動靜脈廔管及腎絲球腎炎之治療策略探討

Abstract

日益增加的末期腎病人大大地增加腎臟取代治療的費用與公共衛生的負擔。如何減少末期腎病的發生，及提升腎臟取代病患的照護品質是面對這一問題的關鍵。本論文針對血液透析病患動靜脈廔管阻塞問題及腎絲球腎炎治療等臨床困境進行研究。自體動靜脈廔管因為感染率較低、生活品質較佳與整體醫療花費較少等因素，是長期血液透析病患建立血管通路的第一選擇，也是使用最廣泛的。然而，自體動靜脈廔管的阻塞率高，且目前仍缺乏有效的藥物或方法來預防阻塞改善通暢率，因此，血液透析病患自體動靜脈廔管功能喪失仍是臨床上未解決的困境。腎絲球腎炎則是導致末期腎病的重要病因之一，腎絲球腎炎的治療目前僅有一些效果有限的免疫調節治療，許多慢性腎絲球腎炎病患最終仍發展至末期腎病卻苦無有效治療，在論文中透過臨床大資料分析探討藥物史塔汀對自體動靜脈廔管阻塞的影響，以及以間質幹細胞治療腎絲球腎炎的大鼠模式，評估其療效與治療機轉，並針對抗氧化機轉深入研究，期許成果能對此醫學難題提供進一步的治療基礎。 動靜脈廔管阻塞的疾病生理特徵是內皮細胞功能失調、血管平滑肌增生與移轉，而史塔汀藥物具有抗發炎、改善內皮細胞功能的特性，具有潛在改善動靜脈廔管阻塞的病理機轉，是預防靜脈廔管阻塞的選擇藥物之一。史塔汀藥物對動靜脈廔管的保護作用已在動物實驗中多次被證實，然而，人類的臨床研究卻無一致性的結論。本論文利用全國性的血液透析世代研究資料庫來分析處方史塔汀類藥物與血液透析病患血管通路通暢度的關係。總共9862對依年齡、性別配對的史塔汀使用者與非使用者，自2000-2008年間75404位新增加的血液透析病患中選出。史塔汀對於自體動靜脈廔管阻塞的影響利用Cox proportional hazards 模式分析。與非使用者相比，史塔汀使用降低18%動靜脈廔管需要進行血管整型術或重建的風險(adjusted hazard ratio=0.82 [95%CI, 0.78-0.87])，減少21%重建風險(風險比=0.79 [95%CI, 0.69-0.80])，這種保護的效果只在自體動靜脈廔管有顯著意義(風險比=0.78[95% CI, 0.73-0.82])，而對使用人工血管的動靜脈廔管無保護效果(風險比=1.10 [95% CI, 0.98-1.24])。本研究的結果顯示在血液透析病患建立動靜脈廔管後使用史塔汀具有保護血管通路通暢的效果。這種效果在不同史塔汀中都出現，而其保護效力與史塔汀的效力強弱有關。使用史塔汀能降低血液透析病患血管通路重建風險，進而降低醫療花費與提昇病患生活品質，這分析的結果可提供將來臨床治療動脈廔管的重要參考依據。 腎絲球腎炎的疾病生理特徵是免疫相關的發炎現象，導致腎小球内繫膜细胞增生與受損，而幹細胞證實具備自我修復與免疫調節的特性提供的腎絲球腎炎符合病理機轉的治療選擇。相較於幹細胞的抗發炎與抗纖維化作用被廣泛探討，氧化壓力的傷害也是腎絲球腎炎重要損傷來源，抗氧化作用雖是幹細胞重要潛在治療機轉，卻是很少研究詳細地探索其作用，本論文透過使用一般濃度氧氣及缺氧狀態培養之間質幹細胞治療anti-Thy1.1引發大鼠腎炎的模式，以尿蛋白、組織化學染色與西方墨點法等方法來確認幹細胞的治療效果與分析其抗氧化的機制。結果發現經腎動脈注射間質幹細胞能緩解腎炎大鼠的蛋白尿、降低腎絲球硬化程度，低氧環境培養可以強化此療效。無論一般濃度氧氣或缺氧狀態培養之間質幹細胞均顯示一致性的減少巨噬細胞/單核球浸潤、壓力指標蛋白表現、自噬作用與凋亡指標蛋白表現，在抗氧化壓力的部分，則能提昇核內的Nrf2表現，減低NFkB的表現。經低氧前處理的幹細胞能強化細胞抗氧化壓力反應組成(ARE anti-oxidative response elements)包括GCLC (glutamate-cysteine ligase catalytic subunit)、GCLM(glutamate-cysteine ligase modifier subunit)、麩胱甘肽過氧化酵素(glutathione peroxidase)、錳超氧化物歧化酶、銅/鋅超氧化物歧化酶的表現。由此結果可知，低氧前處理可藉由活化Nrf2的訊息、加強抗氧化壓力反應組成，提升內在抗氧化能力達到減低傷害的目標，而抗氧化機轉在間質幹細胞治療的腎絲球腎炎上扮演重要角色。 本論文利用臨床大資料分析及動物實驗等方法，針對醫學上仍難以克服兩個的困境，提供史塔汀藥物降低動靜脈廔管阻塞與幹細胞治療腎絲球腎炎的實證基礎。 關鍵字: 動靜脈廔管、間質幹細胞、腎絲球腎炎、氧化壓力、史塔汀The growing end stage renal disease (ESRD) population and expanding cost of renal replacement therapy (RRT) have great burden on both public health and economics. Autologous arteriovenous fistula (AVF) is widely recommended as the first choice of long term vascular access for long term hemodialysis. However, high stenosis rates of AVF remained a medical challenge today. Glomerulonephritis (GN) is still one of the leading causes of ESRD, and has limited immunosuppressive treatment options with non-satisfactory outcomes. This thesis is to explore the potential beneficial effect of statin on AVF and the anti-oxidative therapeutic mechanism of stem cell on GN. The pathophysiology of AVF stenosis is characterized by endothelial dysfunction and vascular smooth muscle proliferation and transmigration. Statins are well known to reduce inflammation and improve endothelial function, thus provide a potential drug of choice for prevention of AVF stenosis. The protective effects of statins against stenosis for permanent hemodialysis access have been repeatedly demonstrated in animal studies, but remain controversial in human studies. This thesis evaluates the association between statin use and permanent hemodialysis access patency using a nationwide hemodialysis cohort. A total of 9862 pairs of statin users and non-users, matched by age and gender, were selected for investigation from 75404 new hemodialysis patients during 2000-2008. The effect