非诺贝特和瘦素对高血压肾脏和腹主动脉损伤的保护作用及机制的研究
|
摘要
背景
在过去10年中,随着人口老龄化及生活水平的提高,高血压患者日益增多,高血压性肾损害发病率也在明显上升。目前,高血压是仅次于糖尿病的第二大致终末期肾病(end-stage renal disease, ESRD)的病因,占所有ESRD的百分之三十。相关统计资料表明,每13个高血压患者就有1例发展成ESRD,在接受降压治疗的高血压患者中,4%-16%的患者蛋白尿排泄异常。虽然近年来高血压的治疗取得了很大的进展,但是高血压所导致的肾损害的发生率却呈逐年上升的趋势,提示临床上应用抗高血压药物治疗的肾保护作用还远远不够。因此,如何降低高血压肾损伤的发病率是目前基础研究和临床实践的重要课题。
高血压肾脏损伤主要表现为蛋白尿、良性肾小球硬化、间质纤维化以及炎症细胞浸润。除了神经内分泌激素,如去甲肾上腺素、血管紧张素Ⅱ以及醛固酮等,氧化应激也是高血压肾病发生和进展的重要因素。氧化应激不但可以通过脂质过氧化等直接损伤肾脏组织,还能作为第二信使激活细胞内信号转导系统,表达相应的活性产物,诱导炎症细胞浸润,引起炎症反应间接损伤肾脏组织。在氧化应激诱导产生的各种活性产物中,TGF-β1起关键性作用。TGF-β1不但可以调节细胞外基质的合成和降解,还可以诱导小管上皮细胞发生上皮-间充质转分化,最终引起细胞外基质的过度沉积。而细胞外基质的过度沉积是高血压肾病的主要特征。
PPARs是1990年发现的核激素受体超家族的新成员,有三种亚型：PPARα、PPARβ/δ和PPARγ。其中PPARα于肾脏、肝脏和心脏中高表达,并在调节糖脂代谢中起重要作用。PPARα激动剂在临床上是常用的降甘油三酯药物。近年来发现PPARα激动剂还有抗氧化的作用,并且能抑制高血压心肌重构,但是PPARα激动剂对高血压肾损伤是否有保护作用及其机制还未有报道。
针对以上问题,我们提出如下假说：PPARα激动剂非诺贝特能够抑制高血压肾损伤的进展；其机制可能是通过抑制氧化应激和MAPK活性从而抑制下游TGF-β1、TIMP-1的表达、间质成纤维细胞的活化以及炎症细胞浸润所引起的炎症反应。
目的
1、观察26周龄SHR肾脏结构和功能的损伤程度；
2、观察PPARα激动剂非诺贝特是否对SHR肾脏损伤具有保护作用；
3、探讨PPARα激动剂非诺贝特对SHR肾脏保护作用的可能机制。
方法
1.实验动物和分组：八周龄雄性SHR大鼠24只,随机分为阳性对照组即SHR组(n=10)和非诺贝特组(n=14)；同周龄WKY大鼠10只作为正常对照组即WKY组(n=10)。非诺贝特组给予非诺贝特(力平之)60mg·kg-1·d-1灌胃18周,WKY组和SHR组以等量生理盐水灌胃。
2.体重和血压：实验过程中,每两周测量体重1次,每两周检测尾动脉血压1次。
3.尿蛋白和肾功能的检测：实验末留取24小时尿检测24小时尿蛋白排泄率；实验末抽取右心室静脉血检测血清肌酐和尿素氮含量。
4.病理形态染色：应用PAS染色观察皮质肾小球硬化；应用Masson染色观察皮质肾小管间质纤维化。光学显微镜采集图像,并用Image ProPlus 5.0作定量分析。
5.免疫组化染色：应用免疫组化染色观察间质成纤维细胞的活化和单核／巨噬细胞在肾脏皮质中的浸润。将处理好的组织切片用含有α-SMA和ED1一抗的1%BSA进行孵育,并用辣根过氧化物酶标记的二抗进行处理。使用DAB显色,光学显微镜采集图像。
6.MDA检测：肾皮质组织匀浆的制备,应用考马斯亮兰蛋白法测定肾皮质组织匀浆蛋白浓度；采用硫代巴比妥酸法检测肾皮质组织MDA的含量。
7.SOD活性检测：肾皮质组织匀浆的制备,应用考马斯亮兰蛋白法测定肾皮质组织匀浆蛋白浓度；采用黄嘌呤氧化酶法检测肾皮质组织T-SOD、Cu/Zn-SOD和Mn-SOD的活性。
8.蛋白印迹分析：使用组织裂解液提取肾皮质组织总蛋白。将含有30μg蛋白的每个标本和蛋白Marker一起分别加入不同的泳道。在10%SDS-聚丙烯酰胺凝胶上电泳,再用一湿电转染仪电转染到聚偏氟乙烯膜(PVDF)上。将膜封闭后,使用一抗孵育,清洗后,再使用HRP结合的二抗孵育。滴加显色液显色。强弱用表达的蛋白和β-actin条带积分光密度的比值表示。
9.实时定量PCR：使用Trizol提取肾皮质组织总RNA。应用逆转录酶将mRNA逆转录成cDNA。使用Applied Biosystems TaqMan 7900HT detection系统进行实时PCR。采用MCP-1、Cu/Zn-SOD和Mn-SOD与β-actin的循环阈值(threshold cycle, Ct)的比值并通过公式：起始模板浓度=2-ΔΔCT。注：ΔΔCT=(干预组目的基因CT值-β-actin CT值)-(对照组目的基因CT值-β-actin CT值),表示mRNA的相对水平。
结果
1. PPARα激动剂非诺贝特降低SHR 24小时尿蛋白排泄率：相比于WKY大鼠,26周龄SHR表现为低体重、蛋白尿和血压升高,而肾功能晚期指标肌酐和尿素氮正常；非诺贝特显著降低SHR 24小时尿蛋白,但是对血压和体重没有明显作用。
2. PPARα激动剂非诺贝特抑制SHR皮质肾小球硬化和间质纤维化：PAS染色结果表明WKY大鼠没有肾小球硬化,而SHR有少量肾小球出现节段性硬化；Masson染色结果表明SHR肾小管间质明显纤维化；免疫组化结果表明SHR肾小球和肾小管间质中α-SMA表达明显增加,说明间质中成纤维细胞活化,合成和分泌细胞外基质的功能增强。非诺贝特显著抑制SHR皮质肾小球硬化、肾小管间质纤维化以及间质中成纤维细胞的活化。
3. PPARα激动剂非诺贝特抑制SHR肾皮质细胞外基质重构：免疫印迹结果表明,相比于WKY大鼠,SHR肾皮质胶原Ⅳ. MMP-9和TIMP-1表达均升高,非诺贝特显著抑制它们在高血压大鼠肾皮质中的表达。
4. PPARα激动剂非诺贝特抑制SHR肾脏间质炎症细胞的浸润：实时定量PCR结果表明,与WKY组大鼠比较,SHR组大鼠肾皮质MCP-1的mRNA表达上调；非诺贝特明显抑制SHR肾皮质MCP-1 mRNA的表达；免疫组化结果表明,相比于WKY大鼠,SHR皮质肾小球和肾小管间质中单核／巨噬细胞明显增多,非诺贝特显著抑制单核巨噬细胞的浸润。说明非诺贝特能够抑制SHR肾皮质内的炎症反应。
5. PPARα激动剂非诺贝特抑制SHR肾皮质组织氧化应激损伤：相比于WKY大鼠,SHR肾皮质组织MDA水平明显升高；非诺贝特显著降低SHR肾皮质组织MDA水平,说明非诺贝特能够抑制SHR肾皮质氧化应激损伤。
6. PPARα激动剂非诺贝特抑制SHR肾皮质NADPH氧化酶亚单位P47phox的表达并增加Cu/Zn-SOD活性和表达：相比于WKY大鼠,SHR肾皮质组织P47phox的蛋白表达明显增加,而Cu/Zn-SOD活性及mRNA水平明显降低,Mn-SOD活性及mRNA水平没有明显改变,提示SHR肾皮质不但有氧化系统激活,而且抗氧化系统减弱,最终导致肾皮质氧化应激损伤；非诺贝特显著抑制P47phox的蛋白表达,并增加Cu/Zn-SOD活性及mRNA的表达,从而减轻肾皮质氧化应激损伤。
7. PPARα激动剂非诺贝特抑制SHR肾皮质组织TGF-β1表达和MAPK活性：免疫印迹结果表明,相比于WKY大鼠,SHR肾皮质组织TGF-β1表达增加,磷酸化的JNK和p38MAPK明显增加,而总JNK和p38MAPK没有明显变化；非诺贝特显著抑制SHR肾皮质组织TGF-β1的表达及JNK和p38MAPK的磷酸化。
结论
1.26周龄SHR肾脏损伤表现为蛋白尿、轻度皮质肾小球硬化、肾小管间质纤维化和炎症细胞浸润,基本符合人类高血压早期肾损伤的病理改变。
2. PPARα激动剂非诺贝特显著降低SHR尿蛋白排泄率,减轻皮质肾小球硬化、肾小管间质纤维化和炎症细胞浸润,对高血压肾脏具有保护作用。
3. PPARα激动剂非诺贝特对SHR的肾保护作用可能是通过抑制氧化应激和MAPK活性从而抑制下游TGF-β1、TIMP-1的表达以及炎症细胞浸润所引起的炎症反应。
4. PPARα激动剂非诺贝特的抗氧化作用对氧化应激性肾脏损伤具有潜在的治疗作用。
背景
腹主动脉瘤(andominal aortic aneurysm, AAA)是局部血管壁膨胀,超过正常血管直径的50%的一种病理状态。AAA的病理学改变主要表现为炎症细胞的侵入,动脉中层弹力蛋白基质结构的破坏和平滑肌细胞减少。在美国60岁以上人群的年发病率约为3%,每年大约46,000人需要手术治疗,导致15,000人死亡。国内的发病率也呈逐年上升的趋势。目前临床上尚没有被证明有效的AAA治疗药物.治疗选择仅限于腔内或有创手术治疗。由于这种高危疾病给人们带来的危害随着生活人类生活水平的提高和人口老龄化而不断增加,关于AAA发病机制和找到可阻断或延缓这一过程的有效药物成为近年来研究的热点。
近年来对AAA的病因及病理生理学已有了崭新的认识,传统的动脉粥样硬化学说并不符合AAA的发病特点,多因素致病,复杂的退行性病变包括慢性炎症浸润、细胞外基质的重塑成为共识。其中多种蛋白水解酶家族对血管壁细胞外基质的降解是多种原因诱发AAA的最终途径,其中基质金属蛋白酶家族是具有最关键作用的蛋白降解酶家族。来自不同团队的多个研究表明人类AAA是一种慢性炎症性疾病,表现为Th2型细胞因子(IL-4)占主导低位,而Th1型细胞因子(尤其是INF-γ)减少。因此,如何抑制Th2型细胞因子在AAA中的表达将是治疗AAA的新的策略。
瘦素(Leptin)是由肥胖(obese, ob)基因编码、脂肪细胞分泌的一种非糖基化的多肽激素,通过与中枢及外周组织的受体结合,在能量代谢、神经内分泌系统、生殖系统、心血管系统以及骨骼形成等方面发挥重要的调节作用,具有重要的生理病理意义。近年来的研究表明,瘦素作为一种细胞因子样激素在免疫系统中所发挥的作用越来越受到关注。瘦素可以促进T细胞的增殖、活化和细胞因子合成,促使Th细胞向Th1方向分化,并抑制Th2型细胞因子的释放。目前已有研究证实瘦素可以通过调节Th1/Th2促进动脉粥样硬化斑块的形成,但是瘦素对动脉瘤的作用目前还未见报道。
针对以上问题,我们提出如下假说：瘦素能够抑制AAA的形成和进展,其作用机制可能是通过诱导Th1型细胞因子,同时抑制Th2型细胞因子来起作用。
目的
1.构建AngⅡ诱导ApoE-/-小鼠高血压AAA模型；
2.观察Leptin是否对AngⅡ诱导的ApoE-/-小鼠高血压AAA具有保护作用；
3.探讨Leptin对AngⅡ诱导ApoE-/-小鼠高血压AAA保护作用的可能机制。
方法
1. AngⅡ诱导的ApoE-/-小鼠高血压AAA模型的构建及Leptin的干预：八周龄雄性ApoE-/-小鼠皮下埋装有AngⅡ或Saline的渗透泵。根据体重调节AngⅡ的浓度,使AngⅡ的泵速维持在1000ng/kg·min,持续输注28天；同时每天腹膜下注射Leptin (0.6μg/g BW)或Saline,输注28天。
2.体重和血压：实验过程中,每周测量体重1次,每周检测尾动脉血压1次。
3.血清脂质的检测：实验末抽取右心室静脉血检测血清甘油三酯、胆固醇、低密度脂蛋白胆固醇、游离脂肪酸的浓度。
4.AAA的测量：通过Image ProPlus 5.0软件测量右肾动脉至最后一个肋间动脉之间,肾上腹主动脉的最大宽度。将腹主动脉最大宽度超过正常小鼠肾上动脉宽度的50%定义为动脉瘤。
5.病理形态染色：应用Verhoeff弹力纤维染色观察AAA的病理改变,光学显微镜采集图像。
6.免疫组化染色：应用免疫组化染色观察Th1 (INF-γ)/Th2 (IL-4)型细胞因子的表达及定位。将处理好的组织使用含有INF-γ或IL-4一抗的1%BSA进行孵育,并用辣根过氧化物酶标记的二抗进行处理。使用DAB显色,光学显微镜采集图像。
7.蛋白印迹分析：使用组织裂解液提取小鼠腹主动脉组织总蛋白。将含有30μg蛋白的每个标本和蛋白Marker一起分别加入不同的泳道。在10% SDS-聚丙烯酰胺凝胶上电泳,再用一湿电转染仪电转染到聚偏氟乙烯膜(PVDF)上。将膜封闭后,使用一抗孵育,清洗后,再使用HRP结合的二抗孵育。滴加显色液显色。强弱用表达的蛋白和β-actin条带积分光密度的比值表示。
结果
1. Leptin对AngⅡ持续输注诱导的高血压没有明显作用：相比于ApoE-/-组小鼠,ApoE-/-+AngⅡ组小鼠血压明显升高；腹腔注射Leptin对ApoE-/-+AngⅡ小鼠血压没有明显作用。
2. Leptin对血清脂质没有明显影响：相比于ApoE-/-组小鼠,ApoE-/-+AngⅡ组小鼠血清胆固醇、低密度脂蛋白胆固醇的含量明显升高,而游离脂肪酸、甘油三酯无明显变化；Leptin对ApoE-/-小鼠血清脂质无明显影响。
3. Leptin降低ApoE-/-+AngⅡ小鼠AAA的发生率并减轻其严重程度：持续输注AngⅡ诱发巨大AAA形成；腹膜腔注射Leptin可以降低输注AngⅡ诱发的AAA的发生率和严重程度。
4. Leptin降低ApoE-/-+AngⅡ小鼠AAA组织中MMP-2和MMP-9蛋白的表达：相比于ApoE-/-组小鼠,ApoE-/-+AngⅡ组小鼠AAA组织中MMP-2和MMP-9蛋白的表达明显升高；腹膜腔注射Leptin可以显著降低ApoE-/-+AngⅡ小鼠AAA组织中MMP-2和MMP-9蛋白的表达。
5. Leptin降低ApoE-/-+AngⅡ小鼠AAA组织中Th2型细胞因子IL-4蛋白的表达:Western blot结果证实ApoE-/-+AngⅡ小鼠AAA组织中IL-4的表达较对照组明显增加；腹膜腔注射Leptin可以显著降低IL-4的表达。免疫组织化学结果与Western blot结果一致。
6. Leptin增加ApoE-/-+AngⅡ小鼠AAA组织中Thl型细胞因子IFN-γ蛋白的表达：Western blot结果证实ApoE-/-+AngⅡ小鼠AAA组织中IFN-γ的表达较对照组无明显增加；腹膜腔注射Leptin可以显著增加IFN-γ的表达。免疫组织化学结果与Western blot结果一致。
结论
1.AngⅡ持续输注能够诱发ApoE-/-小鼠高血压AAA的形成。
2. Leptin降低AngⅡ诱导的ApoE-/-小鼠高血压AAA的发生率及严重程度,对血压和血脂无明显作用。
3. Leptin对高血压AAA的保护作用可能是通过调节Th1/Th2型细胞因子的表达：Leptin促进Thl型细胞因子的表达,抑制Th2型细胞因子的表达,从而抑制其诱导的MMP-2和MMP-9的表达对血管壁的弹性蛋白和胶原蛋白的降解。
4. Leptin的免疫调节作用可能对高血压AAA具有潜在的治疗作用；该结果也支持动脉粥样硬化和动脉瘤是两种不同的血管疾病,具有不同的致病机制的观点。
Backgroud
Hypertension is an important risk factor for the progression of glomerular and tubulointerstitial diseases to chronic renal failure. The progression of hypertensive renal disease displays several characteristics, including proteinuria, inflammatory cell recruitment, and accumulation of extracellular matrix (ECM) proteins in the interstitium. Oxidative stress has been shown to play an important role in the development of renal injury, because it can stimulate the expression of proinflammatory and profibrotic molecules in addition to direct toxic effects. Conversely, antioxidants can attenuate the development of kidney injury.
