Protective Effect Of Sirt1 On 5/6 Nephrectomized Rats And Its Mechanism

PART IResveratrol, an Activator of Sirtl, Ameliorates Renal Damage in 5/6 Nephrectomized Rats Via the Smad PathwayObjective:Fibrosis plays an important role in the pathogenesis and progression of CKD. Resveratrol (RSV) is a polyphenol with antifibrotic properties. It has also been shown to activate Sirtl which has health beneficial effect. In the present study, we investigated the effect of RSV on a 5/6 nephrectomized CKD rat model and TGF-β/Smad signaling pathway.Methods:Fifty male SD rats were randomly divided into three groups:sham operated, 5/6th nephrectomy+vehicle, and 5/6th nephrectomy +RSV 20mg/kg. RSV or vehicle was administered one week after 5/6 nephrectomy surgery. Proteinuria and rat tail artery blood pressure were detected every four weeks. Serum creatinine and blood urea nitrogen were measured after 12 weeks. PAS and MASSON staining were used to observe the renal pathological changes. Immunohistochemisty staining of TGF-β1, CTGF, Fibronectin, Collagen-Ⅰ and a-SMA were used to analysis the changes of renal fibrosis. Western blot and Realtime PCR were used to measure the expression of TGF-β1, Fibronectin, Collagen-Ⅰ, Smad3 and phospho-Smad3 levels. Co-immunoprecipitation was used to detect the interaction between Sirtl and acetylation of Smad3.Results:(1) Compared with the sham operated rats, Subtotal nephrectomy significantly increased proteinuria (152.14±30.48mg/day vs 25.34±7.54 mg/day), blood urea nitrogen (16.75±3.99mmol/L vs 8.03±1.2mmol/L) and serum creatintine (111.6±21.5μmol/L vs 53.9±11.6μmol/L) at 12 weeks. RSV treatment significantly reduced urinary protein excretion (79.87±34.27mg/day), blood urea nitrogen (11.95± 1.88mmol/L) and serum creatinine levels(83± 14.69 μmol/L).5/6th nephrectomy significantly increased tail artery blood pressure at 12 weeks, which were reduced by RSV treatment. (P<0.05). (2) The glomerular sclerosis index (1.56±0.34 vs 0.35±0.08) and tubulointerstitial fibrosis (1.47 ± 0.29 vs 0.18 ± 0.04) were increased in nephrectomized rats, which were significantly reduced by RSV treatment. (3) The expression of TGF-β1, CTGF, Fibronectin, Collagen-Ⅰ and α-SMA measured by immunohistochemisty, Western-blot and Realtime PCR were increased in nephrectomized rats, which were significantly reduced by RSV treatment. (4) Compared with the sham operated rats, Subtotal nephrectomy increased the expression of phospholylation of Smad3, but RSV treatment did not significantly improve the elevated phosphorylation of Smad3. Further study shows that treatment of nephrectomized rats with RSV significantly reduced acetylation levels of Smad3. Immunoprecipitation studies revealed a binding of acetylated Smad3 with silent information regulator 1(Sirt1).Conclusions:RSV attenuates proteinuria, renal pathology and renal fibrosis in 5/6th nephrectomized rats. The renal protective effect is associated with reduced Smad acetylation and TGF-β signaling. These findings indicate that Sirtl may be a potential therapeutic target for the treatment of CKD.PART ⅡProtective Effect of Sirtl on TGF-β Induced Murine Mesangial Cells Via the Smad PathwayObjective:To investigate the protective effect of Sirtl on TGF-β induced murine mesangial cells (MMC) and the Smad pathway.Methods:(1) Cultured MMC were treated with TGF-β (0,1,2,5ng/ml or 0,6,12, 24h) to detect the expression of fibronectin and collagen I by immunoblot and Realtime PCR. MMC were also treated with TGF-β(2ng/ml) and RSV (10,25,50μM) for 24h to detect the expression of fibronectin and collagen I.(2) MMC were treated with TGF-p and RSV for 24-48h after transfected with RNA interference or overexpression plasmid to detect the expression of Sirtl, fibronectin and collagen I by immunoblot.(3) Cultured MMC were treated with TGF-β (0,1,2ng/ml) and RSV (10,25,50μM) for 3h to detect the expression of Smad3, phospho-Smad3 and acetylation of Smad3 levels by immunoblot. Co-immunoprecipitation was used to detect the interaction between Sirtl and Smad3.