Protective Effect Of Resveratrol On Diabetic Nephropathy And Its Mechanism

Background In China, the prevalence and incidence of diabetic nephropathy(DN) have both increased dramatically over the past decade. It is now the second etiology of end-stage renal diseases(ESRD) in our country, and thus has a great threaten on the people's health. Currently, however, the therapeutics of DN is far beyond perfect. There is still a great number of DN patients progressing into ESRD, bringing heavy burden on the medical care of the country. Hence, the study of the pathogenesis and pathophysiology of DN, and thus better management have become extremely urgent. Nowadays, there is a growing body of literatures showing that, vascular endothelial growth factor(VEGF) and its type 2 receptor Flk-1(VEGF-Flk-1) system plays an important role in the pathogenesis of DN. In animal studies, antagonists of VEGF-Flk-1 system could ameliorate DN significantly. Thus, antagonists of VEGF-Flk-1 system might bring new strategy and better therapeutics to the management of DN. On the other hand, however, there are some serious adverse effects in the current antagonists of VEGF-Flk-1 system, thus making them difficult to be used in clinical practices. As a result, how to develop new antagonists of VEGF-Flk-1 system that have few adverse effects, has become extremely important right now. As a natural plant extract, resveratrol has little side effect, and has been used extensively in human health care. There are also many studies showing that resveratrol has great anti-cancer effect, partially due to its anti-VEGF potential. Besides, resveratrol has also been evidenced to have various protective effects in diabetes and its complications through the activation of silent information regulator 1(SIRT1). In addition, SIRT1 has also been shown to have significant inhibition on the expression of VEGF. Hence, we investigated whether resveratrol could suppress the VEGF-Flk-1 system via activation of SIRT1, and thus attenuate DN.Methods 1. Animal study:One week after uninephrectomized, the Sprague Dawley rats were randomly separated them with control group, DN group, and DN plus resveratrol group, respectively. Eight weeks later, urine and kidney samples were collected for the following studies. (1)Measure the body weight, blood glucose, urine albumin excretion rate, creatinine clearance rate, and kidney weight of each group of the rats; Periodic acid Schiff stain was used to determine the diameters of the rats glomeruli and glomerular matrix index, while the thickness of glomerular basement membrane was examined by transmission electronic microscopy; In addition, immunohistochemistry and immnoblot were used to investigate the renal fibrosis of the rats, by measuring the expression levels of typeⅣcollagen, fibronectin, transforming growth factor, connective tissue growth factor-β1, and plasminogen activator inhibitor-1, respectively. (2)Using immunohistochemistry to detect the location of SIRT1 expression in the rats glomeruli; And then immunoblot was used to examine the levels of VEGF, Flk-1, Tie-2 and Angiopoietin-1 expression in each group of the rats kidneys. The Angiopoietin-2 mRNA transcription levels in each group were determined by realtime PCR.2. In vitro study:(1)In the cultured mouse glomerular podocytes, cells were treated with normal glucose, high glucose, and high glucose plus resveratrol, respectively, and then immunoblot, realtime PCR, and enzyme-linked immunosorbent assay were used to measure the expression and secretion of VEGF in each group of the treated cells; In addition, RNA interference using lentivirus as a vector was proceeded to investigate to the possible mediation SIRT1 may involve in. (2) In the cultured mouse glomerular endothelial cells, cells were treated with normal glucose, high glucose, and high glucose plus resveratrol, respectively, and then immunoblot and realtime PCR were usedto measure the expression Flk-1 in each group of the treated cells; In addition, RNA interference using lentivirus as a vector and transfection with over-expressing plasmid were proceeded to investigate to the possible mediation SIRT1 may involve in. (3)In the cultured glomerular endothelial cells, cells were treated with vehicle, VEGF, and VEGF plus resveratrol, respectively, and then immunofluorescence was used to examine the cellular junction of the treated endothelial cells; In addition, an in vitro vascular permeability assay kit was used to determine the permeability of each group of the treated endothelial cells.Results 1. Animal study:(1)No significance was noted in the levels of body weight and blood glucose of the DN rats with and without resveratrol treatment, while both were decreased and increased respectively as compared with the control rats; The urine albumin excretion rate, creatinine clearance rate, kidney weight, glomeurlar diameter, matrix index, and the thickness of glomerular basement membrane were all increased in the DN rats as compared with control rats, while administration with resveratrol reduced these increases in DN rats; In renal fibrosis, the expression levels of typeⅣcollagen, fibronectin, transforming growth factor, connective tissue growth factor-β1, and plasminogen activator inhibitor-1 were all significantly increased in DN rats, while treatment with resveratrol reduced these increases in DN rats. (2) In the rats glomeruli, SIRT1 was expressed in the nuclear of podocytes, mesangial cells, and endothelial cells. The levels of VEGF and Flk-1 expression were increased in DN rats, while administration of resveratrol reduced their increases in DN. However, the expression level of Tie-2 was reduced in DN rats, while treatment of resveratrol increased it. No significance was found in the expression level of Ang-1 between these groups. The Ang-2 mRNA level was significantly increased in DN rats as compared with control group, while treatment with resveratrol attenuated this increase.2. In vitro study:(1)In the cultured glomerular podocytes, resveratrol could significantly inhibit the high glucose induced increased VEGF expression and secretion, in both dose and time dependent manners. However, the resveratrol's inhibition of VEGF expression was significantly reduced after downregulation of SIRT1 expression in podocytes. (2) In the cultured glomerular endothelial cells, resveratrol could significantly inhibit the high glucose induced increased Flk-1 expression, in both dose and time dependent manners. However, the resveratrol's inhibition of Flk-1 expression was significantly reduced after downregulation of SIRT1 expression in endothelial cells, while over-expression of SIRT1 could remarkably suppress the endothelial cells Flk-1 expression. (3)In the cultured glomerular endothelial cells, resveratrol could apparently protect the VEGF-induced disruption of the cellular junction. In addition, resveratrol could also significantly reduce VEGF-induced endothelial cells hyperpermeability.Conclusion Resveratrol could inhibit the VEGF-Flk-1 system via activating SIRT1, thus attenuate diabetic nephropathy.