Peroxisome proliferator-activated receptors (PPARs), which include three membersα,γ, andβ/δ, are ligand-activated transcription factors belonging to the nuclear receptor superfamily. On activation by their ligands, PPARs bind to specific PPAR response elements (PPREs) in the promoter region of their target genes. PPARa, which is highly expressed in kidney, liver, and heart, has been shown to take part in diverse physiological processes, including maintenance of lipid and glucose homeostasis. In addition, PPARa also exerts antioxidant and anti-inflammatory effects. Activation of PPARa inhibits angiotensinⅡ-induced activation of NADPH oxidase and suppressed ROS production in the vascular wall. Furthermore, a PPAR-responsive element (PPRE) has been identified in promoter regions of catalase and Cu/Zn-SOD genes, which are key enzymes that reduce ROS production. Also, PPARa plays crucial roles in the attenuation of inflammatory response in activated mesangial cells through its antagonizing effects on the NF-κB signaling pathway. Furthermore, PPARa-deficient mice show a prolonged response to inflammatory stimuli, suggesting that PPARa is also a modulator of inflammation. These studies have identified PPARa as a naturally occurring regulator of oxidative stress and inflammation. However, little is known about the effect of PPARa on chronic hypertensive renal damage.
Taken together, we hypothesized that PPARa agonist fenofibrate might inhibit hypertensive renal injury by regulating oxidative stress and inflammation. The results from this study indicate that fenofibrate is able to inhibit renal injury in SHRs without affecting blood pressure by ameliorating renal inflammation and TGF-β1 expression via inhibition of oxidative stress and MAPK activity.
Methods
1. Animal models and experimental design:Twenty-four 8-week-old male SHRs and ten 8-week-old male Wistar-Kyoto (WKY) rats were used in this study. SHRs were divided into the following two groups:1) SHRs given normal oral 0.9% saline (n=10); 2) SHRs given oral fenofibrate at a dose of 60 mg·kg-1·d-1 dissolved in 0.9% saline (n=14). The WKY rats (n=10) given 0.9% saline were used as controls. The compound or vehicle was orally administered in 2 ml-kg"1 once a day for 18 weeks.
2. Assessment of body weight and systolic blood pressure:Body weight was determined every two weeks throughout the study. Systolic blood pressure (SBP) was assessed every two weeks throughout the study using a tail-cuff method in conscious rats after prewarming at 38℃for 10 min.
3. Assessment of UAE, BUN and Creatinine:At the end of the study, the animals were housed in metabolic cages for 24 h to collect urine for subsequent measurements of urinary protein excretion by an immunoassay, and then were anesthetized with pentobarbital, blood samples were collected from the right ventricle and serum was stored at -80℃for subsequently analyzing creatinine and blood urea nitrogen concentrations.
4. Histopathological study:Coronal sections of renal tissue (3 to 4-μm-thick) were stained with periodic acid-Schiff (PAS) and Masson trichrome for analysis of glomerular sclerosis and tubulointerstitial fibrosis, and examined by light microscopy in a blinded fashion. A semiquantitative morphometric score index was used to evaluate the degree of glomerulosclerosis. Tubulointerstitial fibrosis was assessed semiquantitatively.
5. Immunohistochemical staining:Four micrometer-thick sections of 10% formalin fixed tissues were stained with antibodies as follows:mouse anti-α-SMA antibody and mouse anti-ED1 antibody. Briefly, sections were deparaffinized, washed with PBS, and incubated with 3%H2O2 in methanol to block endogenous peroxidase activity. Then, Sections were incubated overnight with the anti-a-SMA and anti-ED1 antibody in a humidified chamber at 4℃. The localization of the first antibody was visualized by an indirect immunoperoxidase method. All of these sections were examined in a masked manner using a light microscopy.
6. Assessment of MDA levels in renal cortex:MDA levels were detected by the thiobarbituric acid method. Absorbance was measured at 532 nm by spectrometry. All protein concentrations of renal tissue homogenate samples were determined with the coomassie blue method.
7. Assessment of superoxide dismutase activity:SOD activity was measured by the xanthine oxidase method. Absorbance was determined at 550 nm by spectrometry. All protein concentrations of renal tissue homogenate samples were determined with the coomassie blue method.
8. Western blot analysis:Tissue samples from the kidneys were prepared with lysis buffer. Protein samples (30μg per lane) were subjected to SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were blocked, treated with primary antibody, washed, and then incubated with the secondary horseradish peroxidase-labeled antibody. Bands were visualized with Enhanced Chemiluminescence. The expression of protein was demonstrated by the ratio of integral optical density (IOD) between specific protein andβ-actin.
9. Real-time quantitative PCR:Total RNA from Tissue samples was extracted with Trizol, according to the manufacturer's protocol. The mRNAs were reverse-transcripted into cDNAs using cDNA synthesis kit. Real-time PCR was performed using Applied Biosystems TaqMan 7900HT detection system. The mRNA levels were estimated from the value of the threshold cycle (Ct) of the real-time PCR adjusted by that ofβ-actin through the formula 2ΔCt(ΔCt=gene of interest Ct-β-actin Ct)-(gene of control-β-actin Ct)
Results
1. Fenofibrate treatment attenuates the elevation in levels of urine albumin excretion rate without an apparent effect on blood pressure in SHRs:SHRs had an elevation in levels of urine albumin excretion rate compared with WKY rats and fenofibrate treatment significantly prevented the rise in these values. However, no differences in levels of serum creatinine and blood urea nitrogen were observed among these three groups. SHRs with or without fenofibrate treatment had significantly higher blood pressure compared with WKY rats. However, no significant differences in blood pressure were observed between SHRs with and without fenofibrate treatment. SHRs with or without fenofibrate treatment had significantly lower body weights compared with WKY rats.
2. Fenofibrate ameliorates glomerulosclerosis and tubulointerstitial fibrosis in SHRs:PAS staining showed that segmental glomerulosclerosis was present only in a small number of glomeruli in the SHR group and absent in the WKY group. Moreover, Masson's trichrome staining showed significant tubulointerstitial fibrosis in SHRs. Fenofibrate administration significantly reduced the severity of glomerulosclerosis and tubulointerstitial fibrosis in SHRs. These observations were confirmed by quantitative analysis. Immunohistochemical staining for a-SMA showed that myofibroblasts in the glomeruli and tubulointerstitial space were significandly increased in SHRs and were markedly decreased by fenofibrate.
3. Fenofibrate prevents collagen expression and deposition in SHRs: Representative western blots showed that collagenⅣ, MMP-9 and TIMP-1 were significantly increased in the kidney of SHRs. However, fenofibrate treatment reduced the expression of collagenⅣ, MMP-9 and TIMP-1.
4. Fenofibrate treatment reduces inflammatory cell accumulation in SHRs: Comapred with WKY group, MCP-1 mRNA levels were significantly increased in SHRs and was decreased by fenofibrate treatment. And, SHRs had a significant accumulation of ED1-positive cells in the glomeruli and tubulointerstitial space. Fenofibrate treatment markedly attenuated monocytes/macrophages accumulation in the kidney.
5. Fenofibrate attenuates oxidative stress and increases renal SOD activity and mRNA levels in SHRs:Oxidative stress was evaluated by determination of the levels of MDA in the kidney, an index of lipid peroxidation. MDA levels were markedly increased in the kidney of SHRs and were decreased by fenofibrate treatment. Cu/Zn SOD activity and mRNA levels were significantly decreased in SHRs and was increased by fenofibrate treatment. However, Mn-SOD activity and mRNA levels were similar in these three groups. Protein levels of p47phox, a NAD(P)H oxidase subunits, in the kidney paralleled with MDA levels.
6. Fenofibrate reduces TGF-β1 expression and p38 MAPK and JNK phosphorylation in SHRs:The effect of fenofibrate on TGF-β1 Expression and the phosphorylation of p38 MAPK and JNK were investigated. Representative western blots showed that TGF-β1 Expression, p38 MAPK and JNK phosphorylation were significantly increased in the kidney of SHRs and reduced by fenofibrate treatment. However, no differences in protein levels of total p38 MAPK and JNK were observed among these three groups.
Conclusions
1. Early renal injury in SHRs was characterized by mild proteinuria, glomerular sclerosis, tubulointerstitial fibrosis, inflammatory cells recruitment and normal renal function.
2. PPARa agonist fenofibrate exerted renoprotective effects against hypertensive renal injury in SHRs.
3. The renoprotective effects of PPARa agonist fenofibrate may be due to the inhibition of TGF-β1 expression and inflammatory cell recruitment through suppression of NADPH oxidase activity, upregulation of Cu/Zn SOD activity, and, thus, inhibition of phosphorylation of p38 MAPK and JNK.
4. Fenofibrate, as a potent antioxidant, may have therapeutic potential for the treatment of oxidative kidney damage, such as hypertensive renal injury.
Backgroud
Aortic aneurysms are permanent and localized aortic dilations defined as having diameters 1.5-times greater than normal. Important histological features of aneurysms include chronic adventitial and medial inflammatory cell infiltration, elastin fragmentation and degeneration, and medial attenuation. In the United States alone abdominal aortic aneurysms(AAA) affect 3% of individuals 60 years or older, necessitate 46,000 surgical interventions, and cause～15,000 deaths annually.1 Despite considerable descriptive knowledge of the pathomorphology of AAA, insufficient understanding of the molecular mechanisms underlying its pathogenesis currently limits the prevention and treatment of this human disease.
Recent human studies from several groups indicate that human AAAs comprise an inflammatory disease characterized by the predominance of T helper cell type 2 (Th2) cytokine expression, especially IL-4 and the paucity of Th1 cytokines, especially interferon-γ(IFN-γ). Animal studies show that aortic allografts deficient in interferon-γ(IFN-γ) signaling developed AAA correlating with skewed Th2 cytokine environments, suggesting important regulatory roles for Th1/Th2 cytokine balance in modulating matrix remodeling and important implications for the pathophysiology of aortic aneurysm and atherosclerosis.
Leptin, a cytokine-like hormone produced primarily by adipocytes, has been clearly demonstrated to play an important role in body weight regulation through effects on feeding and energy expenditure. In addition to central effects of leptin on appetite and metabolism, several studies have described direct leptin effects on immune cells, including the promotion of T lymphocyte type 1 helper (Th1) response, of potential importance to the process of atherosclerosis. Importantly, ob/ob mice have reduced secretion of IL-2, LFN-γ, TNF and IL-18, and increased production of TH2-type cytokines, such as IL-4 and IL-10, after mitogenic stimulation.
Thus, we hypothesized that leptin, with the effect of regulation of Thl/Th2 cytokines, may have protective effect on the formation of AAA induced by AngⅡin ApoE-/- mice.
Methods
1. Animal models and experimental design:8 weeks old ApoE-/- mice were infused with saline or AngⅡ(1000 ng/kg/min) for 4 weeks and/or intraperitoneal injections daily with 0.6μg/g BW of recombinant murine leptin or vehicle control (n=8 per group).The dose of leptin chosen was based on a protocol used to achieve weight loss and fertility in leptin-deficient mice.
2. Assessment of systolic blood pressure:Systolic blood pressure (SBP) was assessed every weeks throughout the study using a tail-cuff method in conscious rats after prewarming at 38℃for 10 min.
3. Assessment of blood lipid:At the end of the study, the animals were anesthetized with pentobarbital, blood samples were collected from the right ventricle and serum was stored at -80℃for subsequently analyzing TG, TC, LDL-C and FFA.
4、Measurement of abdominal aortic diameters and AAA incidence:Aortic diameters and AAA incidence were measured. The maximum width of abdominal aortas was measured with computerized morphometry. Aneurysm incidence was quantified on the basis of a definition of an external suprarenal aorta width that was increased by 50% or more compared with saline-infused mice.
5. Histopathological study:Coronal sections of renal tissue (3 to 4-μm-thick) were stained with Verhoeff-Van Giessen staining for analysis of elastin band rupture in AAA, and examined by light microscopy in a blinded fashion.
6. Immunohistochemical staining:Four micrometer-thick sections of 10% formalin fixed tissues were stained with antibodies as follows:mouse anti-IL-4 antibody and mouse anti-INF-γantibody. Briefly, sections were deparaffinized, washed with PBS, and incubated with 3% H2O2 in methanol to block endogenous peroxidase activity. Then, Sections were incubated overnight with the anti- IL-4 and anti- INF-γantibody in a humidified chamber at 4℃. The localization of the first antibody was visualized by an indirect immunoperoxidase method. All of these sections were examined in a masked manner using a light microscopy.
7. Western blot analysis:Tissue samples from the kidneys were prepared with lysis buffer. Protein samples (30μg per lane) were subjected to SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were blocked, treated with primary antibody, washed, and then incubated with the secondary horseradish peroxidase-labeled antibody. Bands were visualized with Enhanced Chemiluminescence. The expression of protein was demonstrated by the ratio of integral optical density (IOD) between specific protein andβ-actin.
Results
1. Leptin did not alter the development of hypertension in response to AngⅡ: ApoE-/- mice infused with AngⅡdeveloped moderate increases (25 mmHg) in systolic blood pressure during the 4 weeks of the study. This elevation in blood pressure was apparent within 3 days after pump implantation and was maintained throughout the course of AngⅡinfusion. Leptin did not affect systolic blood pressure in AngⅡ-infused mice.
2. Leptin did not alter the concentrations of serum lipids:ApoE-/- mice infused with AngⅡhad an elevation in levels of serum TC and LDL-C. Leptin had no significantly effect on the rise in these values. No differences in levels of serum TG and FFA were observed among these three groups.
3. Leptin markedly attenuated AngⅡ-induced hypertensive AAAs:The incidence of AAAs in the suprarenal aorta of Angll infused mice was 87.5%. intraperitoneal injections daily with leptin decreased the incidence (85% vs 62.5%, AngⅡalone vs AngⅡ+ leptin, respectively; P<0.01; Table 2) and the severity of aneurysms formed by AngⅡ(Figure 4).
4. Leptin markedly decreased the protein levels of MMP-2 and MMP-9 in AAA:Because matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, play a critical role in AAA formation, we examined MMP protein expression in abdominal aortic tissue homogenates. AngⅡinfusion induced an increase in the protein expression of both MMP-2 and MMP-9 compared with control. Treatment with leptin markedly decreased the protein levels of MMP-2 and MMP-9 in abdominal aortae of AngⅡ-infused mice.
5. Leptin markedly decreased Th2 cytokine IL-4 expression and increased Th1 cytokine INF-γexpression in AAA:Representative western blots showed that IL-4 expression was significantly increased in AAA induced by AngⅡand reduced by leptin treatment. However, INF-γexpression was significantly increased in AAA by by leptin treatment. The result from immunohistochemical staining paralleled with that from western blot.
Conclusions
1. Infusion with Angll induced the formation of hypertensive AAAs in ApoE-/-mice.
2. Leptin markedly attenuated AngⅡ-induced hypertensive AAAs in ApoE-/-mice with no effect on serum lipids and systolic blood pressure.
3. The protective effects of leptin on hypertensive AAAs may be due to the inhibition of Th2 cytokine IL-4 expression and induction of Th1 cytokine INF-γ expression and thus inhibition of the expression of MMP-2 and MMP-9.
4. Leptin, with the effect of regulation of Th1/Th2 cytokines, may have therapeutic potential for the treatment of hypertensive AAA.