(4) MMC were treated with TGF-p for 3h after transfected with lentivirus using a RNA interference to detect the expression of Sirtl. Immunoprecipitation studies were used to detect the interaction between Sirtl and Smad3.(5) MMC were transiently transfected with lentivirus using a RNA interference, pRL-TK plasmid and Smad3/4-responsive promoter p(CAGA)12-Luc. After transfection and serum starving for 16h, TGF-β (2ng/ml) and RSV were added to the cells for 24 hours. The luciferase activities of p(CAGA)12 were analyzed by luciferase reporter gene assay kit according to the manufacturer’s instruction.Results:(1) TGF-β significantly up-regulated fibronectin and type I collagen expression in a dose- and time-dependent manner by immunoblot and Realtime PCR in cultured MMC. RSV treatment significantly down-regulated TGF-P induced fibronectin and type I collagen expression in a dose-dependent manner.(2) Knocking-down Sirtl using a RNAi markedly attenuated these effects of RSV, supporting that Sirtl mediates the effect of RSV. In contrast, forced up-regulation of Sirtl in cultured mesangial cells significantly reduced fibronectin and type I collagen expression.(3) In cultured MMC, RSV treatment significantly down-regulated TGF-β induced acetylation levels of Smad3 and increased the combination between Sirtl and Smad3 by Co-immunoprecipitation. RSV treatment did not significantly affect TGF-β induced phosphorylation levels of Smad3.(4) In cultured mesangial cells, down-regulation of Sirtl using a RNAi increased TGF-β induced acetylation levels of Smad3, consistent with deacetylation effect of Sirt1.(5) In cultured MMC, RSV treatment significantly down-regulated TGF-β induced the Smad3-responsive promoter activity. Knocking-down Sirtl using a RNAi markedly attenuated the effect of RSV.Conclusions:RSV significantly down-regulated TGF-β induced fibronectin and type Ⅰ collagen expression and Smad3-responsive promoter activity. Knocking-down Sirtl markedly attenuated the effect of RSV. The protective effect was associated with activation of Sirt1 and reduced Smad3 acetylation.PART ⅢThe tolerance of heterozygous Sirtl-knockout mice (Sirtl+/") and wild-type mice (Sirtl+/+) on 5/6th nephrectomyObjective:To investigated the effect of 5/6 nephrectomized Sirtl+/- and Sirt1+/+ mice on proteinuria, renal function and renal pathology.Methods:Male mice between 8 and 10 weeks of age were divided into four groups: Sirt1+/+ -sham operated (WT-Sham), Sirtl+/- -sham operated (KO-Sham), Sirtl+/+-5/6th nephrectomy (WT-Nx), and Sirtl+/- -5/6th nephrectomy (KO-Nx). Proteinuria was detected after every four weeks. Blood urea nitrogen was measured after 12 weeks. PAS and MASSON staining were used to observe the renal pathological changes. Immunohistochemisty staining of TGF-β 1, collagen Ⅰ and a-SMA were used to analysis the changes of renal fibrosis. Western blot was used to measure the expression of fibronectin, collagen Ⅰ and Sirt1.Results:(1) Compared with the WT-Sham operated, wild-type subtotal nephrectomy significantly increased proteinuria (8.88±1.16mg/day vs 2.34±0.37 mg/day) and blood urea nitrogen (36.85±1.83mg/dl vs 20.67±1.15 mg/dl) at 12 weeks. Proteinuria (13.06± 1.24mg/day) and blood urea nitrogen (44.03±2.06 mg/dl) level of KO-Nx group increased more significantly than that of WT-Nx group.(2) Compared with the WT-Sham operated, wild-type subtotal nephrectomy significantly increased glomerulosclerosis and tubulointerstitial fibrosis. The renal fibrosis of KO-Nx group was even heavier than that of WT-Sham group.(3) Compared with the WT-Sham operated, the expression of TGF-P, Fibronectin and Collagen-Ⅰ by immunohistochemisty and Western-blot were increased in WT-Nx gorup, which were more significantly increased in KO-Nx group.Conclusions:Genetic ablation of one allele of Sirtl (Sirtl+/-) induced much worse renal damage than wild type (Sirtl+/+) mice after 5/6th nephrectomy, supporting that down-regulation of Sirtl aggravates renal injury in 5/6 nephrectomized mice.