ACM  >> Vol. 9 No. 4 (April 2019)

    探讨慢性肾衰竭的大鼠肾脏UCP2的表达变化与肾脏氧化应激水平变化的关系
    Relationship between Changes of the Expression of UCP2 and Oxidative Stress in the Kidneys of Rats with Chronic Renal Failure

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作者:  

徐亚苓,吴 华,纪玲玲,韩 帅,高弼虎:大连大学附属中山医院,血液净化中心,辽宁 大连

关键词:
慢性肾衰竭氧化应激解偶联蛋白2Chronic Renal Failure Oxidative Stress Uncoupling Protein 2

摘要:

目的:观察慢性肾衰竭大鼠肾脏氧化应激水平的变化与肾脏UCP2表达的变化,探讨二者的相关性。方法:60只雄性SD大鼠随机分为假手术组(sham)和肾衰组(NX),肾衰组行5/6肾大部切除术,假手术组仅行肾包膜剥离术。分别在第4、8、12周时处死大鼠死,留取血液及肾组织,采用ELISA方法检测血清生化指标肌酐(SCr)及尿素氮(BUN),肾组织匀浆中超氧化物气化酶(SOD)、谷胱甘肽过氧化物酶(GSH-px)及丙二醛(MDA),通过Western-blot方法检测肾组织UCP2的表达水平。结果:① 第4、8、12周时,肾衰组大鼠SCr及BUN、水平均显著高于假手术组(P < 0.05)。② 第4、8、12周时,肾衰组大鼠肾组织匀浆中SOD及GSH-px低于假手术组,MDA高于假手术组(P < 0.05)。比较肾衰组,第4和12周时SOD及GSH-px高于第8周、MDA低于第8周(P < 0.05)。③ 第4周时,两组大鼠肾脏UCP2的表达差异无统计学意义(P > 0.05),第8、12周时,肾衰组UCP2的表达水平高于假手术组(P < 0.05),12周时最显著(P < 0.05)。结论:随着病程的进展,慢性肾衰竭大鼠肾脏氧化应激状态愈加严重,肾脏UCP2表达的增加可能是应对氧化应激的一种代偿机制,同时减弱氧化应激状态,在慢性肾衰竭发生过程中起到保护作用。

Objective: To observe the changes of renal oxidative stress level and the expression of UCP2 in the kidney of rats with chronic renal failure, and to explore the potential correlation between them. Methods: Sixty male SD rats were randomly divided into sham operation group (sham) and chronic renal failure group (NX). Chronic renal failure group underwent 5/6 partial nephrectomy while the sham group only underwent renal capsule dissection. At the 4th, 8th and 12th week, rats were sacrificed to sample blood and kidney tissue. Serum Cr (Scr) and BUN, SOD, GSH-px and MDA levels in kidney were detected by using ELISA. The expression of UCP2 in renal tissues was detected by using Western-blotting. Results: 1) At the 4th, 8th, and 12th week, the levels of SCr and BUN in the chronic renal failure groups were significantly higher than that in the sham groups (P < 0.05). 2) At the 4th, 8th and 12th week, the SOD and GSH-px in the renal tissue homogenate of the rats with renal failure were lower than that of the sham operation groups, and the MDA was higher than that of the sham operation groups (P < 0.05). Compared with the renal failure groups, at 4th and 12th week, SOD and GSH-px were higher than at 8th week, and MDA was lower than 8th week (P < 0.05). 3) At the 4th week, there was no significant difference in the expression of UCP2 in the kidney between the two groups (P > 0.05). At the 8th and 12th week, the expression of UCP2 in the renal failure groups was higher than that in the sham operation groups (P < 0.05). The difference was the most significant at the 12th week. Conclusion: With the progress of the disease, the oxidative stress in the kidney of rats with chronic renal failure is more serious. The increase of renal UCP2 expression may be a compensatory mechanism for oxidative stress, and at meanwhile the lower level of oxidative stress plays a protective role in the development of chronic renal failure.