在过去10年中,随着人口老龄化及生活水平的提高,高血压患者日益增多,高血压性肾损害发病率也在明显上升。目前,高血压是仅次于糖尿病的第二大致终末期肾病(end-stage renal disease, ESRD)的病因,占所有ESRD的百分之三十。相关统计资料表明,每13个高血压患者就有1例发展成ESRD,在接受降压治疗的高血压患者中,4%-16%的患者蛋白尿排泄异常。虽然近年来高血压的治疗取得了很大的进展,但是高血压所导致的肾损害的发生率却呈逐年上升的趋势,提示临床上应用抗高血压药物治疗的肾保护作用还远远不够。因此,如何降低高血压肾损伤的发病率是目前基础研究和临床实践的重要课题。
高血压肾脏损伤主要表现为蛋白尿、良性肾小球硬化、间质纤维化以及炎症细胞浸润。除了神经内分泌激素,如去甲肾上腺素、血管紧张素Ⅱ以及醛固酮等,氧化应激也是高血压肾病发生和进展的重要因素。氧化应激不但可以通过脂质过氧化等直接损伤肾脏组织,还能作为第二信使激活细胞内信号转导系统,表达相应的活性产物,诱导炎症细胞浸润,引起炎症反应间接损伤肾脏组织。在氧化应激诱导产生的各种活性产物中,TGF-β1起关键性作用。TGF-β1不但可以调节细胞外基质的合成和降解,还可以诱导小管上皮细胞发生上皮-间充质转分化,最终引起细胞外基质的过度沉积。而细胞外基质的过度沉积是高血压肾病的主要特征。
PPARs是1990年发现的核激素受体超家族的新成员,有三种亚型：PPARα、PPARβ/δ和PPARγ。其中PPARα于肾脏、肝脏和心脏中高表达,并在调节糖脂代谢中起重要作用。PPARα激动剂在临床上是常用的降甘油三酯药物。近年来发现PPARα激动剂还有抗氧化的作用,并且能抑制高血压心肌重构,但是PPARα激动剂对高血压肾损伤是否有保护作用及其机制还未有报道。
针对以上问题,我们提出如下假说：PPARα激动剂非诺贝特能够抑制高血压肾损伤的进展；其机制可能是通过抑制氧化应激和MAPK活性从而抑制下游TGF-β1、TIMP-1的表达、间质成纤维细胞的活化以及炎症细胞浸润所引起的炎症反应。
目的
1、观察26周龄SHR肾脏结构和功能的损伤程度；
2、观察PPARα激动剂非诺贝特是否对SHR肾脏损伤具有保护作用；
3、探讨PPARα激动剂非诺贝特对SHR肾脏保护作用的可能机制。
方法
1.实验动物和分组：八周龄雄性SHR大鼠24只,随机分为阳性对照组即SHR组(n=10)和非诺贝特组(n=14)；同周龄WKY大鼠10只作为正常对照组即WKY组(n=10)。非诺贝特组给予非诺贝特(力平之)60mg·kg-1·d-1灌胃18周,WKY组和SHR组以等量生理盐水灌胃。
2.体重和血压：实验过程中,每两周测量体重1次,每两周检测尾动脉血压1次。
3.尿蛋白和肾功能的检测：实验末留取24小时尿检测24小时尿蛋白排泄率；实验末抽取右心室静脉血检测血清肌酐和尿素氮含量。
4.病理形态染色：应用PAS染色观察皮质肾小球硬化；应用Masson染色观察皮质肾小管间质纤维化。光学显微镜采集图像,并用Image ProPlus 5.0作定量分析。
5.免疫组化染色：应用免疫组化染色观察间质成纤维细胞的活化和单核／巨噬细胞在肾脏皮质中的浸润。将处理好的组织切片用含有α-SMA和ED1一抗的1%BSA进行孵育,并用辣根过氧化物酶标记的二抗进行处理。使用DAB显色,光学显微镜采集图像。
6.MDA检测：肾皮质组织匀浆的制备,应用考马斯亮兰蛋白法测定肾皮质组织匀浆蛋白浓度；采用硫代巴比妥酸法检测肾皮质组织MDA的含量。
7.SOD活性检测：肾皮质组织匀浆的制备,应用考马斯亮兰蛋白法测定肾皮质组织匀浆蛋白浓度；采用黄嘌呤氧化酶法检测肾皮质组织T-SOD、Cu/Zn-SOD和Mn-SOD的活性。
8.蛋白印迹分析：使用组织裂解液提取肾皮质组织总蛋白。将含有30μg蛋白的每个标本和蛋白Marker一起分别加入不同的泳道。在10%SDS-聚丙烯酰胺凝胶上电泳,再用一湿电转染仪电转染到聚偏氟乙烯膜(PVDF)上。将膜封闭后,使用一抗孵育,清洗后,再使用HRP结合的二抗孵育。滴加显色液显色。强弱用表达的蛋白和β-actin条带积分光密度的比值表示。
9.实时定量PCR：使用Trizol提取肾皮质组织总RNA。应用逆转录酶将mRNA逆转录成cDNA。使用Applied Biosystems TaqMan 7900HT detection系统进行实时PCR。采用MCP-1、Cu/Zn-SOD和Mn-SOD与β-actin的循环阈值(threshold cycle, Ct)的比值并通过公式：起始模板浓度=2-ΔΔCT。注：ΔΔCT=(干预组目的基因CT值-β-actin CT值)-(对照组目的基因CT值-β-actin CT值),表示mRNA的相对水平。
结果
1. PPARα激动剂非诺贝特降低SHR 24小时尿蛋白排泄率：相比于WKY大鼠,26周龄SHR表现为低体重、蛋白尿和血压升高,而肾功能晚期指标肌酐和尿素氮正常；非诺贝特显著降低SHR 24小时尿蛋白,但是对血压和体重没有明显作用。
2. PPARα激动剂非诺贝特抑制SHR皮质肾小球硬化和间质纤维化：PAS染色结果表明WKY大鼠没有肾小球硬化,而SHR有少量肾小球出现节段性硬化；Masson染色结果表明SHR肾小管间质明显纤维化；免疫组化结果表明SHR肾小球和肾小管间质中α-SMA表达明显增加,说明间质中成纤维细胞活化,合成和分泌细胞外基质的功能增强。非诺贝特显著抑制SHR皮质肾小球硬化、肾小管间质纤维化以及间质中成纤维细胞的活化。
3. PPARα激动剂非诺贝特抑制SHR肾皮质细胞外基质重构：免疫印迹结果表明,相比于WKY大鼠,SHR肾皮质胶原Ⅳ. MMP-9和TIMP-1表达均升高,非诺贝特显著抑制它们在高血压大鼠肾皮质中的表达。
4. PPARα激动剂非诺贝特抑制SHR肾脏间质炎症细胞的浸润：实时定量PCR结果表明,与WKY组大鼠比较,SHR组大鼠肾皮质MCP-1的mRNA表达上调；非诺贝特明显抑制SHR肾皮质MCP-1 mRNA的表达；免疫组化结果表明,相比于WKY大鼠,SHR皮质肾小球和肾小管间质中单核／巨噬细胞明显增多,非诺贝特显著抑制单核巨噬细胞的浸润。说明非诺贝特能够抑制SHR肾皮质内的炎症反应。
5. PPARα激动剂非诺贝特抑制SHR肾皮质组织氧化应激损伤：相比于WKY大鼠,SHR肾皮质组织MDA水平明显升高；非诺贝特显著降低SHR肾皮质组织MDA水平,说明非诺贝特能够抑制SHR肾皮质氧化应激损伤。
6. PPARα激动剂非诺贝特抑制SHR肾皮质NADPH氧化酶亚单位P47phox的表达并增加Cu/Zn-SOD活性和表达：相比于WKY大鼠,SHR肾皮质组织P47phox的蛋白表达明显增加,而Cu/Zn-SOD活性及mRNA水平明显降低,Mn-SOD活性及mRNA水平没有明显改变,提示SHR肾皮质不但有氧化系统激活,而且抗氧化系统减弱,最终导致肾皮质氧化应激损伤；非诺贝特显著抑制P47phox的蛋白表达,并增加Cu/Zn-SOD活性及mRNA的表达,从而减轻肾皮质氧化应激损伤。
7. PPARα激动剂非诺贝特抑制SHR肾皮质组织TGF-β1表达和MAPK活性：免疫印迹结果表明,相比于WKY大鼠,SHR肾皮质组织TGF-β1表达增加,磷酸化的JNK和p38MAPK明显增加,而总JNK和p38MAPK没有明显变化；非诺贝特显著抑制SHR肾皮质组织TGF-β1的表达及JNK和p38MAPK的磷酸化。
结论
1.26周龄SHR肾脏损伤表现为蛋白尿、轻度皮质肾小球硬化、肾小管间质纤维化和炎症细胞浸润,基本符合人类高血压早期肾损伤的病理改变。
2. PPARα激动剂非诺贝特显著降低SHR尿蛋白排泄率,减轻皮质肾小球硬化、肾小管间质纤维化和炎症细胞浸润,对高血压肾脏具有保护作用。
3. PPARα激动剂非诺贝特对SHR的肾保护作用可能是通过抑制氧化应激和MAPK活性从而抑制下游TGF-β1、TIMP-1的表达以及炎症细胞浸润所引起的炎症反应。
4. PPARα激动剂非诺贝特的抗氧化作用对氧化应激性肾脏损伤具有潜在的治疗作用。
背景
腹主动脉瘤(andominal aortic aneurysm, AAA)是局部血管壁膨胀,超过正常血管直径的50%的一种病理状态。AAA的病理学改变主要表现为炎症细胞的侵入,动脉中层弹力蛋白基质结构的破坏和平滑肌细胞减少。在美国60岁以上人群的年发病率约为3%,每年大约46,000人需要手术治疗,导致15,000人死亡。国内的发病率也呈逐年上升的趋势。目前临床上尚没有被证明有效的AAA治疗药物.治疗选择仅限于腔内或有创手术治疗。由于这种高危疾病给人们带来的危害随着生活人类生活水平的提高和人口老龄化而不断增加,关于AAA发病机制和找到可阻断或延缓这一过程的有效药物成为近年来研究的热点。
近年来对AAA的病因及病理生理学已有了崭新的认识,传统的动脉粥样硬化学说并不符合AAA的发病特点,多因素致病,复杂的退行性病变包括慢性炎症浸润、细胞外基质的重塑成为共识。其中多种蛋白水解酶家族对血管壁细胞外基质的降解是多种原因诱发AAA的最终途径,其中基质金属蛋白酶家族是具有最关键作用的蛋白降解酶家族。来自不同团队的多个研究表明人类AAA是一种慢性炎症性疾病,表现为Th2型细胞因子(IL-4)占主导低位,而Th1型细胞因子(尤其是INF-γ)减少。因此,如何抑制Th2型细胞因子在AAA中的表达将是治疗AAA的新的策略。
瘦素(Leptin)是由肥胖(obese, ob)基因编码、脂肪细胞分泌的一种非糖基化的多肽激素,通过与中枢及外周组织的受体结合,在能量代谢、神经内分泌系统、生殖系统、心血管系统以及骨骼形成等方面发挥重要的调节作用,具有重要的生理病理意义。近年来的研究表明,瘦素作为一种细胞因子样激素在免疫系统中所发挥的作用越来越受到关注。瘦素可以促进T细胞的增殖、活化和细胞因子合成,促使Th细胞向Th1方向分化,并抑制Th2型细胞因子的释放。目前已有研究证实瘦素可以通过调节Th1/Th2促进动脉粥样硬化斑块的形成,但是瘦素对动脉瘤的作用目前还未见报道。
针对以上问题,我们提出如下假说：瘦素能够抑制AAA的形成和进展,其作用机制可能是通过诱导Th1型细胞因子,同时抑制Th2型细胞因子来起作用。
目的
1.构建AngⅡ诱导ApoE-/-小鼠高血压AAA模型；
2.观察Leptin是否对AngⅡ诱导的ApoE-/-小鼠高血压AAA具有保护作用；
3.探讨Leptin对AngⅡ诱导ApoE-/-小鼠高血压AAA保护作用的可能机制。
方法
1. AngⅡ诱导的ApoE-/-小鼠高血压AAA模型的构建及Leptin的干预：八周龄雄性ApoE-/-小鼠皮下埋装有AngⅡ或Saline的渗透泵。根据体重调节AngⅡ的浓度,使AngⅡ的泵速维持在1000ng/kg·min,持续输注28天；同时每天腹膜下注射Leptin (0.6μg/g BW)或Saline,输注28天。
2.体重和血压：实验过程中,每周测量体重1次,每周检测尾动脉血压1次。
3.血清脂质的检测：实验末抽取右心室静脉血检测血清甘油三酯、胆固醇、低密度脂蛋白胆固醇、游离脂肪酸的浓度。
4.AAA的测量：通过Image ProPlus 5.0软件测量右肾动脉至最后一个肋间动脉之间,肾上腹主动脉的最大宽度。将腹主动脉最大宽度超过正常小鼠肾上动脉宽度的50%定义为动脉瘤。
5.病理形态染色：应用Verhoeff弹力纤维染色观察AAA的病理改变,光学显微镜采集图像。
6.免疫组化染色：应用免疫组化染色观察Th1 (INF-γ)/Th2 (IL-4)型细胞因子的表达及定位。将处理好的组织使用含有INF-γ或IL-4一抗的1%BSA进行孵育,并用辣根过氧化物酶标记的二抗进行处理。使用DAB显色,光学显微镜采集图像。
7.蛋白印迹分析：使用组织裂解液提取小鼠腹主动脉组织总蛋白。将含有30μg蛋白的每个标本和蛋白Marker一起分别加入不同的泳道。在10% SDS-聚丙烯酰胺凝胶上电泳,再用一湿电转染仪电转染到聚偏氟乙烯膜(PVDF)上。将膜封闭后,使用一抗孵育,清洗后,再使用HRP结合的二抗孵育。滴加显色液显色。强弱用表达的蛋白和β-actin条带积分光密度的比值表示。
结果
1. Leptin对AngⅡ持续输注诱导的高血压没有明显作用：相比于ApoE-/-组小鼠,ApoE-/-+AngⅡ组小鼠血压明显升高；腹腔注射Leptin对ApoE-/-+AngⅡ小鼠血压没有明显作用。
2. Leptin对血清脂质没有明显影响：相比于ApoE-/-组小鼠,ApoE-/-+AngⅡ组小鼠血清胆固醇、低密度脂蛋白胆固醇的含量明显升高,而游离脂肪酸、甘油三酯无明显变化；Leptin对ApoE-/-小鼠血清脂质无明显影响。
3. Leptin降低ApoE-/-+AngⅡ小鼠AAA的发生率并减轻其严重程度：持续输注AngⅡ诱发巨大AAA形成；腹膜腔注射Leptin可以降低输注AngⅡ诱发的AAA的发生率和严重程度。
4. Leptin降低ApoE-/-+AngⅡ小鼠AAA组织中MMP-2和MMP-9蛋白的表达：相比于ApoE-/-组小鼠,ApoE-/-+AngⅡ组小鼠AAA组织中MMP-2和MMP-9蛋白的表达明显升高；腹膜腔注射Leptin可以显著降低ApoE-/-+AngⅡ小鼠AAA组织中MMP-2和MMP-9蛋白的表达。
5. Leptin降低ApoE-/-+AngⅡ小鼠AAA组织中Th2型细胞因子IL-4蛋白的表达:Western blot结果证实ApoE-/-+AngⅡ小鼠AAA组织中IL-4的表达较对照组明显增加；腹膜腔注射Leptin可以显著降低IL-4的表达。免疫组织化学结果与Western blot结果一致。
6. Leptin增加ApoE-/-+AngⅡ小鼠AAA组织中Thl型细胞因子IFN-γ蛋白的表达：Western blot结果证实ApoE-/-+AngⅡ小鼠AAA组织中IFN-γ的表达较对照组无明显增加；腹膜腔注射Leptin可以显著增加IFN-γ的表达。免疫组织化学结果与Western blot结果一致。
结论
1.AngⅡ持续输注能够诱发ApoE-/-小鼠高血压AAA的形成。
2. Leptin降低AngⅡ诱导的ApoE-/-小鼠高血压AAA的发生率及严重程度,对血压和血脂无明显作用。
3. Leptin对高血压AAA的保护作用可能是通过调节Th1/Th2型细胞因子的表达：Leptin促进Thl型细胞因子的表达,抑制Th2型细胞因子的表达,从而抑制其诱导的MMP-2和MMP-9的表达对血管壁的弹性蛋白和胶原蛋白的降解。
4. Leptin的免疫调节作用可能对高血压AAA具有潜在的治疗作用；该结果也支持动脉粥样硬化和动脉瘤是两种不同的血管疾病,具有不同的致病机制的观点。
Backgroud
Hypertension is an important risk factor for the progression of glomerular and tubulointerstitial diseases to chronic renal failure. The progression of hypertensive renal disease displays several characteristics, including proteinuria, inflammatory cell recruitment, and accumulation of extracellular matrix (ECM) proteins in the interstitium. Oxidative stress has been shown to play an important role in the development of renal injury, because it can stimulate the expression of proinflammatory and profibrotic molecules in addition to direct toxic effects. Conversely, antioxidants can attenuate the development of kidney injury.
Peroxisome proliferator-activated receptors (PPARs), which include three membersα,γ, andβ/δ, are ligand-activated transcription factors belonging to the nuclear receptor superfamily. On activation by their ligands, PPARs bind to specific PPAR response elements (PPREs) in the promoter region of their target genes. PPARa, which is highly expressed in kidney, liver, and heart, has been shown to take part in diverse physiological processes, including maintenance of lipid and glucose homeostasis. In addition, PPARa also exerts antioxidant and anti-inflammatory effects. Activation of PPARa inhibits angiotensinⅡ-induced activation of NADPH oxidase and suppressed ROS production in the vascular wall. Furthermore, a PPAR-responsive element (PPRE) has been identified in promoter regions of catalase and Cu/Zn-SOD genes, which are key enzymes that reduce ROS production. Also, PPARa plays crucial roles in the attenuation of inflammatory response in activated mesangial cells through its antagonizing effects on the NF-κB signaling pathway. Furthermore, PPARa-deficient mice show a prolonged response to inflammatory stimuli, suggesting that PPARa is also a modulator of inflammation. These studies have identified PPARa as a naturally occurring regulator of oxidative stress and inflammation. However, little is known about the effect of PPARa on chronic hypertensive renal damage.
Taken together, we hypothesized that PPARa agonist fenofibrate might inhibit hypertensive renal injury by regulating oxidative stress and inflammation. The results from this study indicate that fenofibrate is able to inhibit renal injury in SHRs without affecting blood pressure by ameliorating renal inflammation and TGF-β1 expression via inhibition of oxidative stress and MAPK activity.
Methods
1. Animal models and experimental design:Twenty-four 8-week-old male SHRs and ten 8-week-old male Wistar-Kyoto (WKY) rats were used in this study. SHRs were divided into the following two groups:1) SHRs given normal oral 0.9% saline (n=10); 2) SHRs given oral fenofibrate at a dose of 60 mg·kg-1·d-1 dissolved in 0.9% saline (n=14). The WKY rats (n=10) given 0.9% saline were used as controls. The compound or vehicle was orally administered in 2 ml-kg"1 once a day for 18 weeks.
2. Assessment of body weight and systolic blood pressure:Body weight was determined every two weeks throughout the study. Systolic blood pressure (SBP) was assessed every two weeks throughout the study using a tail-cuff method in conscious rats after prewarming at 38℃for 10 min.
3. Assessment of UAE, BUN and Creatinine:At the end of the study, the animals were housed in metabolic cages for 24 h to collect urine for subsequent measurements of urinary protein excretion by an immunoassay, and then were anesthetized with pentobarbital, blood samples were collected from the right ventricle and serum was stored at -80℃for subsequently analyzing creatinine and blood urea nitrogen concentrations.
4. Histopathological study:Coronal sections of renal tissue (3 to 4-μm-thick) were stained with periodic acid-Schiff (PAS) and Masson trichrome for analysis of glomerular sclerosis and tubulointerstitial fibrosis, and examined by light microscopy in a blinded fashion. A semiquantitative morphometric score index was used to evaluate the degree of glomerulosclerosis. Tubulointerstitial fibrosis was assessed semiquantitatively.
