【作者】 云宇；

【导师】 王殿华； 沈志强；

【作者基本信息】 昆明医学院 ， 外科学， 2010， 博士

【摘要】 [研究背景和目的]肾脏是高灌流、对缺血-再灌注易受损伤的器官。缺血-再灌注损伤(ischemia reperfusion injury,IRI)在许多肾脏疾病,例如:肾移植、肾部分切除术、肾动脉血管成形术、动脉分流术、意外创伤、肾积水和输尿管手术过程中均会发生,是临床上导致急性肾功能衰竭的主要原因之一,死亡率较高,且存活的患者也会发生不同程度的慢性肾功能损害。因此,寻找防治肾脏缺血-再灌注损伤的方法与措施一直是近年来研究的热点的问题。缺血预处理(ischemic preconditioning,IPreC)是目前公认的一种有效的抗器官组织缺血-再灌注损伤的机械性干预措施。1999年,Toosy等证实给予肾脏几个短暂的停灌/再灌处理,可以对后来发生的长时间的缺血损伤有比较明显的保护作用。但是,缺血预处理必须在IRI发生之前进行,这对于许多无法预期的肾脏缺血性疾病是不可能实现的,例如:在肾移植中,肾源的供给许多是无法预料的,也不可能有进行缺血预处理的时机,使缺血预处理的临床应用受到很大的限制。缺血后处理(ischemic postconditioning,IPostC)是2003年由Vinten-Johansen’s研究小组提出了一种新的机械性干预措施,即在心肌缺血发生之后,再灌注开始时给予3个短暂循环的再灌/停灌的处理。这种新的干预措施可以缩小心梗面积、保护内皮功能、减轻细胞水肿、减少缺血梗死区中性粒细胞的聚集和改善心功能,对心肌缺血-再灌注损伤有比较明显的保护作用,且作用程度与缺血预处理相似。与缺血预处理不同,缺血后处理是在组织器官缺血之后才实施,在临床上应用具有“简单、可行、有效”三大特点,目前被认为是一种防治缺血性疾病的新的机械性抗损伤措施,广泛地被应用于抗器官组织缺血-再灌注损伤的防治。因此,本研究探讨了缺血后处理对肾脏IRI的保护作用,通过与缺血预处理相比较,从整体、细胞和分子水平上阐明缺血后处理抗大鼠肾脏IRI的作用机制,本研究可为外科抗肾脏缺血-再灌注损伤提供新的策略,也可为缺血后处理防治肾脏缺血-再灌注损伤的应用提供理论和实验依据。[方法]SPF级SD雄性大鼠,体重:180～220 g,随机分为①假手术组(Sham组):仅行开腹右肾切除,左肾蒂分离,不夹闭;②缺血-再灌注组(I/R组):开腹右肾切除,左肾蒂分离,无创血管夹夹闭45 min;③缺血预处理组(IPreC组):开腹右肾切除,左肾蒂分离,行3个循环夹闭2 min/再灌5 min预处理后,无创血管夹夹闭45 min;④缺血后处理组(IPostC组):开腹右肾切除,左肾蒂分离,无创血管夹夹闭45 min后,行6个循环夹闭10 sec/再灌10 sec后处理,各组再灌24 h。本研究分两个部分:1.缺血后处理抗大鼠肾脏缺血-再灌注损伤作用应用分光分析法测定血清肌酐(Cr)、尿素氮(BUN)评价肾功能;HE染色观察肾组织病理形态学改变,评价缺血后处理抗大鼠肾脏缺血-再灌注损伤作用。2.缺血后处理抗大鼠肾缺血-再灌注损伤的机制研究分别从以下3方面进行探讨:A从抗氧化途径研究缺血后处理抗大鼠肾脏缺血-再灌注损伤作用机制应用分光分析法测定肾组织丙二醛(MDA)、还原性谷胱甘肽(GSH)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活性:采用Real Time RT-PCR和Western blotting法分别测定SOD mRNA和蛋白表达情况,探讨缺血后处理抗大鼠肾脏缺血-再灌注损伤作用机制。B从抗炎途径研究缺血后处理抗大鼠肾脏缺血-再灌注损伤作用的机制应用分光分析法测定肾组织中性粒细胞髓过氧化物酶(MPO)活性,采用Real Time RT-PCR和Western blotting法分别测定环氧酶(COX-1,COX-2)mRNA和蛋白表达情况,探讨缺血后处理抗大鼠肾脏缺血-再灌注损伤作用的机制。C从抗细胞凋亡途径研究缺血后处理抗大鼠肾脏缺血-再灌注损伤作用机制应用DNA琼脂糖凝胶电泳法和末端脱氧核苷酸转移酶介导dUTP缺口末端标记法(TUNEL法)检测大鼠肾组织细胞凋亡发生情况;采用Real TimeRT-PCR和Western blotting法分别测定Bcl-2、Bax mRNA和蛋白的表达情况,探讨缺血后处理抗大鼠肾脏缺血-再灌注损伤作用的机制。[结果]1.缺血后处理抗大鼠肾脏缺血-再灌注损伤作用①肾功能的评价与Sham组比较,I/R、IPreC和IPostC组的Cr及BUN水平明显升高(P＜0.05);与I/R组比较,IPreC和IPostC组的Cr及BUN水平明显降低(P＜0.05),IPreC和IPostC组Cr及BUN水平无明显差异(P＞0.05)。②肾组织病理学观察Sham组肾小球、肾小管没有发现明显的形态学改变,I/R组肾小管上皮细胞肿胀,出现水样变性和空泡变性,肾小管扩张,内可见管型和坏死脱落细胞,少量管腔内有蛋白性液体积聚,部分肾小管结构毁损,间质充血水肿,大量炎性细胞浸润,管周血管明显扩张淤血;IPreC和IPostC组肾小球轻度淤血,少量肾小管上皮细胞轻度水肿,空泡变性,少见管型,间质充血水肿,有少量炎性细胞浸润,管周稍有淤血。2.从抗氧化途径研究缺血后处理抗大鼠肾脏缺血-再灌注损伤作用机制①各氧化指标的变化与Sham组比较,I/R、IPreC和IPostC组的MDA浓度明显升高,GSH浓度、SOD、CAT和GSH-Px活性明显降低(P＜0.05);与I/R组比较,IPreC和IPostC组的MDA浓度明显降低,GSH水平SOD、CAT和GSH-Px活性明显升高(P＜0.05),IPreC和IPostC组各个指标水平无明显差异(P＞0.05)。②SOD mRNA和蛋白表达与Sham组比较,I/R、IPreC和IPostC组的SOD mRNA和蛋白表达明显下降(P＜0.05),与I/R组比较,IPreC和IPostC组的SOD mRNA和蛋白表达相对升高,IPreC和IPostC相比,SOD mRNA和蛋白表达无明显差异(P＞0.05)。3.从抗炎途径研究缺血后处理抗大鼠肾脏缺血-再灌注损伤作用的机制①MPO活性的变化与Sham组比较,I/R、IPreC和IPostC组的MPO活性明显升高(P＜0.05);与I/R组比较,IPreC和IPostC组的MPO活性明显降低(P＜0.05)。②COX蛋白表达各组COX-1 mRNA和蛋白表达无统计学差异。