of statins on permanent hemodialysis access patency was evaluated using Cox proportional hazards models. Compared with non-users, statin users had an overall 18% risk reduction in the composite endpoint in which angioplasty and recreation were combined (adjusted hazard ratio=0.82 [95%CI, 0.78-0.87]) and 21% in recreation of permanent hemodialysis access (adjusted hazard ratio=0.79 [95%CI, 0.69-0.80]). Specifically, the protective effect was found for arteriovenous fistula (adjusted hazard ratio= 0.78[95% CI, 0.73-0.82] for composite endpoint and 0.74 [95% CI, 0.69-0.80] for vascular recreation), but not for arteriovenous grafts (adjusted hazard ratio= 1.10 [95% CI, 0.98-1.24] and 0.94 [95% CI, 0.83-1.07]). These findings suggest that statins use after permanent HD access creation possess a dose-responsive effect of protecting AVF from stenosis for patients undertaking hemodialysis. The beneficial effect on permanent HD access outcomes is a universal class effect and the effect size is associated with the statins’ potency. The use of statin may reduce failure of AVF, therefore, promoting patient outcomes and reducing health-care costs. These results have important therapeutic implications for future prospective randomized control studies. Pathophysiologic feature of GN is immune mediated inflammation related mesangial proliferation and damage. Stem cells had been proved the potential of self-renewal and the ability of immune modulation and provide one potential therapeutic option to the unmet need of GN. The anti-oxidative ability of stem cells is one potential mechanism in the therapeutic application, but scarcely studied. This study aimed to investigate the mechanisms of the anti-oxidative effects involved in the use of normoxic and hypoxic conditions treated mesenchymal stem cells to treat anti-Thy1.1 induced rat glomerulonephritis. Proteinuria, histochemical staining and western blotting were used to explore the therapeutic effects and anti-oxidative mechanisms. Mesenchymal stem cell transplantation ameliorated proteinuria and glomerulosclerosis glomerulonephritis rats. Hypoxic conditioning significantly enhanced these therapeutic effects. Normoxic and hypoxic mesenchymal stem cells demonstrated a consistent reduction in macrophage/monocyte infiltration, levels of stress index proteins, autophagy and apoptosis. In addition, they both promoted intranuclear nuclear factor (erythroid-derived 2)-like expression and ameliorated elevated nuclear factor kappa B expression in diseased kidneys. Hypoxic preconditioning treated mesenchymal stem cells significantly enhanced the expression of anti-oxidative response elements including glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, glutathione peroxidase, catalase, Mn and Cu/Zn superoxide dismutase. Anti-oxidative mechanisms play a role in the therapeutic effect of mesenchymal stem cells in glomerulonephritis. Hypoxic preconditioning is one effective strategy to activate intrinsic anti-oxidative defense systems by promoting the Nrf2 pathway signaling, rescuing ROS scavengers and increasing anti-oxidative responsive element proteins. The results of this thesis provide evidence supporting therapeutic options to permanent hemodialysis vascular access failure and glomerulonephritis. Key words:arteriovenous fistula, mesenchymal stem cell, glomerulonephritis, oxidative stress, statin