5. Immunohistochemical staining:Four micrometer-thick sections of 10% formalin fixed tissues were stained with antibodies as follows:mouse anti-α-SMA antibody and mouse anti-ED1 antibody. Briefly, sections were deparaffinized, washed with PBS, and incubated with 3%H2O2 in methanol to block endogenous peroxidase activity. Then, Sections were incubated overnight with the anti-a-SMA and anti-ED1 antibody in a humidified chamber at 4℃. The localization of the first antibody was visualized by an indirect immunoperoxidase method. All of these sections were examined in a masked manner using a light microscopy.
6. Assessment of MDA levels in renal cortex:MDA levels were detected by the thiobarbituric acid method. Absorbance was measured at 532 nm by spectrometry. All protein concentrations of renal tissue homogenate samples were determined with the coomassie blue method.
7. Assessment of superoxide dismutase activity:SOD activity was measured by the xanthine oxidase method. Absorbance was determined at 550 nm by spectrometry. All protein concentrations of renal tissue homogenate samples were determined with the coomassie blue method.
8. Western blot analysis:Tissue samples from the kidneys were prepared with lysis buffer. Protein samples (30μg per lane) were subjected to SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were blocked, treated with primary antibody, washed, and then incubated with the secondary horseradish peroxidase-labeled antibody. Bands were visualized with Enhanced Chemiluminescence. The expression of protein was demonstrated by the ratio of integral optical density (IOD) between specific protein andβ-actin.
9. Real-time quantitative PCR:Total RNA from Tissue samples was extracted with Trizol, according to the manufacturer's protocol. The mRNAs were reverse-transcripted into cDNAs using cDNA synthesis kit. Real-time PCR was performed using Applied Biosystems TaqMan 7900HT detection system. The mRNA levels were estimated from the value of the threshold cycle (Ct) of the real-time PCR adjusted by that ofβ-actin through the formula 2ΔCt(ΔCt=gene of interest Ct-β-actin Ct)-(gene of control-β-actin Ct)
Results
1. Fenofibrate treatment attenuates the elevation in levels of urine albumin excretion rate without an apparent effect on blood pressure in SHRs:SHRs had an elevation in levels of urine albumin excretion rate compared with WKY rats and fenofibrate treatment significantly prevented the rise in these values. However, no differences in levels of serum creatinine and blood urea nitrogen were observed among these three groups. SHRs with or without fenofibrate treatment had significantly higher blood pressure compared with WKY rats. However, no significant differences in blood pressure were observed between SHRs with and without fenofibrate treatment. SHRs with or without fenofibrate treatment had significantly lower body weights compared with WKY rats.
2. Fenofibrate ameliorates glomerulosclerosis and tubulointerstitial fibrosis in SHRs:PAS staining showed that segmental glomerulosclerosis was present only in a small number of glomeruli in the SHR group and absent in the WKY group. Moreover, Masson's trichrome staining showed significant tubulointerstitial fibrosis in SHRs. Fenofibrate administration significantly reduced the severity of glomerulosclerosis and tubulointerstitial fibrosis in SHRs. These observations were confirmed by quantitative analysis. Immunohistochemical staining for a-SMA showed that myofibroblasts in the glomeruli and tubulointerstitial space were significandly increased in SHRs and were markedly decreased by fenofibrate.
3. Fenofibrate prevents collagen expression and deposition in SHRs: Representative western blots showed that collagenⅣ, MMP-9 and TIMP-1 were significantly increased in the kidney of SHRs. However, fenofibrate treatment reduced the expression of collagenⅣ, MMP-9 and TIMP-1.
4. Fenofibrate treatment reduces inflammatory cell accumulation in SHRs: Comapred with WKY group, MCP-1 mRNA levels were significantly increased in SHRs and was decreased by fenofibrate treatment. And, SHRs had a significant accumulation of ED1-positive cells in the glomeruli and tubulointerstitial space. Fenofibrate treatment markedly attenuated monocytes/macrophages accumulation in the kidney.
5. Fenofibrate attenuates oxidative stress and increases renal SOD activity and mRNA levels in SHRs:Oxidative stress was evaluated by determination of the levels of MDA in the kidney, an index of lipid peroxidation. MDA levels were markedly increased in the kidney of SHRs and were decreased by fenofibrate treatment. Cu/Zn SOD activity and mRNA levels were significantly decreased in SHRs and was increased by fenofibrate treatment. However, Mn-SOD activity and mRNA levels were similar in these three groups. Protein levels of p47phox, a NAD(P)H oxidase subunits, in the kidney paralleled with MDA levels.
6. Fenofibrate reduces TGF-β1 expression and p38 MAPK and JNK phosphorylation in SHRs:The effect of fenofibrate on TGF-β1 Expression and the phosphorylation of p38 MAPK and JNK were investigated. Representative western blots showed that TGF-β1 Expression, p38 MAPK and JNK phosphorylation were significantly increased in the kidney of SHRs and reduced by fenofibrate treatment. However, no differences in protein levels of total p38 MAPK and JNK were observed among these three groups.
Conclusions
1. Early renal injury in SHRs was characterized by mild proteinuria, glomerular sclerosis, tubulointerstitial fibrosis, inflammatory cells recruitment and normal renal function.
2. PPARa agonist fenofibrate exerted renoprotective effects against hypertensive renal injury in SHRs.
3. The renoprotective effects of PPARa agonist fenofibrate may be due to the inhibition of TGF-β1 expression and inflammatory cell recruitment through suppression of NADPH oxidase activity, upregulation of Cu/Zn SOD activity, and, thus, inhibition of phosphorylation of p38 MAPK and JNK.
4. Fenofibrate, as a potent antioxidant, may have therapeutic potential for the treatment of oxidative kidney damage, such as hypertensive renal injury.
Backgroud
Aortic aneurysms are permanent and localized aortic dilations defined as having diameters 1.5-times greater than normal. Important histological features of aneurysms include chronic adventitial and medial inflammatory cell infiltration, elastin fragmentation and degeneration, and medial attenuation. In the United States alone abdominal aortic aneurysms(AAA) affect 3% of individuals 60 years or older, necessitate 46,000 surgical interventions, and cause～15,000 deaths annually.1 Despite considerable descriptive knowledge of the pathomorphology of AAA, insufficient understanding of the molecular mechanisms underlying its pathogenesis currently limits the prevention and treatment of this human disease.
Recent human studies from several groups indicate that human AAAs comprise an inflammatory disease characterized by the predominance of T helper cell type 2 (Th2) cytokine expression, especially IL-4 and the paucity of Th1 cytokines, especially interferon-γ(IFN-γ). Animal studies show that aortic allografts deficient in interferon-γ(IFN-γ) signaling developed AAA correlating with skewed Th2 cytokine environments, suggesting important regulatory roles for Th1/Th2 cytokine balance in modulating matrix remodeling and important implications for the pathophysiology of aortic aneurysm and atherosclerosis.
Leptin, a cytokine-like hormone produced primarily by adipocytes, has been clearly demonstrated to play an important role in body weight regulation through effects on feeding and energy expenditure. In addition to central effects of leptin on appetite and metabolism, several studies have described direct leptin effects on immune cells, including the promotion of T lymphocyte type 1 helper (Th1) response, of potential importance to the process of atherosclerosis. Importantly, ob/ob mice have reduced secretion of IL-2, LFN-γ, TNF and IL-18, and increased production of TH2-type cytokines, such as IL-4 and IL-10, after mitogenic stimulation.
Thus, we hypothesized that leptin, with the effect of regulation of Thl/Th2 cytokines, may have protective effect on the formation of AAA induced by AngⅡin ApoE-/- mice.
Methods
1. Animal models and experimental design:8 weeks old ApoE-/- mice were infused with saline or AngⅡ(1000 ng/kg/min) for 4 weeks and/or intraperitoneal injections daily with 0.6μg/g BW of recombinant murine leptin or vehicle control (n=8 per group).The dose of leptin chosen was based on a protocol used to achieve weight loss and fertility in leptin-deficient mice.
2. Assessment of systolic blood pressure:Systolic blood pressure (SBP) was assessed every weeks throughout the study using a tail-cuff method in conscious rats after prewarming at 38℃for 10 min.
3. Assessment of blood lipid:At the end of the study, the animals were anesthetized with pentobarbital, blood samples were collected from the right ventricle and serum was stored at -80℃for subsequently analyzing TG, TC, LDL-C and FFA.
4、Measurement of abdominal aortic diameters and AAA incidence:Aortic diameters and AAA incidence were measured. The maximum width of abdominal aortas was measured with computerized morphometry. Aneurysm incidence was quantified on the basis of a definition of an external suprarenal aorta width that was increased by 50% or more compared with saline-infused mice.
5. Histopathological study:Coronal sections of renal tissue (3 to 4-μm-thick) were stained with Verhoeff-Van Giessen staining for analysis of elastin band rupture in AAA, and examined by light microscopy in a blinded fashion.
6. Immunohistochemical staining:Four micrometer-thick sections of 10% formalin fixed tissues were stained with antibodies as follows:mouse anti-IL-4 antibody and mouse anti-INF-γantibody. Briefly, sections were deparaffinized, washed with PBS, and incubated with 3% H2O2 in methanol to block endogenous peroxidase activity. Then, Sections were incubated overnight with the anti- IL-4 and anti- INF-γantibody in a humidified chamber at 4℃. The localization of the first antibody was visualized by an indirect immunoperoxidase method. All of these sections were examined in a masked manner using a light microscopy.
7. Western blot analysis:Tissue samples from the kidneys were prepared with lysis buffer. Protein samples (30μg per lane) were subjected to SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were blocked, treated with primary antibody, washed, and then incubated with the secondary horseradish peroxidase-labeled antibody. Bands were visualized with Enhanced Chemiluminescence. The expression of protein was demonstrated by the ratio of integral optical density (IOD) between specific protein andβ-actin.
Results
1. Leptin did not alter the development of hypertension in response to AngⅡ: ApoE-/- mice infused with AngⅡdeveloped moderate increases (25 mmHg) in systolic blood pressure during the 4 weeks of the study. This elevation in blood pressure was apparent within 3 days after pump implantation and was maintained throughout the course of AngⅡinfusion. Leptin did not affect systolic blood pressure in AngⅡ-infused mice.
2. Leptin did not alter the concentrations of serum lipids:ApoE-/- mice infused with AngⅡhad an elevation in levels of serum TC and LDL-C. Leptin had no significantly effect on the rise in these values. No differences in levels of serum TG and FFA were observed among these three groups.
3. Leptin markedly attenuated AngⅡ-induced hypertensive AAAs:The incidence of AAAs in the suprarenal aorta of Angll infused mice was 87.5%. intraperitoneal injections daily with leptin decreased the incidence (85% vs 62.5%, AngⅡalone vs AngⅡ+ leptin, respectively; P<0.01; Table 2) and the severity of aneurysms formed by AngⅡ(Figure 4).
4. Leptin markedly decreased the protein levels of MMP-2 and MMP-9 in AAA:Because matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, play a critical role in AAA formation, we examined MMP protein expression in abdominal aortic tissue homogenates. AngⅡinfusion induced an increase in the protein expression of both MMP-2 and MMP-9 compared with control. Treatment with leptin markedly decreased the protein levels of MMP-2 and MMP-9 in abdominal aortae of AngⅡ-infused mice.
5. Leptin markedly decreased Th2 cytokine IL-4 expression and increased Th1 cytokine INF-γexpression in AAA:Representative western blots showed that IL-4 expression was significantly increased in AAA induced by AngⅡand reduced by leptin treatment. However, INF-γexpression was significantly increased in AAA by by leptin treatment. The result from immunohistochemical staining paralleled with that from western blot.
Conclusions
1. Infusion with Angll induced the formation of hypertensive AAAs in ApoE-/-mice.
2. Leptin markedly attenuated AngⅡ-induced hypertensive AAAs in ApoE-/-mice with no effect on serum lipids and systolic blood pressure.
3. The protective effects of leptin on hypertensive AAAs may be due to the inhibition of Th2 cytokine IL-4 expression and induction of Th1 cytokine INF-γ expression and thus inhibition of the expression of MMP-2 and MMP-9.
4. Leptin, with the effect of regulation of Th1/Th2 cytokines, may have therapeutic potential for the treatment of hypertensive AAA.
引文
1. S. Adler and H. Huang. Oxidant stress in kidneys of spontaneously hypertensive rats involves both oxidase overexpression and loss of extracellular superoxide dismutase. Am J Physiol Renal Physiol,2004,287:F907-F913.
2. Hiroyuki Kobori, Yuri Ozawa, Yuki Suzaki, and Akira Nishiyama. Enhanced Intrarenal Angiotensinogen Contributes to Early Renal Injury in Spontaneously Hypertensive Rats. J Am Soc Nephrol.2005; 16:2073-2080.
3. Hao Xue, Ying-Li Zhang, Guo-Shu Liu, and Hai Wang. A New ATP-Sensitive Potassium Channel Opener Protects the Kidney from Hypertensive Damage in Spontaneously Hypertensive Rats. JPET,2005,315:501-509.
4. Edna D. Lekgabe, Helen Kiriazis, Chongxin Zhao, Qi Xu, Xiao Lei Moore, Yidan Su, Ross A.D. Bathgate, Xiao-Jun Du, Chrishan S. Samuel. Relaxin Reverses Cardiac and Renal Fibrosis in Spontaneously Hypertensive Rats. Hypertension. 2005; 46:412-418.
5.许顶立,任昊.高血压病肾脏损害的诊断与防治.中华心血管病杂志,2004,32：190-192.
6. Ruilope LM.The kidney as a sensor of cardiovascular risk in essential hypertension.J Am Soc Nephrol,2002,13:165.
7. Porush JG. Hypertension and chronic renal failure:the use of ACE inhibitors. Am J Kidney Dis,1998;31:177.
8. Ritz E, Nowicki It Fliser D et al. Proteinuria and hypertension. Kidney Int,1994; 46:76-80.
9.刘力生.高血压.第二版.北京：人民卫生出版社,2001.
10. N Takahashi and O Smithies. Human genetics, animal models and computer simulations for studying hypertension. Trends Genet,2004; 20:136-45.
11. H Kim, JH Krege, KD Kluckman, JR Hagaman, JB Hodgin, CF Best, C Jennette, TM Coffman, N Maeda, and O Smithies. Genetic Control of Blood Pressure and the Angiotensinogen Locus. PNAS,1995; 92:2735-2739.
12. JJ Mullins, J Peters, and D Ganten. Fulminant hypertension in transgenic rats harbouring the mouse Ren-2 gene. Nature,1990; 344:541-4.
13. Z Yang, DR Sibley, and PA Jose. D5 dopamine receptor knockout mice and hypertension. J Recept Signal Transduct Res,2004; 24:149-64.
14. Paula M. Oliver, Jennifer E. Fox, Ron Kim, Howard A. Rockman, Hyung-Suk Kim, Robert L. Reddick, Kailash N. Pandey, Sharon L. Milgram, Oliver Smithies, and Nobuyo Maeda. Hypertension, cardiac hypertrophy, and sudden death in mice lacking natriuretic peptide receptor A. PNAS,1997; 94:14730-14735.
15. Y Kurihara, H Kurihara, H Suzuki, T Kodama, K Maemura, R Nagai, H Oda, T Kuwaki, WH Cao, and N Kamada. Elevated blood pressure and craniofacial abnormalities in mice deficient in endothelin-1. Nature,1994; 368:703-10.
16. W Li, S Mital, C Ojaimi, A Csiszar, G Kaley, and TH Hintze. Premature death and age-related cardiac dysfunction in male eNOS-knockout mice. J Mol Cell Cardiol, 2004; 37:671-80.
17. Yigal M Pinto, Martin Paul, and Detlev Ganten. Lessons from rat models of hypertension:from Goldblatt to genetic engineering. Cardiovasc Res,1998; 39: 77-88.
18. Lilach O. Lerman, Karl A. Nath, Martin Rodriguez-Porcel, James D. Krier, Robert S. Schwartz, Claudio Napoli, and J. Carlos Romero. Increased Oxidative Stress in Experimental Renovascular Hypertension. Hypertension,2001; 37:541-546.
19. V Lahera, J Salazar, MG Salom, and JC Romero. Deficient production of nitric oxide induces volume-dependent hypertension. J Hypertens Suppl,1992; 10: S173-7.
20. C Catena, G Giacchetti, M Novello, G Colussi, A Cavarape, and LA Sechi. Cellular mechanisms of insulin resistance in rats with fructose-induced hypertension. Am J Hypertens,2003; 16:973-8.
21. Schreiner GF. Renal toxicity of albumin and other lipoproteins.Curr Opin Nephrol Hypertens,1995; 4:369-373
22. Wheeler DC,Chana RS, Topley N, et al. Oxidation of low density lipoprotein by mesangial cells may promote glomerular injury. Kidney Int,1994; 45:1628-1636
23. BH Rovin and LC Tan. LDL stimulates mesangial fibronectin production and chemoattractant expression. Kidney Int, Jan 1993; 43:218-25.
24. DC Wheeler, RS Chana, N Topley, MM Petersen, M Davies, and JD Williams. Oxidation of low density lipoprotein by mesangial cells may promote glomerular injury. Kidney Int, Jun 1994; 45:1628-36.
25. Madamanchi NR, Runge MS. Mitochondrial dysfunction in atherosclerosis. Circ Res.2007; 100:460-73.
26. Bassenge E, Schneider HT, Daiber A. Oxidative stress and cardiovascular diseases. Dtsch Med Wochenschr.2005; 130:2904-2909.
27. Heistad DD. Oxidative stress and vascular disease. Arterioscler Thromb Vasc Biol. 2005; 12:104.
28. Deshpande NN, Sorescu D, Seshiah P, Ushio-Fukai M, Akers M, Yin Q, Griendling KK. Mechanism of hydrogen peroxide-induced cell cycle arrest in vascular smooth muscle. Antioxid Redox Signal.2002; 4:845-854.
29. Taniyama Y, Griendling KK. Reactive oxygen species in t he vasculature molecular and cellular mechanisms. Hypertension.2003; 42:1075.