与Sham组比较,I/R、IPreC和IPostC组的COX-2 mRNA和蛋白表达明显升高(P＜0.05),与I/R组比较,IPreC和IPostC组COX-2的mRNA和蛋白表达明显降低(P＜0.05),IPreC和IPostC相比,COX-2的mRNA和蛋白表达无明显差异(P＞0.05)。4.从抗细胞凋亡途径研究缺血后处理抗大鼠肾脏缺血-再灌注损伤作用的机制①肾小管上皮细胞凋亡的状况与Sham组比较,I/R、IPreC和IPostC细胞凋亡数明显增加,凋亡指数(AI)明显增高(P＜0.05);与I/R组相比较,IPreC和IPostC细胞凋亡数明显减少,AI明显降低(P＜0.05),IPreC和IPostC组AI无明显差异(P＞0.05)。②Bcl-2、Bax mRNA和蛋白表达与Sham组比较,I/R、IPreC和IPostC组的Bcl-2 mRNA和蛋白表达明显下降,Bax mRNA和蛋白表达相对升高,Bcl-2/Bax下降(P＜0.05);与I/R组比较,IPreC和IPostC组的Bcl-2 mRNA和蛋白表达相对升高,Bax mRNA和蛋白表达相对降低,Bcl-2/Bax明显升高(P＜0.05),IPreC和IPostC相比,Bcl-2和Bax的mRNA和蛋白表达无明显差异(P＞0.05)。[结论]1.肾缺血-再灌注使大鼠肾功能和结构受损,表现为Cr和BUN升高,肾小球、肾小管受损,MDA、MPO升高,GSH、SOD、CAT和GSH-Px活性降低,SOD的mRNA和蛋白表达下降,COX-2 mRNA和蛋白表达升高,肾组织细胞凋亡增加,Bcl-2 mRNA和蛋白表达降低,Bax mRNA和蛋白表达升高。2.缺血后处理能减轻Cr和BUN水平,减轻肾组织结构的损伤,具有显著的抗大鼠肾缺血-再灌注损伤的作用。3.缺血后处理抗大鼠肾缺血-再灌注损伤作用机制与抗氧化有关,可能是通过清除氧自由基,抑制脂质过氧化,升高组织抗氧化物酶的活性和蛋白表达,提高肾组织的抗氧化能力。4.缺血后处理抗大鼠肾缺血-再灌注损伤作用机制与抗炎有关,可能是通过抑制中性粒细胞聚集,抑制COX-2 mRNA和蛋白的表达,从而抑制炎症介质的产生。5.缺血后处理抗大鼠肾缺血-再灌注损伤作用机制与抗细胞凋亡作用有关,可能是通过调控细胞凋亡相关基因Bcl-2/Bax mRNA和蛋白表达。6.缺血后处理抗大鼠肾缺血-再灌注损伤作用及其机制与缺血预处理有相似之处。7.缺血后处理抗大鼠肾缺血-再灌注损伤的作用及机制的研究有助于为防治肾脏缺血-再灌注损伤提供理论和实验依据,为外科抗肾脏缺血-再灌注损伤提供新的策略。更多还原

【Abstract】 Background and objectiveKidney is easily subjected to ischemic reperfusion injury due to its abundance of blood supply. The temporary discontinuation of renal blood supply is encountered in many clinical situations, such as kidney transplantation, partial nephrectomy, renal artery angioplasty, cardiopulmonary bypass, aortic bypass surgery, accidental or iatrogenic trauma, sepsis, hydronephrosis and elective urological operations. Acute renal failure (ARF) resulting from renal ischemia reperfusion injury (IRI) remains a major clinical problem because of its frequent occurrence and high mortality. How to treat the renal IRI is always keeping in attention.Currently, ischemic preconditioning (IPreC) is a well-known method in treating organ IRI. Toosy demonstrated that several short cycles of ischemia and reperfusion before renal ischemia could effectively render the renal resistant to a subsequent ischemic insult, although extensive researches have demonstrated that IPreC reduced renal IRI. However, IPreC is clinically feasible only when the occurrence of ischemia is predictable. It is fail to treat unpredictable accident events. It is need to find out other useful method to amelioration renal IRI.Compared to ischemia, the onset of reperfusion is more predictable. In 2003, Vinten-Johnsen and his colleagues reported that brief episodes of myocardial ischemia and reperfusion employed during reperfusion after a prolonged ischemic insult had been demonstrated to play a cardioprotective role. This phenomenon was termed "Ischemic postconditioning (IPostC)". In a dog model, IPostC limited infarct size, reduced tissue edema and poly morphonuclear neutrophil accumulation