1. 引言

慢性肾衰竭(CRF)是一种严重影响公众健康的全球性难题,各种肾脏病引起的肾脏不可逆性损伤,临床上出现以代谢产物潴留、水、电解质、酸碱平衡紊乱、贫血为主要表现的临床综合征。CRF有证据表明氧化应激是CRF发生发展的重要病理生理机制之一,而CRF患者体内的尿毒素同时又增加了氧化应激状态,二者相互联系、相互影响。氧化应激是指体内活性氧(ROS)生成增加或清除下降导致ROS的生成和清除失去平衡。线粒体是细胞中活性氧的主要来源,UCP2是线粒体内膜上的阴离子载体蛋白,当它被激活时可导致质子漏现象 [1] ,降低质子驱动力,从而减少线粒体ROS的产生。本实验通过5/6肾大部切除术建立慢性肾衰竭大鼠模型,探讨慢性肾衰竭大鼠肾脏UCP2的表达变化和肾脏氧化应激之间可能的相关性。

2. 材料与方法

1) 60只雄性SD大鼠随机分为假手术组(sham)和肾衰组(NX)各30只,肾衰组行5/6肾大部切除术,用10%水合氯醛腹腔注射麻醉后,沿脊柱做切口,切除左侧肾脏上、下极各约1/3的肾组织。右肾用丝线结扎肾门后行右肾全切术。假手术组仅行肾包膜剥离术。分别在第4、8、12周时处死大鼠,留取血液及肾组织,检测血清生化指标肌酐(SCr)及尿素氮(BUN),采用ELISA方法 [2] 检测肾组织匀浆中超氧化物气化酶(SOD)、谷胱甘肽过氧化物酶(GSH-px)及丙二醛(MDA),通过Western-blot方法 [3] 检测肾组织UCP2的表达水平,比较两组大鼠肾组织氧化应激水平的变化及UCP2的表达变化。

统计学方法

采用SPSS22.0版统计学软件进行统计学分析,计量资料采用均数±标准差( X ¯ ± S )表示,组间比较采用单因素方差分析、两独立样本t检验,以P < 0.05为差异具有统计学意义。

3. 结果

3.1. 肾功能

4周时,肾衰组和假手术组大鼠的SCr和BUN比较,SCr (P = 0.001)、BUN (P = 0.000),8周时,SCr (P = 0.000)、BUN (P = 0.000),12周时,SCr (P = 0.000)、BUN (P = 0.000),差异有统计学意义(均P < 0.05)。SCr (P = 0.762)和BUN (P = 0.073)水平在各假手术组间比较,差异均无统计学意义(P > 0.05)。但在各肾衰组间比较,12周时SCr (P = 0.000)和BUN (P = 0.017)显著高于8周、8周时SCr (P = 0.004)和BUN (P = 0.000)显著高于4周,差异有统计学意义(P < 0.05)。见表1

Table 1. Comparison of biochemical parameters among different rat groups ( X ¯ ± S , n = 10)

表1. 各组大鼠生化指标变化( X ¯ ± S , n = 10)

P < 0.05与假手术组比较;P < 0.05与肾衰4周组比较;P < 0.05与肾衰8周组比较。

3.2. 肾组织氧化应激水平

4周时,肾衰组和假手术组大鼠肾组织匀浆中SOD、GSH-px及MDA比较,SOD (P = 0.045)、GSH-px (P = 0.004)、MDA (P = 0.000),8周时,SOD (P = 0.009)、GSH-px (P = 0.000)、MDA (P = 0.000),12周时,SOD (P = 0.001)、GSH-px (P = 0.000)、MDA (P = 0.000),差异有统计学意义(均P < 0.05). SOD (P = 0.946)、GSH-px (P = 0.670)和MDA (P = 0.850)水平在各肾衰组间比较,差异无统计学意义(均P > 0.05)。在各肾衰组间比较,从第4周到12周SOD先下降(P = 0.07)差异无统计学意义(P > 0.05),后上升(P = 0.03)差异有统计学意义(P < 0.05),GSH-px也先下降(P = 0.000)后上升(P = 0.003),MDA先升高(P = 0.016)再下降(P = 0.001),差异具有统计学意义(均P < 0.05)。见表2

3.3. 肾组织UCP2的表达水平

第4周时,肾衰组和假手术组大鼠肾脏UCP2的表达差异无统计学意义(P > 0.05),第8、12周时肾衰组UCP2的表达高于假手术组,差异有统计学意义(P < 0.05)。UCP2水平在假手术组间比较差异无统计学意义(P > 0.05)。在肾衰组间比较,12周显著高于8周,差异有统计学意义(P < 0.05)。见图1图2

Table 2. Comparison of renal oxidative stress among different rat groups ( X ¯ ± S , n = 10)

表2. 各组大鼠肾脏氧化应激水平变化( X ¯ ± S , n = 10)

