抗氧化治療策略在基礎與臨床氧化傷害模式之評估

Abstract

尿毒症患者接受血液透析時，就開始承受心血管疾病的負擔。乃因慢性系統性發炎、貧血、加速動脈硬化及心血管疾病併發症與氧化壓力增加有密切相關。因在血液透析過程中當血液中白血球與人工腎臟接觸時會活化白血球細胞上的NADPH oxidase，使得O2轉為Superoxide(O2.-)，Superoxide(O2.-)需經Superoxide dimutase或Glutathion peroxidase轉為過氧化氫(H2O2)，H2O2再經Catalase轉為H2O。當吞噬細胞被激化時MPO被迅速分泌且在H2O2存在的情況下經MPO催化而氧化產生相對應的次氯酸鹵化物。當產生的氧化劑超過局部的抗氧化能力時，於是乎氧化壓力不斷地進行。將更進一步導致碳水化合物，類脂及存在於此種環境中的蛋白質和DNA 等重要的大分子氧化。 在我們的研究中發現病患在血液透析後血裡的氧化自由基明顯增加，其氧化自由基主要是過氧化氫和較小數量Superoxide(O2.-)及HOCl。 血液透析過程中流失抗氧化劑維生素C、導致血液中維生素C減少，總抗氧化劑狀態和RBC-MFR 活性減少，血漿中及紅血球細胞膜上的phosphatidylcholine hydroperoxide (PCOOH)和methemoglobin增加。靜脈灌注維生素C明顯清除血液透析後引起的氧化壓力，降低了血漿中及紅血球細胞膜上的PCOOH 和methemoglobin 的量和保護RBC-MFR 活性。覆被維他命E的人工腎臟對血液透析後血裡的總氧化自由基的活性只有部分影響，但仍能有效地防止紅血球免於受到氧化壓力的傷害。靜脈灌注維生素C或使用覆被維他命E的人工腎臟能有效地降低血液透析後引發的氧化壓力，如紅血球溶血和類脂超氧化程度的改善，並進一步降低血液透析病患前發炎細胞素的過度表現。 還原電解水(ERW)的保護機制乃源於具還原能力的活性原子氫，這有助于清除氧化自由基，並且參與細胞功能的還原。為了減少血液透析提升的氧化壓力，我們透過新安裝的HD-24K (Nihom Trim Co., Osaka, Japan)所產生的還原電解水運用在血液透析病患。在未接受還原電解水治療的末期腎衰竭病人其血液透析前血漿中單一次血液透析後，氧化壓力明顯增加。單一次血液透析時施予ERW處理將降低75±14 % 的氧化自由基的形成，此意味著血液透析合併ERW處理可能具有降低在尿毒病患與慢性透析患者體內的氧化性大分子的臨床重要性。ERW能降低氧化自由基，特別是H2O2和HOCl，ERW 處理應具有對白血球細胞和內皮細胞氧化損害減到最低的好處。將ERW的應用在透析液裡可以有效地改善血液透析提升的氧化壓力，也降低了血漿中及紅血球細胞膜上的PCOOH 和methemoglobin 的量和保護RBC-MFR 活性。經使用六個月的 ERW 處理，二十六種前發炎細胞素被向下調控。且經六個月ERW處理後在沒有增加紅血球生成素劑量的情況下能明顯改善血比容。總之，我們的研究發現於接受透析過程中使用維他命C或覆被維他命E的人工腎臟可降低後續氧化壓力的增加並減少紅血球的傷害，進而改善貧血，而以電解還原水作為透析用水亦然，但長期於透析過程中使用維他命C，仍有草酸累積的疑慮，而長期於透析過程中以電解還原水作為透析用水則無草酸累積的危險。 除了電解還原水可作為降低氧化壓力的工具，也有報告指出慢性低氧預處理可降低氧化傷害，我們的研究發現慢性低氧預處理透過超氧自由基轉化酵素的活性和總量的向上調控來降低超氧自由基對腎臟引起的機能失調。內毒素調降腎臟的抗氧化狀態。我們假設慢性低氧預處理可以保護腎臟以降低內毒素引起的氧化傷害。在慢性低氧預處理四星期的大鼠[相當5500米高度的低氧環境(每天15小時)]和海平面四星期的大鼠之腹腔內注射內毒素(LPS，4毫克每千克體重)。LPS會提升xanthine oxidase(XO)和gp91phox (NADPH oxidase的亞單位)的表現，同時經lucigenin提升的chemiluminescence可發現海平面大鼠來自腎臟表面和腎靜脈血的superoxide產生有爆發性的表現。且LPS會增加腎臟的IL-1b的產生。當與海平面大鼠相比時，在四星期的慢性低氧預處理後，在腎臟皮層Cu/ZnSOD、MnSOD、catalase顯著增加。以及調降腎臟皮層的XO和腎臟皮層的gp91phox和腎臟髓質的gp91phox 。結合了提升抗氧化蛋白質酵素並調降氧化性蛋白酵素而達到降低LPS引發superoxide產生，調降腎臟XO和gp91phox表現，和腎臟的IL-1b產生的效應。我們確認慢性低氧預處理提升腎內抗氧化性/氧化性蛋白比率而克服了內毒素引起的氧化自由基的形成並進一步降低發炎性細胞素的釋放，而達到保護腎臟免於氧化傷害的後果。 麩胺酸受體(glutamate receptor)不只在中樞神經系統表現亦存在於週邊某些組織內，然而週邊麩胺酸受體的功能卻鮮少知道。己有報告指出glutamate/glycine啟動的N-methyl-D-aspartate (NMDA)感受器位于腎臟上。而NMDA 感受器亜單位NR1已被發現在受損的腎臟上表現增加。Said等學者發現麩胺酸受體的活化是造成肺臟氧化傷害的主因，NMDA感受器在gentamicin nephrotoxicity中也扮演主要角色。我們利用一個腎臟缺血再灌流的氧化傷害模式來評估腎內功能性的NMDA感受器的亞單位NR1的角色。基於NMDA感受器在gentamicin nephrotoxicity裡扮演重要的角色，腎內近端小管裡有相當高的NMDA感受器的表現，以及腎臟的缺血再灌流的氧化傷害在近端小管裡最嚴重，故我們進一步研究NMDA感受器在腎臟缺血再灌流的氧化傷害中是否被活化並且是否在腎臟缺血再灌流引起腎臟氧化傷害中扮演重要的角色。為了驗証是否在腎臟遭受缺血再灌流引起的腎臟氧化傷害中，NMDA 感受器可能在腎臟功能的損害上所扮演的角色，我們利用西方點墨法來分析NMDA 感受器(功能性亞單位NR1)在缺血後老鼠腎臟(左腎動脈經45分鐘缺血封閉後再經24小時再灌流)上的表現。 在評估老鼠腎臟NMDA 感受器被活化時對腎臟血流動力學的影響，我們利用NMDA(NMDAR agonist )經老鼠腎內動脈(ira)注入及NMDA抑制劑AP-5 (2毫微毫升的體積，經老鼠腎內動脈(ira)注入)。利用perivascular 脈搏都普勒來測量腎臟的血流。利用3H-inulin廓清率來測量GFR。在老鼠腎內動脈(ira)注入NMDA或NMDA抑制劑D-AP-5之前及注入之後做測量。在NMDA注入之後會降低GFR，尿量和尿鈉和鉀的的排泄，且此現象與使用NMDA劑量成正相關。而D-AP-5完全拮抗掉NMDA引起的腎臟功能變化。利用西方點墨法來分析正常的老鼠腎臟和缺血後老鼠腎臟NMDA感受器，發現缺血後腎臟NMDA感受器無論在外部髓質及內部髓質和皮層皆顯示向上調控的情況，尤其是缺血後老鼠腎臟的外部髓質。缺血後腎臟上的NMDA感受器表現增加且對在腎內動脈(ira)注入NMDA的反應上表現出GFR 進一步降低和在腎臟的排泄方面其尿量和尿鈉和鉀的的排泄的顯著減少。D-AP-5在缺血再灌流的腎臟對NMDA 感受器的抑制導致的GFR的相當程度的增加並使得尿量和尿鈉排泄量更大。這些發現在老鼠腎臟裡的glutamate的excitotoxic的作用提供了一個分子生物學的基礎。總之，腎臟NMDA感受器的表現可以透過缺血再灌流而被向上調控，並且在腎臟遭受缺血再灌流傷害之後扮演重要的功能上的角色，NMDA感受器可以讓受損的老鼠腎臟降低腎絲球的回應和腎小管的排泄功能。

