The Effect Of Resveratrol On The Expression Of FoxO1 In Kidneys Of Diabetic Rats And The Mechanism

Background & ObjectiveDiabetes is increasing in an epidemic proportion throughout the world and diabetic nephropathy, one of microvascular complications of diabetes, is the leading cause of end-stage renal disease (ESRD). Numerous factors contribute to the pathogenesis and progression of DN. Experimental and clinical evidence indicates that excessive oxidative stress may contribute to the initiation and development of diabetic nephropathy. Transcription factor FoxO family members and FoxO1 in particular play important roles in oxidative stress resistance, which increases its antioxidant target genes (CAT) to reduce oxidative stress.SIRT1 is a mediator of resveratrol action in FoxO1-mediated gene expression, and the dephosphorylated form of FoxO1, which is distributed in the nucleus, is deacetylated by SIRT1. Resveratrol is known to be an activator of sirtuinl (SIRT1). To investigate the effect of FoxO1 on oxidant stress in streptozotocin-induced diabetic rats, we sought to determine the effect of resveratrol on FoxO1. We determined the relationship of FoxO1 and Sirt1 activation to oxidative stress in kidney of DN. Materials & MethodsMale Sprague-Dawley rats (220±20 g) were obtained from Experimental Animal Center of Henan Province. Diabetes was induced by a single intraperitoneal injection of 60 mg/kg STZ (dissolved in 0.05M citrate buffer, pH 4.5) after fasting for 12 h. Blood glucose was measured to confirm the development of diabetes 3 days after STZ injection. Animals in the diabetic group were excluded from the study if plasma glucose was less than 16.7mM. Rats that received an injection of diluent buffer alone served as control. After developed diabetes, rats were divided into two groups: diabetic rats without treatment and diabetic rats treated with resveratrol. At the end of 12-week study,24-hour urine from each rat was collected for 24 hours urinary protein (24 hUPro) and urine albumin (UAIb) examination,then the rats were anesthetized with pentobarbital sodium (50 mg/kg body wt) and blood was collected from abdominal vein for Blood glucose (BG), blood urea nitrogen (BUN) and serum creatinine (Scr) detection. Kidney weight/body weight ratio (KI) of rats were measured and calculated. The right kidney from each rat was fixed in 4% paraformaldehyde and embedded in paraffin for immunohistochemistry and HE stain. The renal cortex of the left kidney from each rat was cut into small pieces (1 mm3) for electron microscope examination and the left cortical tissues from rats kidneys were removed and stored at-70℃for further study. The content of malondiadehyde (MDA) and the activity of superoxide dismutas (SOD) was detected using spectrophotometer.. Expressions of collagen IV and fibronectin in glomeruli were detected by immunohistochemical staining. The expressions of silent information regulator 1 (Sirt1), Foxol and catalase (CAT) mRNA were respectively detected using RT-RCR. The expressions of FoxO1 protein and FoxO1 phosphorylation level were detected using Western blot.Results1. Compared with normal group, BG (28.2±2.7) mmol/L. KI (8.1±0.8)×10-3,UPro/24h (26.8±4.1) mg/24h,UAIb (0.89±0.13) mg/24h,Scr (73.5±10.9)μmol/ L and BUN (15.3±1.4) mmol/L levels were elevated significantly (all P< 0.05), and body weight (250±27) g was decreased in diabetic rats without treatment (P< 0.05). We also observed that KI (5.2±0.5)×10-3,Upro/24h (16.9±2.6) mg/24h, UAIb (0.65±0.11) mg/24h,Scr (56.7±5.9)μmol/Land BUN (11.3±1.1) mmol/L levels were decrease significantly in diabetic rats treated with resveratrol (RSV) (all P< 0.05). But there were no significant differences in BW and BG levels between diabetic rats treated with resveratrol [(271±31) g and (27.8±3.3) mmol/L, respectively] and without treatment (P>0.05).2. Compared with normal group[(1.35±0.29) nmol/mg prot and (65.74±8.71) U/mg prot, respectively], level of MDA generation (3.47±0.24) nmol/mg prot was increased markedly and the activity of SOD (23.51±7.79) U/mg prot was decreased in group of diabetic rats without treatment (all P< 0.05). Similarly, after treated with resveratrol for 12 weeks, level of MDA generation (2.48±0.27) nmol/mg prot was significantly decreased and the activity of SOD (43.31±5.57) U/mg prot in diabetic rats treated with resveratrol (all P< 0.05).3. Compared with normal group (1.09±0.11 and 1.81±0.44, respectively) the expressions of fibronectin (10.65±1.27) and collagen IV (20.11±1.28) in glomeruli were significantly increased in group of diabetic rats without treatment (all P< 0.05). Similarly, after treated with resveratrol for 12 weeks, the expressions of collagenⅣand fibronectin (6.26±1.04 and 10.08±1.01, respectively) in glomeruli were significantly decreased in diabetic rats treated with resveratrol (all P< 0.05).4. The expressions of Sirtl (0.63±0.04), FoxO1 (0.37±0.03) and CAT (0.22±0.03) mRNA in kidneys of diabetic rats without treatment were decreased compared with normal group (0.96±0.08,0.69±0.05 and 0.48±0.04, respectively; all P<0.05).After treated with resveratrol for 12 weeks, the relative amounts of mRNA (0.83±0.05, 0.53±0.04 and 0.40±0.03, respectively; all P<0.05) were higher in diabetic rat treated with resveratrol compared to the diabetic rats without treatment (all P< 0.05).5. Western blot showed that there was no difference FoxO1 protein expression among groups (0.45±0.09,0.48±0.08 and 0.47±0.09, respectively; all P>0.05), but FoxO1 phosphorylation level in group of diabetic rats without treatment (2.15±0.08) was highter than those of normal group (1.14±0.08, P< 0.05), and FoxO1 phosphorylation level in group of diabetic rats treated with resveratrol (1.72±0.07) was lower than those of diabetic rats without treatment (P< 0.05).Conclusions1. The expression of FoxO1 mRNA was decreased, and FoxO1 phosphorylation level was increased in kidney of diabetic rats.The role in defensing against oxidative stress of FoxO1 was decreased in kidney of diabetic rats.2. Resveratrol significantly increased the FoxOl mRNA and decreased FoxO1 phosphorylation levels in kidneys of diabetic rats treated with resveratrol.3. Sirtl activated by resveratrol may increase the expression of FoxO1, which increases its antioxidant target genes (CAT) to reduce ROS level in the kidneys.4. Alterations of the Sirtl/FoxO1 pathway may be considered a potential therapeutic target for DN.