in the area at risk myocardium, and improved endothelial function. It has been suggested that IPostC is as effective as IPreC in limiting infarct size and preserving postischemic endothelial function. Numerous studies demonstrated that IPostC was a simple and harmless method which provided a new tool to protect organ from IRI.Therefore, we investigated the protective role and mechanism of ischemic postconditioning against renal IRI in rats and compared the results with IPreC. Our study may not only provide a useful theoretical and practical evidence for reducing renal IRI, but also provide a novel approach for clinic application.MethodsAdult male Sprague-Dawley rats (180～220 g) were randomly separated into four groups: a) Sham group: the right kidneys were removed only; b) IR group: after right nephrectomy, kidneys were subjected to 45 min of ischemia; c) IPreC group: kidneys were subjected to three cycles of 2 min ischemia separated by 5 min reperfusion periods before 45 min of ischemia; d) IPostC group: kidneys were subjected to 45 min of ischemia followed by six cycles of 10 sec reperfusion separated by 10 sec ischemia. Rats were sacrificed and kidneys were harvested after reperfusion. Each group comprised eight rats. The following two aspects were investgated.1. The protective effects of IPostC on renal IRI in rats.Serum Cr and BUN were evaluated by Cr assay kits and BUN assay kits at 24 h after IRI, respectively. The renal pathologic changes were observed by HE-staining at 24 h after IRI.2. The mechanism associated with renal IRIA The antioxidative mechanism of IPostC against renal IRI in ratSOD, CAT, and GSH-Px activity were investigated. GSH and MDA at 1 h, 3 h, 6 h, 12 h, and 24 h were measured. The mRNA and protein expression of SODat 1 h, 6 h and 24 h were evaluated by Real Time RT-PCR and Western blotting.B The antiinflammatory mechanism of IPostC against renal IRI in ratMPO activity at 1 h, 3 h, 6 h, 12 h and 24 h was investigated.The mRNA andprotein expression of SOD at 1 h, 3 h, 6 h, 12 h and 24 h were evaluated by RealTime RT-PCR and Western blotting.C The antiapoptosis mechanism of IPostC against renal IRI in ratApoptosis was compared by TUNEL method; Real time RT-PCR assay for theBcl-2 and Bax mRNA at 6 h was evaluated; Western blotting assay for the proteinexpression of Bcl-2 and Bax at 6 h was evaluated.Results1. The protective effects of IPostC on renal IRI in ratsThe renal function of rats 24 h after IRI was significantly different between groups. Rats subjected to IRI showed significant increase in serum Cr and BUN compared with Sham group (P < 0.05). The renal function changes caused by IRI were significantly improved by IPreC and IPostC treatments (P < 0.05). There were no significant difference between