30. Budin SB, Othman F, Louis SR, Bakar MA, Das S, Mohamed J. The effects of palm oil tocotrienol-rich fraction supplementation on biochemical parameters, oxidative stress and the vascular wall of streptozotocin-induced diabetic rats. Clinics.2009; 64:235-44.
31. Vaziri ND, Wang XQ, Oveisi F, Rad B. Induction of oxidative stress by glutathione depletion cause severe hypertension in normal rats. Hypertension. 2000; 39:142.
32. Visconti R, Grieco D. New insights on oxidative stress in cancer. Curr Opin Drug Discov Devel.2009; 12:240-5.
33. Tribble DL, Barcellos MH, et al. Ionizing radiation accelerates arotic lesion formation in fat-efd mice via SOD-inhibitable processes. Arterioscler Thromb Vasc Biol,1999,19:1387-1392.
34. Young IS, Woodside jv. Antioxidants in health and disease. J Clin Pathol,2001, 54:176-186.
35. WilmerWA, Dixon CL, Hebert C, et al. Chronic exposure of human mesangial cells to high glucose environment activates the p38 MAPK pathway. Kidney Int, 2001,60:858-871.
36. Vsui M, Egashira K, Kitamoto S, et al. Pathogenic role of oxidative stress in vascular angiotensin-converting enzyme activation in long-term blockade of nitric oxide synthesis in rats. Hypertension,1999,34:546-551.
37. Vega GW, Roson MI, Beliver A, et al. Nitric oxide and superoxide anions in vascular reactivity of renovascular hypertensive rats. Clin Exp Hypertens,1995, 17:817-835.
38. Lerman LO, Nath KA, Rodriguez PM, et al. Increased oxidative stress in experimental renovascular hypertension. Hypertension,2001,37:541-546.
39. Vaziri ND,Oveisi F,Ding Y. Role of incresaed oxygen free radical activity in the pathogenesis of uremic hypertension. Kidney Int,1998,53:1748-1754.
40. Vaziri ND,Liang K,Ding Y Increased nitric oxide inactivation by reactive oxygen species in Lead-induced hypertension. Kidney Int,1999,56:1492-1498.
41. Bapats, Post JA,Braam B, et al. Visualizing tubular lipid peroxidation in intact renal tissue in hypertensive rats. J Am Soc Nephrol,2002,13:2990-2996.
42. Bernardo Rodriguez-Iturbe, Atilio Ferrebuz, Valentina Vanegas, Yasmir Quiroz, Sergio Mezzano, and Nosratola D. Vaziri. Early and Sustained Inhibition of Nuclear Factor-κB Prevents Hypertension in Spontaneously Hypertensive Rats. J. Pharmacol. Exp. Ther., Oct 2005; 315:51-57.
43. Mayerly Nava, Yasmir Quiroz, Nosratola Vaziri, and Bernardo Rodriguez-Iturbe. Melatonin reduces renal interstitial inflammation and improves hypertension in spontaneously hypertensive rats. Am J Physiol Renal Physiol,2003; 284:447.
44. Bernardo Rodriguez-Iturbe, Chang-De Zhan, Yasmir Quiroz, Ram K. Sindhu, and Nosratola D. Vaziri. Antioxidant-Rich Diet Relieves Hypertension and Reduces Renal Immune Infiltration in Spontaneously Hypertensive Rats. Hypertension, 2003; 41:341-346.
45. Li JM, Shah AM. ROS Generation by Nonphagocytic NADPH Oxidase:Potential Relevance in Diabetic Nephropathy. J Am Soc Nephrol,2003,14:S221-S226.
46. Evans JL, Goldfine ID, Maddux BA, et al. Oxidative stress and stress-activated signaling pathways:a unifying hypothesis of type 2 diabetes. Endocrine Rev,2002, 23:599-622.
47. Han CH, Lee MH. Expression and characterization of the flavoprotein domain of gp91phox. J Vet Sci,2000,1:19-26.
48. Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase:role in cardiovascular biology and disease. Circ Res,2000,86:494-501.
49. Lavigne MC, Malech H 乙 Holland SM, et al. Genetic demonstration of p47phox—dependent superoxide anion production in murine vascular smooth muscle cells. Circulation,2001,104:79-84.
50. Shiose A, Kuroda J, Tsuruya K, et al. A novel superoxide-producing NAD(P)H oxidase in kidney. J Biol Chem,2001,276:1417-1423.
51. Asaba K, Tojo A, Onozato ML, et al. Effects of NADPH oxidase inhibitor in diabetic nephropathy. Kidney Int,2005,67:1890-1898.
52. Niu Tian, Rebecca S. Moore, William E. Phillips, Lin Lin, Sharkeshia Braddy, Janelle S. Pryor, Rachel L. Stockstill, Michael D. Hughson, and R. Davis Manning, Jr. NADPH oxidase contributes to renal damage and dysfunction in Dahl salt-sensitive hypertension. Am J Physiol Regulatory Integrative Comp Physiol, 2008; 295:R1858-R1865.
53. Adler S, Huang H. Oxidant stress in kidneys of spontaneously hypertensive rats involves both oxidase overexpression and loss of extracellular superoxide dismutase. Am J Physiol Renal Physiol,2004,287:907-913.
54.Adam Whaley-Connell, Javad Habibi, Ravi Nistala, Shawna A. Cooper, Poorna R. Karuparthi, Melvin R. Hayden, Nathan Rehmer, Vincent G DeMarco, Bradley T. Andresen, Yongzhong Wei, Carlos Ferrario, and James R. Sowers. Attenuation of NADPH Oxidase Activation and Glomerular Filtration Barrier Remodeling With Statin Treatment. Hypertension,2008; 51:474-480.
55. Ohishi K, Carmines PK. Superoxide dismutase restores the influence of nitric oxide on renal arterioles in diabetes mellitus. J Am Soc Nephrol,1995,5: 1559-66.
56. Ha H, Lee HB. Reactive oxygen species and matrix remodeling in diabetic kidney. J Am Soc Nephrol,2003,14:S246-S249.
57. Zhang B, Liang X, Shi W, et al. Role of impaired peritubular capillary and hypoxia in p rogressive interstitial fibrosis after 5/6 subtotal nephrectomy of rats. Nephrology,2005,10:351-357.
58. Rhyu DY, Yang Y, Ha H, et al. Role of reactive oxygen species in TGF-β1 induced mitogen-activated protein kinase activation and epithelial-mesenchymal transition in renal tubular epithelial cells. J Am Soc Nephrol,2005,16:667-675.
59. G. H. Tesch. MCP-1/CCL2:a new diagnostic marker and therapeutic target for progressive renal injury in diabetic nephropathy. Am J Physiol Renal Physiol, 2008; 294:F697-F701.
60. Hunjoo Ha, Mi Ra Yu, Yoon Jin Choi, Masanori Kitamura, and Hi Bahl Lee. Role of High Glucose-Induced Nuclear Factor-κB Activation in Monocyte Chemoattractant Protein-1 Expression by Mesangial Cells. J. Am. Soc. Nephrol., 2002; 13:894-902.
61. Wenzel UO. Chemokines and renal disease. J. Am Kidney Dis,1995; 26:982-995
62. KF Hilgers, A Hartner, M Porst, M Mai, M Wittmann, C Hugo, D Ganten, H Geiger, R Veelken, and JF Mann. Monocyte chemoattractant protein-1 and macrophage infiltration in hypertensive kidney injury. Kidney Int,2000; 58: 2408-19.
63. Sarah F. Knight, Jeffrey E. Quigley, Jianghe Yuan, Siddhartha S. Roy, Ahmed Elmarakby, and John D. Imig. Endothelial Dysfunction and the Development of Renal Injury in Spontaneously Hypertensive Rats Fed a High-Fat Diet. Hypertension,2008; 51:352-359.
64. Marshall CB, Shankland SJ. Cell cycle and glomerular disease:a minkeview. Nephron Exp Nephrol,2006,102:e39-48.
65. Yamagata K, Hagiwara M. Pathological roles of mitochondrial dysfunction in podocyte injury. Nippon Jinzo Gakkai Shi,2007,49:82-7.
66. Nam-Ho Kim, Hernan Rincon-Choles, Basant Bhandari, Goutam Ghosh Choudhury, Hanna E. Abboud, and Yves Gorin. Redox dependence of glomerular epithelial cell hypertrophy in response to glucose. Am J Physiol Renal Physiol, 2006; 290:F741-F751.
67. Jing Dong, Sampath Ramachandiran, Kulbhushan Tikoo, Zhe Jia, Serrine S. Lau, and Terrence J. Monks. EGFR-independent activation of p38 MAPK and EGFR-dependent activation of ERK1/2 are required for ROS-induced renal cell death. Am J Physiol Renal Physiol,2004; 287:F1049-F1058.
1. Michael W. Manning, Lisa A. Cassis, Alan Daugherty. Differential Effects of Doxycycline, a Broad-Spectrum Matrix Metalloproteinase Inhibitor, on Angiotensin II-Induced Atherosclerosis and Abdominal Aortic Aneurysms. Arterioscler Thromb Vasc Biol.2003;23:483-488
2. Manesh Thomas; Dan Gavrila; Michael L. McCormick; Francis J. Miller, Jr; Alan Daugherty; Lisa A. Cassis; Kevin C. Dellsperger; Neal L. Weintraub. Deletion of p47phox Attenuates Angiotensin II-Induced Abdominal Aortic Aneurysm Formation in Apolipoprotein E-Deficient Mice. Circulation.2006;114:404-413.
3. Victoria L. King; Alexander Y. Lin; Fjoralba Kristo; Thomas J.T. Anderson; Neil Ahluwalia; Gregory J. Hardy; A. Phillip Owens Ⅲ; Deborah A. Howatt; Dongxiao Shen; Andrew M. Tager; Andrew D. Luster; Alan Daugherty; Robert E. Gerszten. Interferon-y and the Interferon-Inducible Chemokine CXCL10 Protect Against Aneurysm Formation and Rupture. Circulation.2009; 119:426-435.
4. Kimio Satoh, Patrizia Nigro, Tetsuya Matoba, Michael R O'Dell, Zhaoqiang Cui, Xi Shi, Amy Mohan, Chen Yan, Jun-ichi Abe, Karl A Illig & Bradford C Berk. Cyclophilin A enhances vascular oxidative stress and the development of angiotensin II-induced aortic aneurysms. Nat Med.2009; 15:649-56.
5. KW Johnston, RB Rutherford, MD Tilson, DM Shah, L Hollier, and JC Stanley. Suggested standards for reporting on arterial aneurysms. Subcommittee on Reporting Standards for Arterial Aneurysms, Ad Hoc Committee on Reporting Standards, Society for Vascular Surgery and North American Chapter, International Society for Cardiovascular Surgery. J Vasc Surg,1991; 13:452-8.
6. Beckman JA. Aortic aneurysms:pathophysiology, epidemiology, and prognosis. In: Creager MA, Dzau VJ, Loscalzo J, eds. Vascular Medicine. Philadelphia, Pa: Saunders Elsevier Inc; 2006.
7. Beckman JA, Creager MA. Aortic aneurysms:Clinical evaluation. In:Creager MA, Dzau VJ, Loscalzo J, eds. Vascular Medicine. Philadelphia, Pa:Saunders Elsevier Inc; 2006.
8. H Bengtsson, B Sonesson, and D Bergqvist. Incidence and prevalence of abdominal aortic aneurysms, estimated by necropsy studies and population screening by ultrasound. Ann N Y Acad Sci,1996; 800:1-24.
9. J W Hallett, Jr. Management of abdominal aortic aneurysms. Mayo Clin. Proc, 2000; 75:395-399.
10. A Hallin, D Bergqvist, and L Holmberg. Literature review of surgical management of abdominal aortic aneurysm. Eur J Vasc Endovasc Surg,2001; 22:197-204.
11. M M Thompson and P R F Bell. ABC of arterial and venous disease:Arterial aneurysms.BMJ,2000; 320:1193-1196.
12. A Lopez-Candales, DR Holmes, S Liao, MJ Scott, SA Wickline, and RW Thompson. Decreased vascular smooth muscle cell density in medial degeneration of human abdominal aortic aneurysms.Am. J. Pathol.,1997; 150:993-1007.
13. Tim Freestone, Robert J. Turner, Andrew Coady, Dan J. Higman, Roger M. Greenhalgh, and Janet T. Powell. Inflammation and Matrix Metalloproteinases in the Enlarging Abdominal Aortic Aneurysm. Arterioscler Thromb Vasc Biol,1995; 15:1145-1151.
14. N Diehm, F Dick, T Schaffner, J Schmidli, C Kalka, S Di Santo, J Voelzmann, and I Baumgartner. Novel insight into the pathobiology of abdominal aortic aneurysm and potential future treatment concepts.. Prog Cardiovasc Dis,2007; 50:209-17.
15. Valerie Davis, Raisa Persidskaia, Lisa Baca-Regen, Yoshifumi Itoh, Hideaki Nagase, Yuri Persidsky, Anuja Ghorpade, and B. Timothy Baxter. Matrix Metalloproteinase-2 Production and Its Binding to the Matrix Are Increased in Abdominal Aortic Aneurysms. Arterioscler Thromb Vasc Biol,1998; 18:1625-1633.
16. RW Thompson, DR Holmes, RA Mertens, S Liao, MD Botney, RP Mecham, HG Welgus, and WC Parks. Production and localization of 92-kilodalton gelatinase in abdominal aortic aneurysms. An elastolytic metalloproteinase expressed by aneurysm-infiltrating macrophages. J Clin Invest,1995; 96:318-26.
17. JA Curci, S Liao, MD Huffman, SD Shapiro, and RW Thompson. Expression and localization of macrophage elastase (matrix metalloproteinase-12) in abdominal aortic aneurysms. J Clin Invest,1998; 102:1900-10.
18.I Sinha, KK Hannawa, JL Eliason, G Ailawadi, MP Deogracias, S Bethi, JW Ford, KJ Roelofs, V Grigoryants, PK Henke, JC Stanley, and GR Upchurch Jr. Early MT-1 MMP expression following elastase exposure is associated with increased cleaved MMP-2 activity in experimental rodent aortic aneurysms. Surgery, Aug 2004; 136:176-82.
19. Helena Kuivaniemi, MD, PhD; Chris D. Platsoucas, PhD; M. David Tilson Ⅲ, MD. Aortic Aneurysms An Immune Disease With a Strong Genetic Component. Circulation 2008; 117; 242-252
20. Schonbeck U, Sukhova GK, Gerdes N, Libby P. T(H)2 predominant immune responses prevail in human abdominal aortic aneurysm. Am J Pathol.2002; 161: 499-506.
21. Davis VA, Persidskaia RN, Baca-Regen LM, Fiotti N, Halloran BG, Baxter BT. Cytokine pattern in aneurysmal and occlusive disease of the aorta. J Surg Res. 2001; 101:152-156.
22. Shimizu K, Shichiri M, Libby P, Lee RT, Mitchell RN. Th2-predominant inflammation and blockade of IFN-gamma signaling induce aneurysms in allografted aortas. J Clin Invest.2004; 114:300-308.
23. Nordskog BK, Fields WR, Hellmann GM. Kinetic analysis of cytokine response to cigarette smoke condensate by human endothelial and monocytic cells. Toxicology.2005; 212:87-97.
24. Ouyang Y, Virasch N, Hao P, Aubrey MT, Mukerjee N, Bierer BE, Freed BM. Suppression of human IL-1beta, IL-2, IFN-gamma, and TNF-alpha production by cigarette smoke extracts. J Allergy Clin Immunol.2000; 106:280-287.
25. Russell RE, Culpitt SV, DeMatos C, Donnelly L, Smith M, Wiggins J, Barnes PJ. Release and activity of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 by alveolar macrophages from patients with chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol.2002; 26:602-609.
26. Lavigne MC, Eppihimer MJ. Cigarette smoke condensate induces MMP-12 gene expression in airway-like epithelia. Biochem Biophys Res Commun.2005; 330: 194-203.
27. Tanabe K, Okuya S, Tanizawa Y,et al. Leptin induces proliferation of pancreatic beta cell line MIN6 through activation of mitogen-activated protein[J]. Biochem Biophys Res Commun.1997; 241:765-768.
28. Doneray H, Orbak Z, Yildiz L. The relationship between breast milk leptin and neonatal weight gain. Acm Paediatr.2009; 98:643.647.
29. Rajakumar A, Parrott MS, Powers RW. Leptin Affects System A Amino Acid Transport Activity in the Human Placenta:Evidence for STAT3 Dependent Mechanisms. Placenta.2009; 30:361-367.
30. Wu MH, Chou YC, Chou WY. Circulating levels of leptin, adiposity and breast cancer risk. Br J Cancer.2009; 100:578-582.
31. Sattar N, Wannamethee G Sarwar N et al. Leptin and coronary heart disease: prospective study and systematic review. J Am Coil Cardiol.2009; 53:167—175.
32. G Matarese, A La Cava, V Sanna, GM Lord, RI Lechler, S Fontana, and S Zappacosta. Balancing susceptibility to infection and autoimmunity:a role for leptin? Trends Immunol,2002; 23:182-7.
33. IS Farooqi, G Matarese, GM Lord, JM Keogh, E Lawrence, C Agwu, V Sanna, SA Jebb, F Perna, S Fontana, RI Lechler, AM DePaoli, and S O'Rahilly. Beneficial effects of leptin on obesity, T cell hyporesponsiveness, and neuroendocrine/metabolic dysfunction of human congenital leptin deficiency. J Clin Invest,2002; 110:1093-103.
