氧化壓力在急性與慢性腎臟疾病中的角色

The Roles of Oxidative Stress in Acute and Chronic Renal Disease

徐世平(Shih-Ping Hsu)

指導教授 ： 蔡敦仁 ； 鄭劍廷

國立臺灣大學/醫學院/臨床醫學研究所/博士(2010年)

https://doi.org/10.6342/NTU.2010.00013

摘要

氧化壓力是活性氧化物 (reactive oxygen species, ROS) 生成和抗氧化防禦兩者失衡的結果。氧化壓力除了與許多人類疾病，像是神經疾病、心血管疾病、癌症及老化等有關聯外，在急性與慢性腎臟疾病中的角色也是值得研究的課題。
在各種急性腎損傷中，氧化壓力被認為在致病機轉中有其重要角色。含碘對比劑是引起急性腎損傷常見且重要的原因，主要的可能致病機轉除了改變腎臟血流後造成的缺血與缺氧，也包括上述對腎小管細胞的直接毒性，如氧化壓力傷害。而N-乙醯基半胱氨酸 (N-acetylcysteine, NAC) 被認為可藉由其抗氧化力而預防對比劑腎病變的發生，並已在臨床上使用，惟動物實驗的佐證尚待補強。
另一方面，慢性腎病 (chronic kidney disease, CKD) 是全球性的重要健康問題，對國人健康的影響也很大。慢性腎病包括末期腎病病患的主要死因是心血管疾病，其中部分原因歸結於慢性腎病病患其體內的氧化壓力較常人大，而氧化壓力、發炎反應標記與發炎前驅物質彼此又互相關聯。所以，以抗氧化劑治療或預防末期腎病患者氧化壓力相關的危險因子與併發症的效果，也是值得深入研究。
為了釐清在急性腎損傷中，是否氧化壓力愈大，腎臟受傷愈嚴重，所以使用含碘對比劑引起急性腎損傷的大鼠模型來加以探討。在大鼠實驗中，觀察了含碘對比劑對腎臟傳出神經活動（renal efferent nerve activity, RENA），腎血流動力學，血漿中腎素活性 (plasma renin activity, PRA) 的影響，ROS生成和大鼠腎小管細胞的傷害。在以urethane麻醉的母Wistar大鼠靜脈注射4 類含碘對比劑 (1600 mg/Kg body weight)，分別為Ioxitalamate (高滲透壓，離子性)，Ioxaglate (低滲透壓，離子性)，Iohexol (低滲透壓，非離子)，Iodixanol (等滲透壓，非離子)。以electrophysiologic記錄技術測量RENA，以都卜勒超音波測量腎動脈血流，以radioimmunoassay 法測定PRA (plasma rennin activity)，和透過一種偵測在活體內chemiluminescence 的方法來計算ROS的產生。另外，在 hematoxylin 和eosin染色下，將含碘對比劑引起的腎皮質小管細胞空泡變化依嚴重度分級，並以terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method (TUNEL) 法分析在腎外部髓質腎小管細胞的apoptosis。而且，又觀察了靜脈投予β-adrenoceptor 阻斷劑propranolol (10 mg /Kg body weight)、NAC (100 mg/Kg body weight) 前處置或雙側去腎臟神經後，對上述含碘對比劑可能引起的病生理變化的影響。結果發現，Ioxitalamate在一個小時內顯著增加了RENA和腎血管阻力，腎臟ROS的產生，並伴隨著在第2個小時時的血漿中腎素活性、腎小管細胞裡的空泡和TUNEL-apoptosis形成的增加；其他含碘對比劑對這些參數影響較少。另一方面，以propranolol或NAC做前處置，或者去腎臟神經都能緩和預期因Ioxitalamate所增加的RENA、PRA、ROS產生、在腎小管細胞空泡和TUNEL-apoptosis 的形成。
這些結果驗證了高滲透壓性對比劑Ioxitalamate誘發的氧化壓力最大，增加腎血管阻力最大，所造成的腎小管細胞凋亡程度也最嚴重。另外，也發現Ioxitalamate 可以透過刺激RENA和隨後的腎血管收縮，腎素表現和ROS的產生引起急性腎小管損傷。其次，給予β-adrenergic blockers前處置可能可以經由抑制RENA刺激來防止Ioxitalamate 引起急性腎小管損傷。第三，NAC不僅可以透過直接減少ROS的產生也可以透過減輕RENA激活和其後果，來減輕Ioxitalamate 引起的急性腎小管損傷。