IPreC and IPostC (P > 0.05).Renal IRI resulted in significant renal injury as evidenced by tubular necrosis, medullary hemorrhage, congestion, and development of proteinaceous casts. In contrast, treatments of IPreC and IPostC ameliorated these severe renal damages. According to Jablonski scores, 45 min renal ischemia followed by 24 h reperfusion resulted in severe acute tubular necrosis. Quantitative analysis showed a dramatically decreased score in both IPreC group and IPostC group compared with IRI group (P < 0.05). There were no significant difference between IPreC and IPostC (P> 0.05).2. The antioxidative mechanism of IPostC against renal IRI in ratCompared with Sham group, the I/R group resulted in significant down-regulation of the activities of SOD, CAT, and GSH-Px in renal at 1.5 h, 3 h, 6 h, 12 h and 24 h (P < 0.05); while compared with I/R group, IPreC and IPostC treatments resulted in significant up-regulation of their activities at each time (P < 0.05); compared with Sham group, the IRI rats resulted in significant increases of the levels of MDA, and decreases of GSH in renal tissue at 1 h, 3 h, 6 h, 12 h and 24 h. While compared with I/R group, IPreC and IPostC treatments resulted in significant decreases of the levels of MDA, and increases of GSH at each time in renal tissue (P < 0.05). There were no significant difference between IPreC and IPostC (P> 0.05).The mRNA and protein expression of SOD was evaluated by Real Time RT-PCR and Western blotting. Compared with Sham group, the IRI rats resulted in significant down-regulation of SOD mRNA and protein expression in renal (P < 0.05); while compared with I/R group, IPreC and IPostC treatments resulted in significant up-regulation of SOD mRNA and protein expression at 1 h, 6 h, and 24 h (P < 0.05). There were no significant difference between IPreC and IPostC (P > 0.05).3. The antiinflammatory mechanism of IPostC on renal IRI in ratscompared with Sham group, the IRI rats resulted in significant increases ofthe MPO activity in renal tissue at 1 h, 3 h, 6 h, 12 h and 24 h (P < 0.05); while compared with I/R group, IPreC and IPostC treatments resulted in significant decreases of the MPO activitiy at each time (P < 0.05). There were no significant difference between IPreC and IPostC (P > 0.05).The mRNA and protein expression of COX-1 and COX-2 were evaluated by Real Time RT-PCR and Western blotting. Compared with Sham group, the IRI rats resulted in significant up-regulation of COX-2 expression in renal (P < 0.05); while Compared with I/R group IPreC and IPostC treatments resulted in significant down-regulation of COX-2 expression at 1.5 h, 3 h, 6 h, 12 h and 24 h (P < 0.05). There were no significant difference between IPreC and IPostC (P > 0.05).4. The antiapoptosis mechanism