34. Giuseppe Matarese, Antonio Di Giacomo, Veronica Sanna, Graham M. Lord, Jane K. Howard, Antonino Di Tuoro, Stephen R. Bloom, Robert I. Lechler, Serafino Zappacosta, and Silvia Fontana. Requirement for Leptin in the Induction and Progression of Autoimmune Encephalomyelitis. J. Immunol.,2001; 166:5909-5916.
35. Peter F. Bodary, Shufang Gu, Yuechun Shen, Alyssa H. Hasty, Joshua M. Buckler, and Daniel T. Eitzman. Recombinant Leptin Promotes Atherosclerosis and Thrombosis in Apolipoprotein E-Deficient Mice. Arterioscler Thromb Vasc Biol, 2005; 25:1634.
36. Soraya Taleb, Olivier Herbin, Hafid Ait-Oufella, Wim Verreth, Pierre Gourdy, Veronique Barateau, Regine Merval, Bruno Esposito, Karine Clement, Paul Holvoet, Alain Tedgui, and Ziad Mallat. Defective Leptin/Leptin Receptor Signaling Improves Regulatory T Cell Immune Response and Protects Mice From Atherosclerosis. Arterioscler Thromb Vasc Biol,2007; 27:2691-2698.
37. Peter F. Bodary, Shufang Gu, Yuechun Shen, Alyssa H. Hasty, Joshua M. Buckler, and Daniel T. Eitzman. Recombinant Leptin Promotes Atherosclerosis and Thrombosis in Apolipoprotein E-Deficient Mice. Arterioscler Thromb Vasc Biol, 2005; 25:1634.
[1]J.F. Mann, K.F. Hilgers, R. Veelken, H. Geiger, R.E. Schmieder, F.C. Luft, Effect of antihypertensive therapy on the progression of non-diabetic renal disease, Clin. Nephrol.38(1992) S74-S77.
[2]A.A. Eddy, Molecular basis of renal fibrosis, Pediatr. Nephrol.15 (2000) 290-301.
[3]B. Rodriguez-Iturbe, N.D. Vaziri, J. Herrera-Acosta, R.J. Johnson, Oxidative stress, renal infiltration of immune cells, and salt-sensitive hypertension:all for one and one for all, Am. J. Physiol. Renal. Physiol.286 (2004) F606-F616.
[4]N. Tian, K.D. Thrasher, P.D. Gundy, M.D. Hughson, R. Davis Manning, Antioxidant treatment prevents renal damage and dysfunction and reduces arterial pressure in salt-sensitive hypertension, Hypertension.45 (2005) 934-939.
[5]K. Schoonjans, B. Staels, J. Auwerx, The peroxisome proliferators activated receptors (PPARs) and their effects on lipid metabolism and adipocyte differentiation, Biochim. Biophys. Acta.1302 (1996) 93-109.
[6]O. Braissant, F. Foufelle, C. Scotto, M. Dauca, W. Wahli, Differential expression of peroxisome proliferator-activated receptors (PPARs):Tissue distribution of PPAR-alpha,-beta,-gamma in the adult rat, Endocrinology.137 (1996) 354-366.
[7]M. Guerre-Millo, P. Gervois, E. Raspe, L. Madsen, P. Poulain, B. Derudas, J.M. Herbert, D.A. Winegar, T.M. Willson, J.C. Fruchart, R.K. Berge, B. Staels, Peroxisome proliferator-activated receptor alpha activators improve insulin sensitivity and reduce adiposity, J. Biol. Chem.275 (2000) 16638-16642.
[8]Q.N. Diep, F. Amiri, R.M. Touyz, J.S. Cohn, D. Endemann, M. Fritsch Neves, E.L. Schiffrin, PPARalpha activator effects on Ang II-induced vascular oxidative stress and inflammation, Hypertension.40 (2002) 866-871.
[9]G.D. Girnun, F.E. Domann, S.A. Moore, M.E.C. Robbins, Identification of a functional peroxisome proliferator-activated receptor response element in the rat catalase promoter, Mol. Endocrinol.16 (2002) 2793-2801.
[10]K. Kono, Y. Kamijo, K. Hora, K. Takahashi, M. Higuchi, K. Kiyosawa, H. Shigematsu, F.J. Gonzalez, T. Aoyama, PPARa attenuates the proinflammatory response in activated mesangial cells, Am. J. Physiol. Renal. Physiol.296 (2009) F328-F336.
[11]P.R. Devchand, H. Keller, J.M. Peters, M. Vazquez, F.J. Gonzalez, W. Wahli, The PPAR-alpha leukotoriene B4 pathway to inflammation control, Nature.384 (1996) 39-43.
[12]L.J. Ma, S. Nakamura, J.C. Aldigier, M. Rossini, H. Yang, X. Liang, I. Nakamura, C. Marcantoni, A.B. Fogo, Regression of glomerulosclerosis with high-dose angiotensin inhibition is linked to decreased plasminogen activator inhibitor-1, J. Am. Soc. Nephrol.16 (2005)966-976.
[13]X. Wei, H. Liu, X. Sun, Hydroxysafflor yellow A protects rat brains against ischemia-reperfusion injury by antioxidant action, Neurosci. Lett.386 (2005) 58-62.
[14]H. Kobori, Y. Ozawa, Y. Suzaki, A. Nishiyama, Enhanced Intrarenal Angiotensinogen Contributes to Early Renal Injury in Spontaneously Hypertensive Rats, J. Am. Soc. Nephrol.16 (2005) 2073-2080.
[15]M. Nangaku, T. Miyata, T. Sada, M. Mizuno, R. Inagi, Y. Ueda, N. Ishikawa, H. Yuzawa, H. Koike, C. van Ypersele de Strihou, K. Kurokawa, Anti-hypertensive agents inhibit in vivo the formation of advanced glycation end products and improve renal damage in a type 2 diabetic nephropathy rat model, J. Am. Soc. Nephrol.14 (2003) 1212-22.
[16]S. Matsuo, Y. Morita, S. Maruyama, L. Manchang, Y. Yuzawa, Proteinuria and tubulointerstitial injury:the causative factors for the progression of renal diseases, Contrib. Nephrol.139 (2003) 20-31.
[17]G. Derosa, A. D'Angelo, L. Ciccarelli, M.N. Piccinni, F. Pricolo, S. Salvadeo, L. Montagna, A. Gravina, I. Ferrari, S. Galli, S. Paniga, C. Tinelli, A.F. Cicero, Matrix metalloproteinase-2,-9, and tissue inhibitor of metalloproteinase-1 in patients with hypertension, Endothelium.13 (2006) 227-231.
[18]R. Kalluri, E.G. Neilson, Epithelial-mesenchymal transition and its implications for fibrosis, J. Clin. Invest.112 (2003) 1776-1784.
[19]S. Racasan, B. Braam, D.M. van der Giezen, R. Goldschmeding, P. Boer, H.A. Koomans, J.A. Joles, Perinatal L-Arginine and Antioxidant Supplements Reduce Adult Blood Pressure in Spontaneously Hypertensive Rats, Hypertension.44 (2004) 83-88.
[20]Y. Ishikawa, T. Nishikimi, K. Akimoto, K. Ishimura, H. Ono, H. Matsuoka, Long-Term Administration of Rho-Kinase Inhibitor Ameliorates Renal Damage in Malignant Hypertensive Rats, Hypertension.47 (2006) 1075-1083.
[21]U. Landmesser, H. Cai, S. Dikalov, L. McCann, J. Hwang, H. Jo, S.M. Holland, D.G. Harrison, Role of p47(phox) in vascular oxidative stress and hypertension caused by angiotensin Ⅱ, Hypertension.40 (2002) 511-515.
[22]T. Chabrashvili, A. Tojo, M. Lika Onozato, C. Kitiyakara, M.T. Quinn, T. Fujita, W.J. Welch, C.S. Wilcox, Expression and cellular localization of classic NADPH oxidase subunits in the spontaneously hypertensive rat kidney, Hypertension.39 (2002) 269-274.
[23]A. Buczynski, Z.B. Wachowic, K. Kedziora-Kornatowska, W. Tkaczewski, J. Kedziora, Changes in antioxidant enzymes activities, aggregability and malonyl dialdehyde concentration in blood platelets from patients with coronary heart disease, Atherosclerosis.100 (1993) 223-228.
[24]S. Meng, L.J. Roberts, G.W. Cason, T.S. Curry, R.D. Manning, Superoxide dismutase and oxidative stress in Dahl salt-sensitive and-resistant rats, Am. J. Physiol. Regul. Integ.r Comp. Physiol.283 (2002) R732-R738.
[25]G. Pearson, F. Robinson, T. Beers Gibson, B. Xu, M. Karandikar, K. Berman, M.H. Cobb, Mitogen-activated protein kinase pathways:regulation and physiological functions, Endocr. Rev.22 (2001) 153-183.
[26]B. Gallego, M.A. Arevalo, O. Flores, J.M. Lopez-Novoa, F. Perez-Barriocanal, Renal fibrosis in diabetic and aortic-constricted hypertensive rats, Am. J. Physiol. Regulatory. Integrative. Comp. Physiol.280 (2001) 1823-1829.
[27]G.X. Zhang, X.M. Lu, S. Kimura, A. Nishiyama, Role of mitochondria in angiotensin II-induced reactive oxygen species and mitogen-activated protein kinase activation, Cardiovasc. Res.76 (2007) 204-212.
[28]A. Nishiyama, Y. Yoshizumi, M. Rahman, H. Kobori, D.M. Seth, A. Miyatake, G.X. Zhang, L. Yao, H. Hitomi, T. Shokoji, H. Kiyomoto, S. Kimura, T. Tamaki, M. Kohno, Y. Abe, Effects of AT1 receptor blockade on renal injury and mitogen-activated protein activity in Dahl salt-sensitive rats, Kidney. Int.65 (2004) 972-81.
[29]M. Awazu, K. Ishikura, M. Hida, M. Hoshiya, Mechanisms of mitogen-activated protein kinase activation in experimental diabetes, J. Am. Soc. Nephrol.10 (1999) 738-745.
[30]C. Stambe, R.C. Atkins, GH. Tesch, T. Masaki, GF. Schreiner, D.J. Nikolic-Paterson, The Role of p38a Mitogen-Activated Protein Kinase Activation in Renal Fibrosis, J. Am. Soc. Nephrol.15 (2004) 370-379.
[31]F.Y. Ma, R.S. Flanc, G.H. Tesch, Y. Han, R.C. Atkins, B.L. Bennett, GC. Friedman, J.H. Fan, D.J. Nikolic-Paterson, A Pathogenic Role for c-Jun Amino-Terminal Kinase Signaling in Renal Fibrosis and Tubular Cell Apoptosis, J. Am. Soc. Nephrol.18 (2007) 472-484.
1. Johnston KW, Rutherford RB, Tilson MD, Shah DM, Hollier L, Stanley JC. Suggested standards for reporting on arterial aneurysms. Subcommittee on Reporting Standards for Arterial Aneurysms, Ad Hoc Committee on Reporting Standards, Society for Vascular Surgery and North Am Chapter, International Society for Cardiovascular Surgery. J Vasc Surg.1991;13:452-458.
2. Kniemeyer HW, Kessler T, Reber PU, Ris HB, Hakki H, Widmer MK. Treatment of ruptured abdominal aortic aneurysm, a permanent challenge or a waste of resources? Prediction of outcome using a multi-organ-dysfunction score. Eur J Vasc Endovasc Surg.2000;19:190-196.
3. Stanley JC, Barnes RW, Ernst CB, Hertzer NR, Mannick JA, Moore WS:Vascular surgery in the United States:workforce issues. Report of the Society for Vascular Surgery and the International Society for Cardiovascular Surgery, North American Chapter, Committee on Workforce Issues. J Vasc Surg 1996,23:172-181
4. Patel MI, Hardman DT, Fisher CM, Appleberg M:Current views on the pathogenesis of abdominal aortic aneurysms. J Am Coll Surg 1995,181:371-382
5. Powell J, Greenhalgh RM:Cellular, enzymatic, and genetic factors in the pathogenesis of abdominal aortic aneurysms. J Vasc Surg 1989,9:297-304
6. Uwe Schonbeck, Galina K. Sukhova, Norbert Gerdes, and Peter Libby. TH2 Predominant Immune Responses Prevail in Human Abdominal Aortic Aneurysm. Am. J. Pathol., Aug 2002; 161:499-506.
7. K Shimizu, M Shichiri, P Libby, RT Lee, and RN Mitchell. Th2-predominant inflammation and blockade of IFN-gamma signaling induce aneurysms in allografted aortas. J Clin Invest, Jul 2004; 114(2):300-8.
8. Kwang Kon Koh, Sang Min Park, and Michael J. Quon. Leptin and Cardiovascular Disease:Response to Therapeutic Interventions. Circulation, Jun 2008; 117:3238-3249.
9. M. Otero, R. Lago, R. Gomez, C. Dieguez, F. Lago, J. Gomez-Reino, and O. Gualillo. Towards a pro-inflammatory and immunomodulatory emerging role of leptin. Rheumatology, Aug 2006; 45:944-950.
10. Crowther M, Goodall S, Jones JL, Bell PR, Thompson MM. Localization of matrix metalloproteinase 2 within the aneurysmal and normal aortic wall. Br J Surg. 2000;87:1391-1400.
11. Elmore JR, Keister BF, Franklin DP, Youkey JR, Carey DJ. Expression of matrix metalloproteinases and TIMPs in human abdominal aortic aneurysms. Ann Vase Surg. 1998;12:221-228.
12. Thompson RW, Holmes DR, Mertens RA, Liao S, Botney MD, Mecham RP, Welgus HG, Parks WC. Production and localization of 92-kilodalton gelatinase in abdominal aortic aneurysms:an elastolytic metalloproteinase expressed by aneurysm-infiltrating macrophages. J Clin Invest.1995;96:318-326.
13. Curci JA, Liao SX, Huffman MD, Shapiro SD, Thompson RW. Expression and localization of macrophage elastase (matrix metalloproteinase-12) in abdominal aortic aneurysms. J Clin Invest.1998; 102:1900-1910.
14. Tamarina NA, McMillan WD, Shively VP, Pearce WH. Expression of matrix metalloproteinases and their inhibitors in aneurysms and normal aorta. Surgery. 1997; 122:264-271.
15. Mao D, Lee JK, Van Vickle SJ, Thompson RW. Expression of collagenase-3 (MMP-13) in human abdominal aortic aneurysms and vascular smooth muscle cells in culture. Biochem Biophys Res Commun.1999; 261:904-910.
16. Nollendorfs A, Greiner TC, Nagase H, Baxter BT. The expression and localization of membrane type-1 matrix metalloproteinase in human abdominal aortic aneurysms. J Vase Surg.2001;34:316-322.
17. Pyo R, Lee JK, Shipley JM, Curci JA, Mao D, Ziporin SJ, Ennis TL, Shapiro SD, Senior RM, Thompson RW. Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms. J Clin Invest.2000;105:1641-1649.
18. Longo GM, Xiong W, Greiner TC, Zhao Y, Fiotti N, Baxter BT. Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms. J Clin Invest.2002; 110:625-632.
19. Carmeliet P, Moons L, Lijnen R, Baes M, Lemaitre V, Tipping P, Drew A, Eeckhout Y, Shapiro S, Lupu F, Collen D. Urokinase-generated plasmin activates matrix metalloproteinases during aneurysm formation. Nat Genet. 1997; 17:439-444.
20. Wang YX, Martin-McNulty B, Freay AD, Sukovich DA, Halks-Miller M, Li WW, Vergona R, Sullivan ME, Morser J, Dole WP, Deng GG. Angiotensin II increases urokinase-type plasminogen activator expression and induces aneurysm in the abdominal aorta of apolipoprotein E-deficient mice. Am J Pathol. 2001;159:1455-1464.
21. Deng GG, Martin-McNulty B, Sukovich DA, Freay A, Halks-Miller M, Thinnes T, Loskutoff DJ, Carmeliet P, Dole WP, Wang YX. Urokinase-type plasminogen activator plays a critical role in angiotensin II-induced abdominal aortic aneurysm. Circ Res.2003;92:510-517.
22. Sasaguri T, Arima N, Tanimoto A, Shimajiri S, Hamada T, Sasaguri Y:A role for interleukin 4 in production of matrix metalloproteinase 1 by human aortic smooth muscle cells. Atherosclerosis 1998,138:247-253
23. Chizzolini C, Rezzonico R, De Luca C, Burger D, Dayer JM:Th2 cell membrane factors in association with IL-4 enhance matrix metalloproteinase-1 (MMP-1) while decreasing MMP-9 production by granulocyte-macrophage colony-stimulating factor-differentiated human monocytes. J Immunol 2000,164:5952-5960
24. Borghaei RC, Rawlings JR PL, Mochan E:Interleukin-4 suppression of interleukin-1-induced transcription of collagenase (MMP-1) andstromelysin 1 (MMP-3) in human synovial fibroblasts. Arthritis Rheum 1998,41:1398-1406
25. Nemoto O, Yamada H, Kikuchi T, Shinmei M, Obata K, Sato H, Seiki M, Shimmei M:Suppression of matrix metalloproteinase-3 synthesis by interleukin-4 in human articular chondrocytes. J Rheumatol 1997,24:1774-1779
26. Frostegard J, Ulfgren AK, Nyberg P, Hedin U, Swedenborg J, Andersson U, Hansson GK:Cytokine expression in advanced human atherosclerotic plaques: dominance of pro-inflammatory (Th1) and macrophage-stimulating cytokines. Atherosclerosis 1999,145:33-43
27. Soraya Taleb, Olivier Herbin, Hafid Ait-Oufella, Wim Verreth, Pierre Gourdy, Veronique Barateau, Regine Merval, Bruno Esposito, Karine Clement, Paul Holvoet, Alain Tedgui, and Ziad Mallat. Defective Leptin/Leptin Receptor Signaling Improves Regulatory T Cell Immune Response and Protects Mice From Atherosclerosis. Arterioscler Thromb Vasc Biol, Dec 2007; 27:2691-2698.