而為了驗證長期補充抗氧化劑是否可改善血液透析病患體內的氧化壓力，則是直接進行臨床試驗，而第一個試驗的抗氧化劑是上述的NAC。另一方面，由於綠茶不僅是國人喜愛飲用的茶品，其中所含有的兒茶素 (catechins) 也具抗氧化能力，但是兒茶素在血液透析病患尚無臨床使用的報告，所以第二個試驗的抗氧化劑是兒茶素。
在試用NAC 抗氧化療效（efficacy）的non-randomized，open-label的臨床試驗開始時，符合收案資格的血液透析門診病患共325人。其中同意服用NAC的49人在研究的前3個月中每天服用NAC的200毫克三次，而沒服用NAC的276位病患也同時被觀察。結果發現，有11位服用NAC的病患退出，但沒有嚴重的副作用；而49位沒服用NAC的病患有負面影響的干擾事件 (negative confounding events)。 因此，只利用剩下病患，NAC組38人與沒服用NAC組的227人，的數據來分析補充NAC的療效。兩組的人口統計和實驗室數據在基線是相似的。與基線相比，當erythropoietin劑量穩定時，只有NAC 組的hematocrit有顯著增加。而NAC組血漿中8－isoprostane及氧化低密度脂蛋白 (oxidized LDL） 也較基線值降低。即使用nested case-control study法分析，也發現補充NAC是尿毒性貧血有正面結果（positive outcome）的一個重要的預測因子。所以結論是，口服補充NAC有潛力可輔助性治療血液透析病患尿毒性貧血和氧化壓力。
在以兒茶素作為抗氧化劑補充療法的臨床試驗中，則使用去咖啡因綠茶萃取物 (兒茶素)，並觀察其對於血液透析-誘發之ROS、動脈粥樣硬化危險因子和促發炎細胞激素 (proinflammatory cytokine) 的影響。我們共招募了6位健康志願者和54位血液透析病人來研究。首先，在健康志願者 (n = 6) 和血液透析病人 (n = 10) 之間，分別給予一個口服劑量（455毫克）之兒茶素來比較其血漿中藥物的濃度。接著，在10位血液透析病人於接受血液透析前，給予3種不同之兒茶素口服劑量 (分別為0、455和910毫克) 或口服維生素C (500毫克) 後，比較其抗氧化的效果。第三部分，在另外44位血液透析病人進行為期7個月之介入研究，其中30位病人於整個研究期間均給安慰劑，而另外14位病人在第三到第五個月期間給予每天455毫克口服劑量之兒茶素。與健康志願者相比，在給一個口服劑量之後，血液透析病人 (n = 10) 的血漿中兒茶素濃度達到最高點比較遲而衰退也較慢。在10位血液透析病人中，兒茶素之給予會使因血液透析增加之血漿中次氯酸 (hypochlorous acid, HOCl) 活性減低，且比給予安慰劑或維生素C更為有效。比較使用455毫克或910毫克兒茶素，其間使血漿中次氯酸活性之減少並無明顯之差異。兒茶素也可將血液透析所增加之促發炎細胞激素的表現明顯地與以降低。在為期7個月之介入研究中，與30位只接受安慰劑之血液透析病人比較， 14位病人在每天口服兒茶素的3個月中，有較低之透析前血漿過氧化氫 (hydrogen peroxide, H2O2) 活性、較低之次氯酸活性和較低之磷脂質過氧化氫 (phosphatidylcholine hydroperoxide)、C-反應蛋白 (C-reactive protein) 以及較低之促發炎細胞激素之濃度。所以結論是，兒茶素具有降低由血液透析-誘發所產生之過氧化氫及次氯酸、動脈粥樣硬化危險因子和促發炎作用。
整理以上兩個臨床試驗的結果，可發現口服補充NAC可能對那些能服用的血液透析病患而言，是一種安全和有效治療尿毒性貧血的輔助性方法；而長期服用兒茶素可以減少因血液透析所增加的氧化壓力、心血管疾病風險與發炎反應。也就是說，這些臨床研究結果驗證了長期補充抗氧化劑，NAC或兒茶素，可改善血液透析病患體內的氧化壓力及相關危險因子。
所以，綜合以上動物實驗與臨床試驗的結果，說明了在急性腎損傷中，氧化壓力愈大，腎臟受傷愈嚴重。另外，長期補充抗氧化劑可改善血液透析病患體內的氧化壓力。而這些研究成果已充分有效地運用於目前正在進行的相關動物實驗與臨床試驗中。