Hemodialysis (HD) is used for removal of excessive toxins, metabolic products, and blood components from patients with end-stage renal diseases (ESRD). However, the interaction of blood with non-biological materials of the extracorporeal circuit can activate polymorphonuclear leukocytes (PMNs) to produce quantities of reactive oxygen species (ROS). Increased oxidative stress in ESRD patients may oxidize macromolecules and impair neighboring tissues/cells (including RBCs) and evoke an inflammatory response, then consequently lead to cardiovascular events during chronic hemodialysis. There is increasing evidence that oxidative stress plays a key role in the genesis and severity of dialysis anemia. It reduces RBC survival, impairs the effect of erythropoietin, and increases the susceptibility to hemolysis due to inflammatory, infectious and mechanical stimuli. During the process of oxidative stress, RBCs are subject to membrane lipid peroxidation and susceptible to destruction. Increased ROS can oxidize oxyhemoglobin (oxyHb) to yield H2O2 and methemoglobin (metHb). A trans-plasma membrane electron transport system is present on RBC membranes and plays a role to reduce cytotoxic ferricyanide/metHb to functional ferrocyanide/oxyhemoglobin (oxyHb). We demonstrated intravenous vitamin C (VC) or vitamin E (VE)-coated dialyzer can improve HD-enhanced erythrocyte lipid peroxidation and hemolysis via the preservation of NADH-ferricyanide reductase and NADH-methemoglobin reductase. The protective mechanism of electrolyzed reducing water (ERW) obtained by electrolysis results from active atomic hydrogen with high reducing ability, which can contribute to ROS scavenging activity, and may participate in the redox regulation of cellular function. Therefore, for decreasing hemodialysis-enhanced oxidative stress, we administered ERW by a new setup of HD-24K (Nihom Trim Co., Osaka, Japan) to the patients during HD course. ERW administration diminished HD-enhanced H2O2 and HOCl activity, minimized atherosclerotic, oxidized and inflammatory markers, and partly restored total antioxidant status during one-month treatment. We evaluated oxidative stress in blood and plasma, erythrocyte methemoglobin/ferricyanide reductase (RBC-MFR) activity, plasma methemoglobin, and proinflammatory cytokines in the chronic HD patients without treatment or with vitamin C, vitamin E-coated dialyzer, or ERW treatment during an HD course. The patients showed marked increases in blood ROS, mostly H2O2, and in lesser amounts, O2-. and HOCl after HD without any treatment. HD also resulted in decreased plasma vitamin C, total