組蛋白去乙醯基酶SIRT1在糖尿病腎病變的角色

Roles of Histone Deacetylase SIRT1 in Diabetic Nephropathy

梁家瑋(Chia-Wei Liang)

指導教授 ： 莊麗月

高雄醫學大學/醫學院/生物化學研究所/碩士(2010年)

https://doi.org/10.6832/KMU.2010.00132

摘要

糖尿病腎病變是糖尿病嚴重的併發症與死因之一，其致病機轉主要是當細胞處於高糖的環境之下，促進高度糖化終產物 (advanced glycation end products，AGE) 的形成，促進 protein kinase C 的活化及 reactive oxygen species (ROS) 產生，並活化乙型轉型生長因子 (TGF-β)而造成腎臟細胞肥大、細胞外間質堆積、增加上皮細胞轉變成間質細胞 (Epithelial-to-Mesenchymal Transition, EMT) 與細胞凋亡 (apoptosis) 的情形，最後導致腎臟的纖維化與末期腎病變。 Silent information regulator T1 (SIRT1) 是一種組蛋白去乙醯酶 (class III histone deacetylase) ，可將組蛋白去乙醯化而抑制基因表現，亦可使非組蛋白 (non histone ptoteins) 去乙醯化而改變其活性。 SIRT1之酵素活性會受到 NAD+/NADH 比率的調節而調控代謝相關基因表現。SIRT1的活化劑白藜蘆醇 (Resveratrol) 可以改善肥胖與糖尿病鼠的血糖與代謝狀態、抑制發炎反應而延長糖尿病鼠的壽命。因此本研究的目的在探討SIRT1在糖尿病腎病變的角色。利用高糖刺激正常大鼠腎臟纖維母細胞株NRK-49F與腎小管上皮細胞株NRK-52E，發現在近端腎小管 (NRK-52E) 細胞中，高糖會降低SIRT1蛋白質的表現,也會降低 SIRT1 promoter activity。外加 LY294002 (PI3K inhibitor) 與 SB431542 (TGF-β receptor Ι inhibitor ) 則可逆轉SIRT1 protein 在高糖刺激之下的表現。將 SIRT1 plasmid 轉染入細胞中使其過度表現並加入高糖刺激兩天能逆轉高糖所抑制的細胞增生，亦可逆轉高糖對細胞肥大的促進現象。SIRT1也會抑制 TGF-β promoter activity 與TGF-β bioactivity ，進而降低p21WAF1、p27kip1 蛋白質的表現。SIRT1亦可減弱高糖所促進的發炎相關分子COX-2與細胞EMT的指標蛋白MMP9和α-SMA增加的現象。此外，以Streptozotocin (STZ) 誘導的糖尿病大白鼠與單側輸尿管阻塞 (unilateral ureteral obstruction, UUO) 的腎臟纖維化模式動物實驗中，利用免疫組織化學染色法發現SIRT1表現量減少，p21WAF1、p27kip1、COX-2、E-cadherin、MMP9和α-SMA等蛋白質的表現量則增加。綜合以上結果，在NRK-52E細胞株中高糖會透過活化PI3-K訊息路徑降低SIRT1表現，致使SIRT1無法抑制其下游基因表現或將下游蛋白去乙醯化而修飾其活性，導致糖尿病腎病變之各種效應。

關鍵字

糖尿病腎病變 ； 高糖

並列摘要

Diabetic nephropathy (DN) is a common complication of diabetes mellitus that affects glomerular,tubular,and interstitial cells of the kidneys. Typical events that can be ascribed to the renal cells include accentuated activity of polyol and hexosamine pathways and formation of advanced glycation end products (AGE), which lead to an increased activity of protein kinase C and generation of reactive oxygen species (ROS). Both protein kinase C and ROS, directly or indirectly, are capable of initiating
TGF-β-Smad signaling pathway than induced cell hypertrophy, increased synthesis and accumulation of extracellular matrix, promoted epithelial-to-mesenchymal transition (EMT), and increased cell apoptosis. The final common pathways are renal fibrosis and end-stage renal disease (ESRD).Silent information regulator T1 (SIRT1) belongs to the class III histone deacetylase, it deacetylates histone proteins to silence gene expression. SIRT1 also deacetylates nonhistone proteins to change their function. SIRT1 deacetylase activity is controlled by the NAD+/NADH ratio to mediate gene expression in response to metabolic changes. Previous studies found that resveratrol (a SIRT1 activator) improves metabolic condition and insulin sensitivity with anti-inflammatory effects in the diabetic rats. Thus, we studied the roles of SIRT1 in diabetic nephropathy in vitro and in vivo. We found that high glucose suppressed SIRT1 protein expression and SIRT1 promoter activity in NRK-52E cells (normal rat kidney proximal tubular epithelial cells), but not in NRK-49F cells (normal rat kidney fibroblast). LY294002 (PI3K inhibitor) or SB431542 (TGF-β receptor Ι inhibitor) attenuated high glucose-inhibited SIRT1 protein expression in NRK-52E cells. Therefore, high glucose decreased SIRT1 protein expression via the PI3 kinase and TGF-β pathways. SIRT1 plasmids attenuated high glucose-inhibited cell proliferation and high glucose-induced cell hypertrophy and EMT. SIRT1 plasmids also attenuated high glucose-induced TGF-β promoter activity and bioactivity and attenuated high glucose-induced p21WAF1 and p27kip1 protein expression. In addition, SIRT1 plasmids attenuated high glucose-induced acetylation of p53 and NF-κB and attenuated high glucose-induced COX-2 protein expression. SIRT1 protein was decreased in the kidneys of streptozotocin (STZ)-diabetic rats and unilateral ureteral obstruction rats. In conclusion, SIRT1 plays important roles in the pathogenesis of diabetic nephropathy in vitro and in vivo.

並列關鍵字

Diabetic Nephropathy ； SIRT1

參考文獻

Alcendor, R.R., Gao, S., Zhai, P., Zablocki, D., Holle, E., Yu, X., Tian, B., Wagner, T., Vatner, S.F., and Sadoshima, J. (2007). Sirt1 Regulates Aging and Resistance to Oxidative Stress in the Heart. Circ Res 100, 1512-1521.

Baricos, W.H., Cortez, S.L., El-Dahr, S.S., and Schnaper, H.W. (1995). ECM degradation by cultured human mesangial cells is mediated by a PA//plasmin//MMP-2 cascade. Kidney Int 47, 1039-1047.

Baur, J.A., Pearson, K.J., Price, N.L., Jamieson, H.A., Lerin, C., Kalra, A., Prabhu, V.V., Allard, J.S., Lopez-Lluch, G., Lewis, K., Pistell, P.J., Poosala, S, Becker, K.G., Boss, O., Gwinn, D., Wang, M., Ramaswamy S., Fishbein K.W., Spencer, R.G., Lakatta, E.G., Le Couteur, D., Shaw, R.J., Navas, P., Puigserver, P., Ingram, D.K., de Cabo R., Sinclair, D.A. (2006). Resveratrol improves health and survival of mice on a high-calorie diet. Nature 444, 337-342.

Border, W.A., and Noble, N.A. (1994). Transforming Growth Factor {beta} in Tissue Fibrosis. N Engl J Med 331, 1286-1292.

Brodin, G., ten Dijke, P., Funa, K., Heldin, C.-H., and Landström, M. (1999). Increased Smad Expression and Activation Are Associated with Apoptosis in Normal and Malignant Prostate after Castration. Cancer Research 59, 2731-2738.