28. Peter F. Bodary, Shufang Gu, Yuechun Shen, Alyssa H. Hasty, Joshua M. Buckler, and Daniel T. Eitzman. Recombinant Leptin Promotes Atherosclerosis and Thrombosis in Apolipoprotein E-Deficient Mice. Arterioscler Thromb Vasc Biol, Aug 2005; 25:1634.
2. Hiroyuki Kobori, Yuri Ozawa, Yuki Suzaki, and Akira Nishiyama. Enhanced Intrarenal Angiotensinogen Contributes to Early Renal Injury in Spontaneously Hypertensive Rats. J Am Soc Nephrol.2005; 16:2073-2080.
3. Hao Xue, Ying-Li Zhang, Guo-Shu Liu, and Hai Wang. A New ATP-Sensitive Potassium Channel Opener Protects the Kidney from Hypertensive Damage in Spontaneously Hypertensive Rats. JPET,2005,315:501-509.
4. Edna D. Lekgabe, Helen Kiriazis, Chongxin Zhao, Qi Xu, Xiao Lei Moore, Yidan Su, Ross A.D. Bathgate, Xiao-Jun Du, Chrishan S. Samuel. Relaxin Reverses Cardiac and Renal Fibrosis in Spontaneously Hypertensive Rats. Hypertension. 2005; 46:412-418.
5.许顶立,任昊.高血压病肾脏损害的诊断与防治.中华心血管病杂志,2004,32：190-192.
6. Ruilope LM.The kidney as a sensor of cardiovascular risk in essential hypertension.J Am Soc Nephrol,2002,13:165.
7. Porush JG. Hypertension and chronic renal failure:the use of ACE inhibitors. Am J Kidney Dis,1998;31:177.
8. Ritz E, Nowicki It Fliser D et al. Proteinuria and hypertension. Kidney Int,1994; 46:76-80.
9.刘力生.高血压.第二版.北京：人民卫生出版社,2001.
10. N Takahashi and O Smithies. Human genetics, animal models and computer simulations for studying hypertension. Trends Genet,2004; 20:136-45.
11. H Kim, JH Krege, KD Kluckman, JR Hagaman, JB Hodgin, CF Best, C Jennette, TM Coffman, N Maeda, and O Smithies. Genetic Control of Blood Pressure and the Angiotensinogen Locus. PNAS,1995; 92:2735-2739.
12. JJ Mullins, J Peters, and D Ganten. Fulminant hypertension in transgenic rats harbouring the mouse Ren-2 gene. Nature,1990; 344:541-4.
13. Z Yang, DR Sibley, and PA Jose. D5 dopamine receptor knockout mice and hypertension. J Recept Signal Transduct Res,2004; 24:149-64.
14. Paula M. Oliver, Jennifer E. Fox, Ron Kim, Howard A. Rockman, Hyung-Suk Kim, Robert L. Reddick, Kailash N. Pandey, Sharon L. Milgram, Oliver Smithies, and Nobuyo Maeda. Hypertension, cardiac hypertrophy, and sudden death in mice lacking natriuretic peptide receptor A. PNAS,1997; 94:14730-14735.
15. Y Kurihara, H Kurihara, H Suzuki, T Kodama, K Maemura, R Nagai, H Oda, T Kuwaki, WH Cao, and N Kamada. Elevated blood pressure and craniofacial abnormalities in mice deficient in endothelin-1. Nature,1994; 368:703-10.
16. W Li, S Mital, C Ojaimi, A Csiszar, G Kaley, and TH Hintze. Premature death and age-related cardiac dysfunction in male eNOS-knockout mice. J Mol Cell Cardiol, 2004; 37:671-80.
17. Yigal M Pinto, Martin Paul, and Detlev Ganten. Lessons from rat models of hypertension:from Goldblatt to genetic engineering. Cardiovasc Res,1998; 39: 77-88.
18. Lilach O. Lerman, Karl A. Nath, Martin Rodriguez-Porcel, James D. Krier, Robert S. Schwartz, Claudio Napoli, and J. Carlos Romero. Increased Oxidative Stress in Experimental Renovascular Hypertension. Hypertension,2001; 37:541-546.
19. V Lahera, J Salazar, MG Salom, and JC Romero. Deficient production of nitric oxide induces volume-dependent hypertension. J Hypertens Suppl,1992; 10: S173-7.
20. C Catena, G Giacchetti, M Novello, G Colussi, A Cavarape, and LA Sechi. Cellular mechanisms of insulin resistance in rats with fructose-induced hypertension. Am J Hypertens,2003; 16:973-8.
21. Schreiner GF. Renal toxicity of albumin and other lipoproteins.Curr Opin Nephrol Hypertens,1995; 4:369-373
22. Wheeler DC,Chana RS, Topley N, et al. Oxidation of low density lipoprotein by mesangial cells may promote glomerular injury. Kidney Int,1994; 45:1628-1636
23. BH Rovin and LC Tan. LDL stimulates mesangial fibronectin production and chemoattractant expression. Kidney Int, Jan 1993; 43:218-25.
24. DC Wheeler, RS Chana, N Topley, MM Petersen, M Davies, and JD Williams. Oxidation of low density lipoprotein by mesangial cells may promote glomerular injury. Kidney Int, Jun 1994; 45:1628-36.
25. Madamanchi NR, Runge MS. Mitochondrial dysfunction in atherosclerosis. Circ Res.2007; 100:460-73.
26. Bassenge E, Schneider HT, Daiber A. Oxidative stress and cardiovascular diseases. Dtsch Med Wochenschr.2005; 130:2904-2909.
27. Heistad DD. Oxidative stress and vascular disease. Arterioscler Thromb Vasc Biol. 2005; 12:104.
28. Deshpande NN, Sorescu D, Seshiah P, Ushio-Fukai M, Akers M, Yin Q, Griendling KK. Mechanism of hydrogen peroxide-induced cell cycle arrest in vascular smooth muscle. Antioxid Redox Signal.2002; 4:845-854.
29. Taniyama Y, Griendling KK. Reactive oxygen species in t he vasculature molecular and cellular mechanisms. Hypertension.2003; 42:1075.
30. Budin SB, Othman F, Louis SR, Bakar MA, Das S, Mohamed J. The effects of palm oil tocotrienol-rich fraction supplementation on biochemical parameters, oxidative stress and the vascular wall of streptozotocin-induced diabetic rats. Clinics.2009; 64:235-44.
31. Vaziri ND, Wang XQ, Oveisi F, Rad B. Induction of oxidative stress by glutathione depletion cause severe hypertension in normal rats. Hypertension. 2000; 39:142.
32. Visconti R, Grieco D. New insights on oxidative stress in cancer. Curr Opin Drug Discov Devel.2009; 12:240-5.
33. Tribble DL, Barcellos MH, et al. Ionizing radiation accelerates arotic lesion formation in fat-efd mice via SOD-inhibitable processes. Arterioscler Thromb Vasc Biol,1999,19:1387-1392.
34. Young IS, Woodside jv. Antioxidants in health and disease. J Clin Pathol,2001, 54:176-186.
35. WilmerWA, Dixon CL, Hebert C, et al. Chronic exposure of human mesangial cells to high glucose environment activates the p38 MAPK pathway. Kidney Int, 2001,60:858-871.
36. Vsui M, Egashira K, Kitamoto S, et al. Pathogenic role of oxidative stress in vascular angiotensin-converting enzyme activation in long-term blockade of nitric oxide synthesis in rats. Hypertension,1999,34:546-551.
37. Vega GW, Roson MI, Beliver A, et al. Nitric oxide and superoxide anions in vascular reactivity of renovascular hypertensive rats. Clin Exp Hypertens,1995, 17:817-835.
38. Lerman LO, Nath KA, Rodriguez PM, et al. Increased oxidative stress in experimental renovascular hypertension. Hypertension,2001,37:541-546.
39. Vaziri ND,Oveisi F,Ding Y. Role of incresaed oxygen free radical activity in the pathogenesis of uremic hypertension. Kidney Int,1998,53:1748-1754.
40. Vaziri ND,Liang K,Ding Y Increased nitric oxide inactivation by reactive oxygen species in Lead-induced hypertension. Kidney Int,1999,56:1492-1498.
41. Bapats, Post JA,Braam B, et al. Visualizing tubular lipid peroxidation in intact renal tissue in hypertensive rats. J Am Soc Nephrol,2002,13:2990-2996.
42. Bernardo Rodriguez-Iturbe, Atilio Ferrebuz, Valentina Vanegas, Yasmir Quiroz, Sergio Mezzano, and Nosratola D. Vaziri. Early and Sustained Inhibition of Nuclear Factor-κB Prevents Hypertension in Spontaneously Hypertensive Rats. J. Pharmacol. Exp. Ther., Oct 2005; 315:51-57.
43. Mayerly Nava, Yasmir Quiroz, Nosratola Vaziri, and Bernardo Rodriguez-Iturbe. Melatonin reduces renal interstitial inflammation and improves hypertension in spontaneously hypertensive rats. Am J Physiol Renal Physiol,2003; 284:447.
44. Bernardo Rodriguez-Iturbe, Chang-De Zhan, Yasmir Quiroz, Ram K. Sindhu, and Nosratola D. Vaziri. Antioxidant-Rich Diet Relieves Hypertension and Reduces Renal Immune Infiltration in Spontaneously Hypertensive Rats. Hypertension, 2003; 41:341-346.
45. Li JM, Shah AM. ROS Generation by Nonphagocytic NADPH Oxidase:Potential Relevance in Diabetic Nephropathy. J Am Soc Nephrol,2003,14:S221-S226.
46. Evans JL, Goldfine ID, Maddux BA, et al. Oxidative stress and stress-activated signaling pathways:a unifying hypothesis of type 2 diabetes. Endocrine Rev,2002, 23:599-622.
47. Han CH, Lee MH. Expression and characterization of the flavoprotein domain of gp91phox. J Vet Sci,2000,1:19-26.
48. Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase:role in cardiovascular biology and disease. Circ Res,2000,86:494-501.
49. Lavigne MC, Malech H 乙 Holland SM, et al. Genetic demonstration of p47phox—dependent superoxide anion production in murine vascular smooth muscle cells. Circulation,2001,104:79-84.
50. Shiose A, Kuroda J, Tsuruya K, et al. A novel superoxide-producing NAD(P)H oxidase in kidney. J Biol Chem,2001,276:1417-1423.
51. Asaba K, Tojo A, Onozato ML, et al. Effects of NADPH oxidase inhibitor in diabetic nephropathy. Kidney Int,2005,67:1890-1898.
52. Niu Tian, Rebecca S. Moore, William E. Phillips, Lin Lin, Sharkeshia Braddy, Janelle S. Pryor, Rachel L. Stockstill, Michael D. Hughson, and R. Davis Manning, Jr. NADPH oxidase contributes to renal damage and dysfunction in Dahl salt-sensitive hypertension. Am J Physiol Regulatory Integrative Comp Physiol, 2008; 295:R1858-R1865.
53. Adler S, Huang H. Oxidant stress in kidneys of spontaneously hypertensive rats involves both oxidase overexpression and loss of extracellular superoxide dismutase. Am J Physiol Renal Physiol,2004,287:907-913.
54.Adam Whaley-Connell, Javad Habibi, Ravi Nistala, Shawna A. Cooper, Poorna R. Karuparthi, Melvin R. Hayden, Nathan Rehmer, Vincent G DeMarco, Bradley T. Andresen, Yongzhong Wei, Carlos Ferrario, and James R. Sowers. Attenuation of NADPH Oxidase Activation and Glomerular Filtration Barrier Remodeling With Statin Treatment. Hypertension,2008; 51:474-480.
55. Ohishi K, Carmines PK. Superoxide dismutase restores the influence of nitric oxide on renal arterioles in diabetes mellitus. J Am Soc Nephrol,1995,5: 1559-66.
56. Ha H, Lee HB. Reactive oxygen species and matrix remodeling in diabetic kidney. J Am Soc Nephrol,2003,14:S246-S249.
57. Zhang B, Liang X, Shi W, et al. Role of impaired peritubular capillary and hypoxia in p rogressive interstitial fibrosis after 5/6 subtotal nephrectomy of rats. Nephrology,2005,10:351-357.
58. Rhyu DY, Yang Y, Ha H, et al. Role of reactive oxygen species in TGF-β1 induced mitogen-activated protein kinase activation and epithelial-mesenchymal transition in renal tubular epithelial cells. J Am Soc Nephrol,2005,16:667-675.
59. G. H. Tesch. MCP-1/CCL2:a new diagnostic marker and therapeutic target for progressive renal injury in diabetic nephropathy. Am J Physiol Renal Physiol, 2008; 294:F697-F701.
60. Hunjoo Ha, Mi Ra Yu, Yoon Jin Choi, Masanori Kitamura, and Hi Bahl Lee. Role of High Glucose-Induced Nuclear Factor-κB Activation in Monocyte Chemoattractant Protein-1 Expression by Mesangial Cells. J. Am. Soc. Nephrol., 2002; 13:894-902.
61. Wenzel UO. Chemokines and renal disease. J. Am Kidney Dis,1995; 26:982-995
62. KF Hilgers, A Hartner, M Porst, M Mai, M Wittmann, C Hugo, D Ganten, H Geiger, R Veelken, and JF Mann. Monocyte chemoattractant protein-1 and macrophage infiltration in hypertensive kidney injury. Kidney Int,2000; 58: 2408-19.
63. Sarah F. Knight, Jeffrey E. Quigley, Jianghe Yuan, Siddhartha S. Roy, Ahmed Elmarakby, and John D. Imig. Endothelial Dysfunction and the Development of Renal Injury in Spontaneously Hypertensive Rats Fed a High-Fat Diet. Hypertension,2008; 51:352-359.
64. Marshall CB, Shankland SJ. Cell cycle and glomerular disease:a minkeview. Nephron Exp Nephrol,2006,102:e39-48.
65. Yamagata K, Hagiwara M. Pathological roles of mitochondrial dysfunction in podocyte injury. Nippon Jinzo Gakkai Shi,2007,49:82-7.
66. Nam-Ho Kim, Hernan Rincon-Choles, Basant Bhandari, Goutam Ghosh Choudhury, Hanna E. Abboud, and Yves Gorin. Redox dependence of glomerular epithelial cell hypertrophy in response to glucose. Am J Physiol Renal Physiol, 2006; 290:F741-F751.
67. Jing Dong, Sampath Ramachandiran, Kulbhushan Tikoo, Zhe Jia, Serrine S. Lau, and Terrence J. Monks. EGFR-independent activation of p38 MAPK and EGFR-dependent activation of ERK1/2 are required for ROS-induced renal cell death. Am J Physiol Renal Physiol,2004; 287:F1049-F1058.
1. Michael W. Manning, Lisa A. Cassis, Alan Daugherty. Differential Effects of Doxycycline, a Broad-Spectrum Matrix Metalloproteinase Inhibitor, on Angiotensin II-Induced Atherosclerosis and Abdominal Aortic Aneurysms. Arterioscler Thromb Vasc Biol.2003;23:483-488
2. Manesh Thomas; Dan Gavrila; Michael L. McCormick; Francis J. Miller, Jr; Alan Daugherty; Lisa A. Cassis; Kevin C. Dellsperger; Neal L. Weintraub. Deletion of p47phox Attenuates Angiotensin II-Induced Abdominal Aortic Aneurysm Formation in Apolipoprotein E-Deficient Mice. Circulation.2006;114:404-413.
3. Victoria L. King; Alexander Y. Lin; Fjoralba Kristo; Thomas J.T. Anderson; Neil Ahluwalia; Gregory J. Hardy; A. Phillip Owens Ⅲ; Deborah A. Howatt; Dongxiao Shen; Andrew M. Tager; Andrew D. Luster; Alan Daugherty; Robert E. Gerszten. Interferon-y and the Interferon-Inducible Chemokine CXCL10 Protect Against Aneurysm Formation and Rupture. Circulation.2009; 119:426-435.
4. Kimio Satoh, Patrizia Nigro, Tetsuya Matoba, Michael R O'Dell, Zhaoqiang Cui, Xi Shi, Amy Mohan, Chen Yan, Jun-ichi Abe, Karl A Illig & Bradford C Berk. Cyclophilin A enhances vascular oxidative stress and the development of angiotensin II-induced aortic aneurysms. Nat Med.2009; 15:649-56.
5. KW Johnston, RB Rutherford, MD Tilson, DM Shah, L Hollier, and JC Stanley. Suggested standards for reporting on arterial aneurysms. Subcommittee on Reporting Standards for Arterial Aneurysms, Ad Hoc Committee on Reporting Standards, Society for Vascular Surgery and North American Chapter, International Society for Cardiovascular Surgery. J Vasc Surg,1991; 13:452-8.
6. Beckman JA. Aortic aneurysms:pathophysiology, epidemiology, and prognosis. In: Creager MA, Dzau VJ, Loscalzo J, eds. Vascular Medicine. Philadelphia, Pa: Saunders Elsevier Inc; 2006.
7. Beckman JA, Creager MA. Aortic aneurysms:Clinical evaluation. In:Creager MA, Dzau VJ, Loscalzo J, eds. Vascular Medicine. Philadelphia, Pa:Saunders Elsevier Inc; 2006.
8. H Bengtsson, B Sonesson, and D Bergqvist. Incidence and prevalence of abdominal aortic aneurysms, estimated by necropsy studies and population screening by ultrasound. Ann N Y Acad Sci,1996; 800:1-24.
9. J W Hallett, Jr. Management of abdominal aortic aneurysms. Mayo Clin. Proc, 2000; 75:395-399.
10. A Hallin, D Bergqvist, and L Holmberg. Literature review of surgical management of abdominal aortic aneurysm. Eur J Vasc Endovasc Surg,2001; 22:197-204.