antioxidant status, and RBC-MFR activity and increased erythrocyte levels of phosphatidylcholine hydroperoxide (PCOOH) and plasma methemoglobin. Antioxidants treatment significantly palliated single HD course-induced oxidative stress, plasma and RBC levels of PCOOH, and plasma methemoglobin levels, and preserved RBC-MFR activity. However, ERW had no side effect of oxalate accumulation easily induced by vitamin C. Six-month ERW treatment increased hematocrit and attenuated proinflammatory cytokines profile in the ESRD patients. ERW treatment administration is effective in palliating HD-evoked oxidative stress, as indicated by lipid peroxidation, hemolysis, and over-expression of proinflammatory cytokines in HD patients. A beneficial consequence of increased hematocrit in the ESRD patients undergoing chronic HD with ERW treatment was also recognized. Except for the reducing power of ERW, chronic hypoxic (CH) preconditioning had been reported to reduce oxidative tissue injury. Chronic hypoxic (CH) preconditioning reduces superoxide-induced renal dysfunction via the upregulation of superoxide dismutase (SOD) activity and contents. Endotoxemia reduces renal antioxidant status. We hypothesize that CH preconditioning might protect the kidney from subsequent endotoxemia-induced oxidative injury. Endotoxemia was induced by intraperitoneal injection of lipopolysaccharide (LPS; 4mg/k) in rats kept at sea level (SL) and rats with CH in an altitude chamber (5500m for 15 h/day) for 4 weeks. LPS enhanced xanthine oxidase (XO) and gp91phox (catalytic subunit of NADPH oxidase) expression associated with burst amount of superoxide production from the SL kidney surface and renal venous blood detected by lucigenin-enhanced chemiluminescence. LPS induced increased renal IL-1β protein in the SL rats. After 4 weeks of induction, CH significantly increased Cu/ZnSOD, MnSOD and catalase expression in renal cortex, and depressed renal cortex XO and renal cortex and medulla gp91phox when compared with SL rats. The combined effect of enhanced antioxidant proteins and depressed oxidative proteins significantly reduced LPS-enhanced superoxide production, renal XO and gp91phox expression, and renal IL-1β production. We conclude that CH treatment enhances the intrarenal antioxidant/oxidative protein ratio to overcome endotoxaemia-induced reactive oxygen species formation and inflammatory cytokine release. Said et al. had reported that excitotoxicity due to overactivation of its N-methyl-D-aspartate (NMDA) receptor may be a key factor in oxidant tissue injury. The NMDA receptor is expressed in the kidney. The receptor plays a major role in gentamicin nephrotoxicity. We assessed the role of the functional subunits NR1 of renal NMDA receptor in a model of renal ischemic-reperfusion injury. Based upon the prominent role the receptor plays in gentamicin nephrotoxicity, the high expression of the NMDA receptor in