關鍵字

氧化壓力 ； 含碘對比劑 ； 腎臟傳出神經活動 ； N-乙醯基半胱氨酸 ； 綠茶萃取物 (兒茶素) ； 血液透析

並列摘要

Oxidative stress, a status of imbalance between the production of reactive oxygen species (ROS) and the antioxidant protection, results in significant damage to cell structures and relates to many human diseases, such as neurological diseases, cardiovascular diseases, cancer and aging. Moreover, it is interesting and worth to clarify the roles of oxidative stress in acute and chronic renal disease.
In acute kidney injury (AKI), oxidative stress is presumed to play certain roles in the pathogenic mechanisms. Iodinated contrast media (CM)-induced nephropathy (CMIN) is a common cause of AKI and a major complication after application of CM. Two main mechanisms have been proposed on the basis of the results from experimental studies in CM-induced renal damage: 1) CM decrease renal perfusion; and, 2) CM directly damage renal tubules, increasing ROS. In recent years, some clinical studies have demonstrated N-acetylcysteine (NAC; an antioxidant) can prevent CMIN. However, evidence from experimental studies is waiting to be demonstrated.
Chronic kidney disease (CKD) is not only threatening the whole world population but also becoming a nation-wide health problem in Taiwan. CVD is the main death cause in patients with CKD, including those undergoing dialysis therapy for end-stage renal disease (ESRD). The high cardiovascular risks in CKD patients are partially related to increased oxidative stress, which links to inflammation and atherosclerosis. Therefore, it is worth to explore if antioxidants could be used to treat or prevent the risks and complications related to the oxidative stress in ESRD patients.
In order to clarify whether the more oxidative stress in AKI, the larger damage to the kidney, we explored the effects of CM on RENA, renal hemodynamics, plasma renin activity (PRA), ROS production and renal injury in rats. Four types of CM including Ioxitalamate (high-osmolar, ionic), Ioxaglate (low-osmolar, ionic), Iohexol (low-osmolar, non-ionic), and Iodixanol (iso-osmolar, non-ionic) were given intravenously (1600 mg I/kg body weight) to urethane-anesthetized female Wistar rats. We measured RENA by electrophysiologic recording techniques, renal blood flow (RBF) with Doppler ultrasound, PRA by radioimmunoassay, and ROS by an in vivo chemiluminescence method. We graded the severity of CM-induced vacuoles in cortical tubular cells stained by hematoxylin and eosin, and apoptosis production in outer medulla by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Besides, the effects of pretreatment with intravenous β-adrenoceptor antagonist propranolol (10 mg/kg body weight), antioxidant NAC (100 mg/kg body weight) and renal denervation on CM-induced pathophysiologic parameters were determined. We found Ioxitalamate significantly increased RENA and renal vascular resistance (RVR), PRA, renal ROS production within one hour, and formation of vacuoles and TUNEL-apoptosis in renal tubular cells 2 hours later; other CM had less effect on these parameters. On the other hand, propranolol, NAC, or renal denervation partially attenuated the Ioxitalamate-aggravated responses on RENA, PRA, ROS production, and vacuole and TUNEL-apoptosis formation in renal tubular cells.
The above findings demonstrated high-osmolar CM, Ioxitalamate, induced the most oxidative stress and caused the most damage to the kidney. Moreover, Ioxitalamate may induce acute tubular injury via aggravation of RENA, adrenergic signaling, PRA, and ROS production. Pretreatment with β-adrenergic blockers may modulate RENA and less Ioxitalamate-induced acute tubular injury. Pretreatment with NAC can also moderate Ioxitalamate-induced acute tubular injury via not only reducing ROS production as expected but also stabilizing RENA.