P < 0.05与假手术组比较;P < 0.05与肾衰4周组比较;P < 0.05与肾衰8周组比较。

Figure 1. Expression of UCP2 in the kidneys of rats in each rat group

图1. 各组大鼠肾脏中UCP2的表达水平

与假手术8周组比较,*P < 0.05;与假手术12周组比较,#P < 0.05。

Figure 2. Western-blot strip absorbance scan results

图2. Western-blot条带吸光度扫描结果

4. 讨论

高血压、蛋白尿、纤维化和氧化应激以复杂的方式交织在一起导致CRF的进展。氧化应激是细胞内氧化还原稳态被破坏的状态,也就是ROS产生过量或抗氧化剂缺乏引起的不平衡。Rubattu [4] 等人研究表明,ROS可能通过激活Smad或MAPK途径在TGF-β1诱导的肾小管上皮细胞转化为间质细胞中发挥作用。游离氧自由基可引起肾小球系膜细胞、足细胞、内皮细胞及肾小管上皮细胞脂质过氧化、蛋白质变性、DNA损伤 [5] ,而抗氧化剂维生素D可降低尿毒症大鼠肾脏的氧化应激 [6],以上研究均提示,氧化应激是加重慢性肾衰竭的重要因素。

慢性肾衰竭时患者氧化应激的增加是多种因素引起的,如营养不良导致维生素的流失,糖尿病及高血压的存在,尿毒症毒素的积累,慢性炎症,肾脏替代治疗 [7] ,肾脏线粒体功能障碍,肾素–血管紧张素–醛固酮和交感神经系统的激活,均可产生大量ROS引起氧化应激反应。此外,慢性肾衰竭患者抗氧化系统功能损害也加速氧化应激损伤 [8] 。MDA是ROS攻击细胞膜多不饱和脂肪酸所形成的脂质过氧化物 [9] 。SOD、GSH-px能够将有毒的过氧化物还原成水和氧气,属于天然抗氧化剂。三者间接反映了机体氧化/抗氧化系统的平衡。本实验发现,第4、8、12周时肾衰组大鼠SOD、GSH-px显著低于假手术组,MDA显著高于假手术组,提示慢性肾衰竭可引起大鼠肾脏氧化/抗氧化失衡,ROS水平逐渐增加。比较假手术组,第4、8、12周时SOD、GSH-px及MDA差异均无统计学意义,说明年龄对肾脏氧化应激的变化影响不大。比较肾衰组,从第4到第8周,氧化应激状态愈加严重,第12周时,氧化应激状态有所缓解。

UCP2是位于线粒体内膜上的阴离子载体蛋白,通过解偶联作用抑制线粒体ROS的产生。UCP2基因敲除小鼠的脾细胞更容易受到病原体感染导致细胞凋亡,且UCP2基因敲除小鼠中高水平的活性氧可能是细胞凋亡的原因 [10] 。小檗碱刺激AMPK活性,从而上调UCP2的表达,导致胰岛素瘤细胞中氧化应激减少 [11] ,以上研究均提示UCP2具有负调控ROS的作用 [12] 。另外,有研究发现,CoQ在分离的线粒体中增加质子传导,需要脂肪酸的参与,并被GDP抑制,CoQ在可能被还原时激活,但在可能被氧化时则不被激活,表明CoQ可能通过产生超氧化物来介导解偶联。用黄嘌呤加黄嘌呤氧化酶(一种产生超氧化物的外源物质)代替CoQ,质子传导速率增加,并且可以被超氧化物歧化酶所抑制,说明超氧化物可刺激线粒体解偶联蛋白2的表达 [13] 。本实验发现,4周时两组大鼠肾脏UCP2的表达差异无统计学意义,8周时肾衰组肾脏UCP2的表达高于假手术组,而此时肾衰组肾脏氧化应激最严重,提示ROS增加到一定程度时可以刺激肾脏UCP2的表达。UCP2水平在假手术组间比较差异无统计学意义,说明年龄对UCP2的表达影响不大。比较肾衰组,12周时UCP2水平最高,而此时肾脏氧化应激较8周时有所缓解,提示UCP2的表达增加可能减弱氧化应激状态,在一定程度上可保护肾功能,本研究与上述研究结果基本一致。

总之,慢性肾衰竭可导致氧化应激状态愈加严重,而UCP2表达增加可能是应对氧化应激的一种代偿机制,同时可以减弱氧化应激状态,在慢性肾衰竭发生过程中起保护作用,但具体机制仍有待进一步研究。

文章引用:
徐亚苓, 吴华, 纪玲玲, 韩帅, 高弼虎. 探讨慢性肾衰竭的大鼠肾脏UCP2的表达变化与肾脏氧化应激水平变化的关系[J]. 临床医学进展, 2019, 9(4): 600-605. https://doi.org/10.12677/ACM.2019.94091

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