the renal proximal tubule, and the high degree of renal ischemic-reperfusion injury found in the proximal tubule, we speculated that the NMDA receptor might be activated by and may play a role in renal injury caused by renal ischemic-reperfusion injury in the part III study. In order to clarify whether NMDA receptors may have a role on functional detriment after kidneys suffered ischemic reperfusion injury, the expression of NMDA receptors (as functional subunit NR1) in the post-ischemic kidney (obtained by 45-min occlusion of left renal artery and reperfusion for 24 h) were examined by Western blot analysis. Renal hemodynamic effects of NMDA-R activation was assessed in rats using intrarenal arterial (ira) infusion of NMDA and NMDA-R inhibition was assessed in rats using a specific antagonist of the NMDA-R (AP-5) before treatment of NMDA. Renal blood flow was measured by perivascular pulse Doppler. GFR was measured by 3H-inulin clearance. Measurements were made before and during intrarenal arterial infusion of NMDA or D-AP5. Surprisingly, NMDA-infusion reduced GFR, urine amount and urinary excretion of sodium and potassium in dose- dependent manner. D-AP5 completely abolished the decreased renal function due to NMDA. Western blots of normal- and post-ischemic kidneys demonstrate that renal NMDA receptor is upregulated on outer medulla, inner medulla and cortex of post-ischemic kidneys, especially the outer medulla. The post-ischemic kidneys after 45-min occlusion of left renal artery and reperfusion for 24 h results in reduced GFR responses and significant decrease in renal excretion due to NMDA-infusion associated with increased renal NMDA receptor expression. The significant increments of GFR and renal excretion due to NMDA receptor inhibition by D-AP5 in the IR kidneys were larger than those of control kidneys except urinary potassium excrection. These findings provide a molecular-biological basis for the excitotoxic actions of glutamate in rat kidneys. In conclusion, kidney NMDA receptor expression is upregulated by ischemic-reperfusion, and this receptor may play an important functional role in detrimental effects on rat kidney glomerular response and excretion function after kidneys suffered ischemic reperfusion injury. Chronic hypoxic preconditioning was reported to reduce significantly the glutamate-induced neuronal injury. In the part II of our study, the benefit of CH-induced HPC is to prevent excessive generation of ROS by shifting the intrarenal redox status towards antioxidative defense. During ischemia/reperfusion injury, overactivation of its N-methyl-D-aspartate (NMDA) receptor is toxic to renal cells, that is excitotoxicity may be a key factor in renal oxidative injury. Therefore application of reducing water or chronic hypoxia will attenuate the renal oxidative injury caused by excitotoxicity due to overactivation of its N-methyl-D-aspartate (NMDA) receptors.