- 學位論文
Sirt1在腎臟中生理及病理的角色
The Physiologic and Pathologic Roles of Sirt1 in the Kidney
摘要
Sirtuin 1(Sirt1)是一個依賴菸鹼醯胺腺嘌呤二核酸以進行作用的去乙醯基酶，已知與許多細胞活動相關。之前的研究顯示sirtuin 1 (Sirt1)具有腎臟保護作用，然而，它在腎臟內的分佈及功能仍未清楚。吾人證實Sirt1在大鼠腎臟主要表現於腎小管及間質細胞的細胞質，且與aquaporin-2有共同位置，顯示它可能參與鈉與水的調節。大鼠禁食24小時後，其Sirt1在腎臟非腎絲球部分的細胞質內表現上升，但在大量飲水(每公斤體重50毫升)或禁止飲水24小時後，腎臟的Sirt1表現量並無改變。給予低鹽食物(0.075% NaCl)或限制60%的熱量攝取7天後，腎臟的Sirt1表現明顯增加；而攝取高鹽食物(8% NaCl)後，腎臟的Sirt1表現無明顯改變。攝取低鹽食物也會使心臟、肌肉、腦部及脂肪組織內Sirt1表現增加。低鹽時大鼠腎臟的飢餓素(ghrelin)表現量也增加，且與Sirt1的分佈型式相似。以集尿管細胞株的體外實驗發現，ghrelin增加Sirt1表現是藉由活化其受體而達成。這個結果指出此”ghrelin-Sirt1系統”可能參與調節遠端腎元的對鈉的重吸收。 另一方面，吾人以單側輸尿管阻塞(unilateral ureteral obstruction, UUO)為模式，探討Sirt1在腎臟發炎和纖維化的角色和機轉。Sirt1在阻塞側腎臟的腎小管及間質細胞表現上升，但在對側腎臟則無變化。Resveratrol是一種Sirt1活化劑，可減少阻塞側腎臟的發炎和纖維化，而Sirt1抑制劑sirtinol使發炎更厲害。此結果顯示Sirt1可能可預防腎小管間質的纖維化。進一步發現，阻塞側腎臟的血管收縮素第一型受體（angiotensin type 1 receptor, AT1R）、核因子活化B細胞κ輕鏈增強子(NF-κB), 單核球趨化蛋白-1 (MCP-1)和纖維連接蛋白(fibronectin)的表現增加，而Resveratrol可減少這些分子的表現量，反之，sirtinol加強這些分子的表現。在腎臟纖維母細胞的體外實驗，過度表現Sirt1會減少AT1R和NF-κB的表現，而減少Sirt1表現量則出現相反結果。Sirtinol會增加AT1R、NF-κB、MCP-1和結締組織生長因子的表現，而resveratrol則減少AT1R的表現。吾人的結果顯示Sirt1在纖維母細胞中抑制AT1R和NF-κB的表現，而此機制可能於Sirt1減少UUO引起傷害的現象中扮演重要角色。
並列摘要
Sirtuin 1(Sirt1) is a nicotinamide adenine dinucleotide-dependent deacetylase known to be associated with many cellular activities. Previous studies have shown that Sirt1 is renoprotective; however, details regarding its distribution and functions in the kidney remain unknown. Our study demonstrated that Sirt1 was mainly expressed in the cytoplasm of tubulointerstitial cells in normal rat kidneys and was co-localized with aquaporin 2, indicating it may be involved in water/salt regulation. After a 24-h fast, renal Sirt1 expression increased in the non-glomerular cytoplasmic portion of the kidney. No significant changes in Sirt1 expression occurred after water loading (50 mL/kg) or 24-h water deprivation. After consuming a low-salt (0.075%) or 60% calorie restriction diet for 7 days, Sirt1 expression in the rat kidney was significantly increased, whereas a high-salt (8%) diet did not change the level of Sirt1 expression. The low-salt diet also increased Sirt1 expression in the heart, muscle, brain, and fat tissues. The increased Sirt1 that was observed in rats on a low-salt diet was associated with increased ghrelin expression in the distal nephron, with both molecules exhibiting similar distribution patterns. An in vitro experiment suggested that ghrelin increases Sirt1 expression in cortical collecting duct cells by activating ghrelin receptors. These results indicated that this ‘ghrelin-Sirt1 system’ may participate in regulating sodium reabsorption in the distal nephron. On the other hand, we explored the roles and mechanisms of Sirt1 on renal inflammation and fibrosis by using unilateral ureteral obstruction (UUO) rat model. Sirt1 expression increased significantly in the obstructed kidney but not in the contralateral kidney and was mainly detected in tubulointerstitial cells. Resveratrol, a Sirt1 activator, decreased UUO-induced inflammation and fibrosis, while sirtinol, a Sirt1 inhibitor, enhanced UUO-induced inflammation. These results suggest that Sirt1 may prevent renal tubulointerstitial fibrosis. In vivo, we evaluated the effects of activating or inhibiting Sirt1 on renal pathology and UUO pathogenesis mediators; in vitro, we evaluated the effects of regulating Sirt1 expression or activity on UUO pathogenesis mediators in renal fibroblasts. UUO increased renal angiotensin type 1 receptor (AT1R), NF-κB, monocyte chemotactic protein 1 (MCP-1), and fibronectin expression. Resveratrol attenuated these UUO-induced changes, whereas sirtinol enhanced them, with the exception of fibronectin. In renal fibroblasts, Sirt1 overexpression reduced AT1R and NF-κB levels, while Sirt1 knockdown had the opposite effects. Sirtinol increased the levels of AT1R, NF-κB, MCP-1, and connective tissue growth factor, while resveratrol reduced AT1R levels. Our results suggested that Sirt1 inhibited AT1R and NF-κB expression in renal fibroblasts and that these mechanisms may play roles in alleviating UUO-induced damages.