In order to realize whether long-term supplement with antioxidants can improve the oxidative stress and related damage in hemodialysis (HD) patients, we first tested NAC in a clinical trial. Besides, we also tried catechins, potent antioxidants extracted from green tea, in HD patients because there was no report yet.
In the trial with NAC, we explored the efficacy of oral NAC supplement in anemia and oxidative stress in HD patients. Of the eligible patients (n = 325) in an outpatient HD unit, 49 received NAC 200 mg orally thrice a day during the first 3 months, while the other 276 patients not receiving NAC were observed. During the 4-month study, 11 patients receiving NAC withdrew but had no severe adverse effects, while 49 patients not receiving NAC had negative confounding events. Thus only the data of the remaining patients, 38 taking NAC and 227 not taking NAC, were analyzed for efficacy. The demographic and laboratory data of both groups were similar at baseline. When the erythropoietin dosage was stable throughout, only the NAC group had a significant increase in hematocrit, accompanied with a decrease in plasma levels of 8-isoprostane and oxidized low-density lipoprotein. Analyzed as in a nested case-control study, NAC supplementation was also found to be one significant predictor of positive outcomes of uremic anemia. In summary, oral NAC supplement may be promising therapy for uremic anemia and oxidative stress in HD patients.
In the trial with catechins, we evaluated the effects of decaffeinated green tea extract (catechins) supplement on HD-induced ROS, atherosclerotic risk factors, and proinflammatory cytokines. We enrolled 6 healthy subjects and 54 HD patients for this study. First, the pharmacokinetics of one oral dose of catechins were compared between the healthy (n = 6) and HD patients (n = 10). Second, in the 10 HD patients, we compared the antioxidant effects of three different doses (0, 455, and 910 mg) of oral catechins to that of oral vitamin C (500 mg) during an HD session. Third, the other 44 HD patients participated in a 7-month interventional study, including 30 patients taking placebo throughout and 14 patients taking catechins (455 mg) daily from the third to the fifth month. Compared to the healthy, the HD patients (n = 10) had later peaks and decay of plasma catechins after one oral dose. In the 10 HD patients, catechins supplement reduced HD-enhanced plasma HOCl activity more effectively than placebo or vitamin C. Between treatments with 455 and 910 mg of catechins, there was no significant difference in reducing plasma HOCl activity. Catechins also significantly reduced pro-inflammatory cytokine expression enhanced by HD. In the 7-month interventional study, in comparison to the 30 HD patients taking placebo, the 14 patients had less predialysis plasma H2O2 and HOCl activities, phosphatidyl choline hydroperoxide, C-reactive protein (CRP), and pro-inflammatory cytokine levels during the 3 months when they took catechins daily. In summary, catechins reduce HD-induced H2O2/HOCl production, atherosclerotic risk factor, and pro-inflammation.
Based on the findings in the above two clinical trials, oral NAC supplement is probably a safe and effective therapy for uremic anemia in those who can tolerate NAC; catechins may reduce HD-induced ROS, atherosclerosis risk, and pro-inflammation. That is, we demonstrated long-term supplementation with antioxidants, like NAC or catechins, may improve the oxidative stress and related damage in HD patients.
In conclusion, the results in the rat experiment and clinical trials clarify: 1) the more oxidative stress in AKI, the larger damage to the kidney; and, 2) long-term supplementation with antioxidants can improve the oxidative stress in HD patients. Further experimental and clinical studies have been conducted on the basis of the above significant findings.

並列關鍵字

oxidative stress ； iodinated contrast media ； renal efferent nerve activity ； N-acetylcysteine ； green tea extract (catechins) ； hemodialysis

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