of IPostC against renal IRI in ratcompared with Sham group, the IRI rats resulted in significant increases of the apoptosis cell in renal tubular at 6 h and apoptosis index (AI) was significantly increased (P < 0.05); while compared with I/R group, IPreC and IPostC treatments resulted in significant decreases of the apoptosis cell and AI was significantly decreased (P < 0.05) in renal tubular at 6 h (P < 0.05). There were no significant difference between IPreC and IPostC (P > 0.05).The mRNA and protein expressiones of Bcl-2 and Bax were evaluated by Real Time RT-PCR and Western blotting. Compared with Sham group, the IRI rats resulted in significant down-regulation of Bcl-2 mRNA and protein expression, and up-regulation of Bax mRNA and protein expression in renal and increase of Bcl-2/Bax (P < 0.05); while compared with I/R group IPreC and IPostC treatments resulted in significant up-regulation of Bcl-2 and down-regulation of Bax expression in renal (P < 0.05) and increase of Bcl-2/Bax. There were no significant difference between IPreC and IPostC (P > 0.05).Conclusions1. The function and structure of renal tissue were damaged by renal ischemic reperfusion in rats with the increase of the level of Cr and BUN, the damage of renal tubula and glomerulum, the elevation of MDA and MPO, the activity decreasing of GSH、SOD、CAT and GSH-Px, the decreasing of SGD mRNA and protein expression, the elevation of COX-2 mRNA and protein expression, the increasing of cell apoptosis, the decreasing of Bcl-2 mRNA and protein expression, and the increasing of Bax mRNA and protein expression.2. Postconditioning was effectively protected kidney against IRI with the decrease of the level of Cr and BUN, and the improvement of the renal morphological features.3. The mechanism of ischemic postconditioning against renal IRI may be related to antioxidative effect on the reducing the free radical, inhibiting lipid peroxidation, increasing the activity of antioxidative enzyme and SOD mRNA and protein expression, which improve antioxidative ability of kidney. 4. The mechanism of ischemic postconditioning against renal IRI may be related to anti-inflammatory effect on the inhibiting of PMN aggregation, the mRNA and protein expression of COX-2, and the producing of inflammatory mediator.5. The mechanism of ischemic postconditioning against renal IRI may be associated to antiapoptosis effect by up-regulating Bcl-2 mRNA and protein expression and down-regulating Bax mRNA and protein expression.6. The protective effect and mechanism of postconditoning against renal IRI were similar to that of ischemic preconditioning.7. This study may not only provide a novel approach for clinic application, but also a useful theoretical and practival evidence for treating renal IRI.更多还原