kidney function After perfusion, the kidneys were taken for routine paraffin and HE staining to observe the renal kidney morphology of paraffin sections. The determination of urine samples were carried out according to instructions of the appropriate kits.2. The effects of high glucose and valsartan on the cytokines expression in IPK: The effects of high glucose and valsartan on the expression of TGF-β1, VEGF and PDGF-BB were determinated by immunohistochemical staining.Part two The effect of high glucose and valsartan on the cytokines expression in the glomerular mesangial cells:The mesangial cells of logarithmic growth phase were inoculated into 24-well plates by 1×104/ml/hole. The culture plate cover slip was put into the hole, lysine-coated, after the cells seeded. After serum-free cultured for 24h, the cells were synchronized in the GO period, and then divided into:control group, high glucose group and valsartan group. After cultured for 0.5h, 1h,2h,4h and 6h, the effects of high glucose and valsartan on the expression of TGF-β1, VEGF and PDGF-BB were determinated by immunocytochemical staining. The effects of high glucose and valsartan on the secretion of TGF-β1 were determinated by ELISA. The effects of high glucose and valsartan on the mRNA expression of TGF-β1, VEGF and PDGF-BB were determinated by RT-PCR.Results:Part one:The effects of high glucose and valsartan on the cytokines expression in the rat isolated perfused kidney1. The effects of high glucose and valsartan on the kidney function1.1 kidney morphology:In high glucose group, the renal morphology was more seriously damaged than the control group, and the damage was aggravated with the extension of perfusion time; in valsartan group, the kidney damage was improved compared with high glucose group.1.2 Detection of renal function parameters:In the control group, urine output, urine creatinine, LDH and kalium ion concentration reduced with the extension of perfusion time; in high glucose group, urine output, urine creatinine, LDH and kalium reduced more obviously compared with the control group, which were improved significantly in valsartan group.2. The effects of high glucose and valsartan on the cytokines expression in IPK2.1 The effects of high glucose and valsartan on the expression of TGF-β1 in IPK: Immunohistochemistry results showed that high glucose can stimulate the protein expression of TGF-β1 in rat IPK. In glucose group, TGF-β1 protein expression had decreased at 4h and recovered at 6h. And TGF-β1 protein expression was inhibited after the intervention of valsartan.2.2 The effects of high glucose and valsartan on the expression of VEGF in IPK: Immunohistochemistry results showed that high glucose could stimulate the protein expression of VEGF in rat IPK, and the expression was significantly higher than control group within the beginning 2h, which decreased at 4h and 6h. And VEGF protein expression was suppressed after the intervention of valsartan.2.3 The effects of high glucose and valsartan on the expression of PDGF-BB in IPK:Immunohistochemistry results showed that in the control group, the expression level of PDGF-BB decreased with the extension of perfusion time; high glucose could improve the protein expression of PDGF-BB in rat IPK, and the expression level had a pick-up at 6h; PDGF-BB protein expression was suppressed after the intervention of valsartan.Part two:The effect of high glucose and valsartan on the cytokines expression in the glomerular mesangial cells1. The effects of high glucose and valsartan on the protein expression of cytokines in GMCs1.1 The effect of high glucose and valsartan on the protein expression of TGF-β1 in GMCs:immunocytochemical results showed that TGF-β1 expression increased with the extension of the culture time in the normal mesangial cells; high glucose could promote TGF-β1 protein expression, and TGF-β1 expression levels decreased at 2h, which had a pick-up at 6h; TGF-β1 protein expression was inhibited after the intervention of valsartan. ELISA results showed that high glucose stimulated the secretion of TGF-β1 of mesangial cells, which was reduced and lower than the control group at 6h; the secretion of TGF-β1 of mesangial cells was significantly reduced after the intervention of valsartan, especially at 2h and 4h.1.2 The effect of high glucose and valsartan on the protein expression of VEGF in GMCs:immunocytochemical results showed that VEGF expression increased with the extension of the culture time in the normal mesangial cells; The protein expression of VEGF upgraded in the first 2h and decreased at 4h, which was higher than control group; VEGF protein expression was inhibited after the intervention of valsartan and lower than the high glucose group, with statistical significance.1.3 The effects of high glucose and valsartan on the protein expression of PDGF-BB in GMCs:immunocytochemical results showed that PDGF-BB expression increased with the extension of the culture time in the normal mesangial cells; high glucose could promote PDGF-BB protein expression, which was slightly higher than control group; and PDGF-BB protein expression was inhibited after the intervention of valsartan2. The effects of high glucose and valsartan on the mRNA expression of cytokines in GMCs:RT-PCR results showed that high glucose can promote TGF-β1, VEGF and PDGF-BB mRNA expression, which could be inhibited by valsartan. In the training start, TGF-β1 mRNA expression was higher than VEGF and PDGF-BB mRNA expression, but as the culture time, VEGF and PDGF-BB mRNA expression gradually strengthened, while the expression of TGF-β1 mRNA has decreased.Conclusion:High glucose could damage the renal function in isolated perfused kidney, in whch results in hypourocrinia, decreasing creatinine clearance rate and the activity of LDH. Valsartan could ameliorate the renal function damaged by high glucose in isolated perfused kidney. High glucose could promote the expression of TGF-β1, VEGF and PDGF-BB in both of isolated perfused kidney and cultured rat glomerular mesangial cells. The expression of TGF-β1 precede that of VEGF and PDGF-BB. Valsartan could inhibite the expression of TGF-β1, VEGF and PDGF-BB induced by high glucose, and reduce the content of TGF-β1 in MCs'enchylema. The effects of high glucose on the expression of TGF-β1, VEGF and PDGF-BB were investigated in this research using rat isolated perfused kidney model and cultured mesangial cells, and the results would be helpful for the prevention and therapy of diabetic nephropathy.