11. M M Thompson and P R F Bell. ABC of arterial and venous disease:Arterial aneurysms.BMJ,2000; 320:1193-1196.
12. A Lopez-Candales, DR Holmes, S Liao, MJ Scott, SA Wickline, and RW Thompson. Decreased vascular smooth muscle cell density in medial degeneration of human abdominal aortic aneurysms.Am. J. Pathol.,1997; 150:993-1007.
13. Tim Freestone, Robert J. Turner, Andrew Coady, Dan J. Higman, Roger M. Greenhalgh, and Janet T. Powell. Inflammation and Matrix Metalloproteinases in the Enlarging Abdominal Aortic Aneurysm. Arterioscler Thromb Vasc Biol,1995; 15:1145-1151.
14. N Diehm, F Dick, T Schaffner, J Schmidli, C Kalka, S Di Santo, J Voelzmann, and I Baumgartner. Novel insight into the pathobiology of abdominal aortic aneurysm and potential future treatment concepts.. Prog Cardiovasc Dis,2007; 50:209-17.
15. Valerie Davis, Raisa Persidskaia, Lisa Baca-Regen, Yoshifumi Itoh, Hideaki Nagase, Yuri Persidsky, Anuja Ghorpade, and B. Timothy Baxter. Matrix Metalloproteinase-2 Production and Its Binding to the Matrix Are Increased in Abdominal Aortic Aneurysms. Arterioscler Thromb Vasc Biol,1998; 18:1625-1633.
16. RW Thompson, DR Holmes, RA Mertens, S Liao, MD Botney, RP Mecham, HG Welgus, and WC Parks. Production and localization of 92-kilodalton gelatinase in abdominal aortic aneurysms. An elastolytic metalloproteinase expressed by aneurysm-infiltrating macrophages. J Clin Invest,1995; 96:318-26.
17. JA Curci, S Liao, MD Huffman, SD Shapiro, and RW Thompson. Expression and localization of macrophage elastase (matrix metalloproteinase-12) in abdominal aortic aneurysms. J Clin Invest,1998; 102:1900-10.
18.I Sinha, KK Hannawa, JL Eliason, G Ailawadi, MP Deogracias, S Bethi, JW Ford, KJ Roelofs, V Grigoryants, PK Henke, JC Stanley, and GR Upchurch Jr. Early MT-1 MMP expression following elastase exposure is associated with increased cleaved MMP-2 activity in experimental rodent aortic aneurysms. Surgery, Aug 2004; 136:176-82.
19. Helena Kuivaniemi, MD, PhD; Chris D. Platsoucas, PhD; M. David Tilson Ⅲ, MD. Aortic Aneurysms An Immune Disease With a Strong Genetic Component. Circulation 2008; 117; 242-252
20. Schonbeck U, Sukhova GK, Gerdes N, Libby P. T(H)2 predominant immune responses prevail in human abdominal aortic aneurysm. Am J Pathol.2002; 161: 499-506.
21. Davis VA, Persidskaia RN, Baca-Regen LM, Fiotti N, Halloran BG, Baxter BT. Cytokine pattern in aneurysmal and occlusive disease of the aorta. J Surg Res. 2001; 101:152-156.
22. Shimizu K, Shichiri M, Libby P, Lee RT, Mitchell RN. Th2-predominant inflammation and blockade of IFN-gamma signaling induce aneurysms in allografted aortas. J Clin Invest.2004; 114:300-308.
23. Nordskog BK, Fields WR, Hellmann GM. Kinetic analysis of cytokine response to cigarette smoke condensate by human endothelial and monocytic cells. Toxicology.2005; 212:87-97.
24. Ouyang Y, Virasch N, Hao P, Aubrey MT, Mukerjee N, Bierer BE, Freed BM. Suppression of human IL-1beta, IL-2, IFN-gamma, and TNF-alpha production by cigarette smoke extracts. J Allergy Clin Immunol.2000; 106:280-287.
25. Russell RE, Culpitt SV, DeMatos C, Donnelly L, Smith M, Wiggins J, Barnes PJ. Release and activity of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 by alveolar macrophages from patients with chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol.2002; 26:602-609.
26. Lavigne MC, Eppihimer MJ. Cigarette smoke condensate induces MMP-12 gene expression in airway-like epithelia. Biochem Biophys Res Commun.2005; 330: 194-203.
27. Tanabe K, Okuya S, Tanizawa Y,et al. Leptin induces proliferation of pancreatic beta cell line MIN6 through activation of mitogen-activated protein[J]. Biochem Biophys Res Commun.1997; 241:765-768.
28. Doneray H, Orbak Z, Yildiz L. The relationship between breast milk leptin and neonatal weight gain. Acm Paediatr.2009; 98:643.647.
29. Rajakumar A, Parrott MS, Powers RW. Leptin Affects System A Amino Acid Transport Activity in the Human Placenta:Evidence for STAT3 Dependent Mechanisms. Placenta.2009; 30:361-367.
30. Wu MH, Chou YC, Chou WY. Circulating levels of leptin, adiposity and breast cancer risk. Br J Cancer.2009; 100:578-582.
31. Sattar N, Wannamethee G Sarwar N et al. Leptin and coronary heart disease: prospective study and systematic review. J Am Coil Cardiol.2009; 53:167—175.
32. G Matarese, A La Cava, V Sanna, GM Lord, RI Lechler, S Fontana, and S Zappacosta. Balancing susceptibility to infection and autoimmunity:a role for leptin? Trends Immunol,2002; 23:182-7.
33. IS Farooqi, G Matarese, GM Lord, JM Keogh, E Lawrence, C Agwu, V Sanna, SA Jebb, F Perna, S Fontana, RI Lechler, AM DePaoli, and S O'Rahilly. Beneficial effects of leptin on obesity, T cell hyporesponsiveness, and neuroendocrine/metabolic dysfunction of human congenital leptin deficiency. J Clin Invest,2002; 110:1093-103.
34. Giuseppe Matarese, Antonio Di Giacomo, Veronica Sanna, Graham M. Lord, Jane K. Howard, Antonino Di Tuoro, Stephen R. Bloom, Robert I. Lechler, Serafino Zappacosta, and Silvia Fontana. Requirement for Leptin in the Induction and Progression of Autoimmune Encephalomyelitis. J. Immunol.,2001; 166:5909-5916.
35. Peter F. Bodary, Shufang Gu, Yuechun Shen, Alyssa H. Hasty, Joshua M. Buckler, and Daniel T. Eitzman. Recombinant Leptin Promotes Atherosclerosis and Thrombosis in Apolipoprotein E-Deficient Mice. Arterioscler Thromb Vasc Biol, 2005; 25:1634.
36. Soraya Taleb, Olivier Herbin, Hafid Ait-Oufella, Wim Verreth, Pierre Gourdy, Veronique Barateau, Regine Merval, Bruno Esposito, Karine Clement, Paul Holvoet, Alain Tedgui, and Ziad Mallat. Defective Leptin/Leptin Receptor Signaling Improves Regulatory T Cell Immune Response and Protects Mice From Atherosclerosis. Arterioscler Thromb Vasc Biol,2007; 27:2691-2698.
37. Peter F. Bodary, Shufang Gu, Yuechun Shen, Alyssa H. Hasty, Joshua M. Buckler, and Daniel T. Eitzman. Recombinant Leptin Promotes Atherosclerosis and Thrombosis in Apolipoprotein E-Deficient Mice. Arterioscler Thromb Vasc Biol, 2005; 25:1634.
[1]J.F. Mann, K.F. Hilgers, R. Veelken, H. Geiger, R.E. Schmieder, F.C. Luft, Effect of antihypertensive therapy on the progression of non-diabetic renal disease, Clin. Nephrol.38(1992) S74-S77.
[2]A.A. Eddy, Molecular basis of renal fibrosis, Pediatr. Nephrol.15 (2000) 290-301.
[3]B. Rodriguez-Iturbe, N.D. Vaziri, J. Herrera-Acosta, R.J. Johnson, Oxidative stress, renal infiltration of immune cells, and salt-sensitive hypertension:all for one and one for all, Am. J. Physiol. Renal. Physiol.286 (2004) F606-F616.
[4]N. Tian, K.D. Thrasher, P.D. Gundy, M.D. Hughson, R. Davis Manning, Antioxidant treatment prevents renal damage and dysfunction and reduces arterial pressure in salt-sensitive hypertension, Hypertension.45 (2005) 934-939.
[5]K. Schoonjans, B. Staels, J. Auwerx, The peroxisome proliferators activated receptors (PPARs) and their effects on lipid metabolism and adipocyte differentiation, Biochim. Biophys. Acta.1302 (1996) 93-109.
[6]O. Braissant, F. Foufelle, C. Scotto, M. Dauca, W. Wahli, Differential expression of peroxisome proliferator-activated receptors (PPARs):Tissue distribution of PPAR-alpha,-beta,-gamma in the adult rat, Endocrinology.137 (1996) 354-366.
[7]M. Guerre-Millo, P. Gervois, E. Raspe, L. Madsen, P. Poulain, B. Derudas, J.M. Herbert, D.A. Winegar, T.M. Willson, J.C. Fruchart, R.K. Berge, B. Staels, Peroxisome proliferator-activated receptor alpha activators improve insulin sensitivity and reduce adiposity, J. Biol. Chem.275 (2000) 16638-16642.
[8]Q.N. Diep, F. Amiri, R.M. Touyz, J.S. Cohn, D. Endemann, M. Fritsch Neves, E.L. Schiffrin, PPARalpha activator effects on Ang II-induced vascular oxidative stress and inflammation, Hypertension.40 (2002) 866-871.
[9]G.D. Girnun, F.E. Domann, S.A. Moore, M.E.C. Robbins, Identification of a functional peroxisome proliferator-activated receptor response element in the rat catalase promoter, Mol. Endocrinol.16 (2002) 2793-2801.
[10]K. Kono, Y. Kamijo, K. Hora, K. Takahashi, M. Higuchi, K. Kiyosawa, H. Shigematsu, F.J. Gonzalez, T. Aoyama, PPARa attenuates the proinflammatory response in activated mesangial cells, Am. J. Physiol. Renal. Physiol.296 (2009) F328-F336.
[11]P.R. Devchand, H. Keller, J.M. Peters, M. Vazquez, F.J. Gonzalez, W. Wahli, The PPAR-alpha leukotoriene B4 pathway to inflammation control, Nature.384 (1996) 39-43.
[12]L.J. Ma, S. Nakamura, J.C. Aldigier, M. Rossini, H. Yang, X. Liang, I. Nakamura, C. Marcantoni, A.B. Fogo, Regression of glomerulosclerosis with high-dose angiotensin inhibition is linked to decreased plasminogen activator inhibitor-1, J. Am. Soc. Nephrol.16 (2005)966-976.
[13]X. Wei, H. Liu, X. Sun, Hydroxysafflor yellow A protects rat brains against ischemia-reperfusion injury by antioxidant action, Neurosci. Lett.386 (2005) 58-62.
[14]H. Kobori, Y. Ozawa, Y. Suzaki, A. Nishiyama, Enhanced Intrarenal Angiotensinogen Contributes to Early Renal Injury in Spontaneously Hypertensive Rats, J. Am. Soc. Nephrol.16 (2005) 2073-2080.
[15]M. Nangaku, T. Miyata, T. Sada, M. Mizuno, R. Inagi, Y. Ueda, N. Ishikawa, H. Yuzawa, H. Koike, C. van Ypersele de Strihou, K. Kurokawa, Anti-hypertensive agents inhibit in vivo the formation of advanced glycation end products and improve renal damage in a type 2 diabetic nephropathy rat model, J. Am. Soc. Nephrol.14 (2003) 1212-22.
[16]S. Matsuo, Y. Morita, S. Maruyama, L. Manchang, Y. Yuzawa, Proteinuria and tubulointerstitial injury:the causative factors for the progression of renal diseases, Contrib. Nephrol.139 (2003) 20-31.
[17]G. Derosa, A. D'Angelo, L. Ciccarelli, M.N. Piccinni, F. Pricolo, S. Salvadeo, L. Montagna, A. Gravina, I. Ferrari, S. Galli, S. Paniga, C. Tinelli, A.F. Cicero, Matrix metalloproteinase-2,-9, and tissue inhibitor of metalloproteinase-1 in patients with hypertension, Endothelium.13 (2006) 227-231.
[18]R. Kalluri, E.G. Neilson, Epithelial-mesenchymal transition and its implications for fibrosis, J. Clin. Invest.112 (2003) 1776-1784.
[19]S. Racasan, B. Braam, D.M. van der Giezen, R. Goldschmeding, P. Boer, H.A. Koomans, J.A. Joles, Perinatal L-Arginine and Antioxidant Supplements Reduce Adult Blood Pressure in Spontaneously Hypertensive Rats, Hypertension.44 (2004) 83-88.
[20]Y. Ishikawa, T. Nishikimi, K. Akimoto, K. Ishimura, H. Ono, H. Matsuoka, Long-Term Administration of Rho-Kinase Inhibitor Ameliorates Renal Damage in Malignant Hypertensive Rats, Hypertension.47 (2006) 1075-1083.
[21]U. Landmesser, H. Cai, S. Dikalov, L. McCann, J. Hwang, H. Jo, S.M. Holland, D.G. Harrison, Role of p47(phox) in vascular oxidative stress and hypertension caused by angiotensin Ⅱ, Hypertension.40 (2002) 511-515.
[22]T. Chabrashvili, A. Tojo, M. Lika Onozato, C. Kitiyakara, M.T. Quinn, T. Fujita, W.J. Welch, C.S. Wilcox, Expression and cellular localization of classic NADPH oxidase subunits in the spontaneously hypertensive rat kidney, Hypertension.39 (2002) 269-274.
[23]A. Buczynski, Z.B. Wachowic, K. Kedziora-Kornatowska, W. Tkaczewski, J. Kedziora, Changes in antioxidant enzymes activities, aggregability and malonyl dialdehyde concentration in blood platelets from patients with coronary heart disease, Atherosclerosis.100 (1993) 223-228.
[24]S. Meng, L.J. Roberts, G.W. Cason, T.S. Curry, R.D. Manning, Superoxide dismutase and oxidative stress in Dahl salt-sensitive and-resistant rats, Am. J. Physiol. Regul. Integ.r Comp. Physiol.283 (2002) R732-R738.
[25]G. Pearson, F. Robinson, T. Beers Gibson, B. Xu, M. Karandikar, K. Berman, M.H. Cobb, Mitogen-activated protein kinase pathways:regulation and physiological functions, Endocr. Rev.22 (2001) 153-183.
[26]B. Gallego, M.A. Arevalo, O. Flores, J.M. Lopez-Novoa, F. Perez-Barriocanal, Renal fibrosis in diabetic and aortic-constricted hypertensive rats, Am. J. Physiol. Regulatory. Integrative. Comp. Physiol.280 (2001) 1823-1829.
[27]G.X. Zhang, X.M. Lu, S. Kimura, A. Nishiyama, Role of mitochondria in angiotensin II-induced reactive oxygen species and mitogen-activated protein kinase activation, Cardiovasc. Res.76 (2007) 204-212.
[28]A. Nishiyama, Y. Yoshizumi, M. Rahman, H. Kobori, D.M. Seth, A. Miyatake, G.X. Zhang, L. Yao, H. Hitomi, T. Shokoji, H. Kiyomoto, S. Kimura, T. Tamaki, M. Kohno, Y. Abe, Effects of AT1 receptor blockade on renal injury and mitogen-activated protein activity in Dahl salt-sensitive rats, Kidney. Int.65 (2004) 972-81.
[29]M. Awazu, K. Ishikura, M. Hida, M. Hoshiya, Mechanisms of mitogen-activated protein kinase activation in experimental diabetes, J. Am. Soc. Nephrol.10 (1999) 738-745.
[30]C. Stambe, R.C. Atkins, GH. Tesch, T. Masaki, GF. Schreiner, D.J. Nikolic-Paterson, The Role of p38a Mitogen-Activated Protein Kinase Activation in Renal Fibrosis, J. Am. Soc. Nephrol.15 (2004) 370-379.
[31]F.Y. Ma, R.S. Flanc, G.H. Tesch, Y. Han, R.C. Atkins, B.L. Bennett, GC. Friedman, J.H. Fan, D.J. Nikolic-Paterson, A Pathogenic Role for c-Jun Amino-Terminal Kinase Signaling in Renal Fibrosis and Tubular Cell Apoptosis, J. Am. Soc. Nephrol.18 (2007) 472-484.
1. Johnston KW, Rutherford RB, Tilson MD, Shah DM, Hollier L, Stanley JC. Suggested standards for reporting on arterial aneurysms. Subcommittee on Reporting Standards for Arterial Aneurysms, Ad Hoc Committee on Reporting Standards, Society for Vascular Surgery and North Am Chapter, International Society for Cardiovascular Surgery. J Vasc Surg.1991;13:452-458.
2. Kniemeyer HW, Kessler T, Reber PU, Ris HB, Hakki H, Widmer MK. Treatment of ruptured abdominal aortic aneurysm, a permanent challenge or a waste of resources? Prediction of outcome using a multi-organ-dysfunction score. Eur J Vasc Endovasc Surg.2000;19:190-196.
3. Stanley JC, Barnes RW, Ernst CB, Hertzer NR, Mannick JA, Moore WS:Vascular surgery in the United States:workforce issues. Report of the Society for Vascular Surgery and the International Society for Cardiovascular Surgery, North American Chapter, Committee on Workforce Issues. J Vasc Surg 1996,23:172-181
4. Patel MI, Hardman DT, Fisher CM, Appleberg M:Current views on the pathogenesis of abdominal aortic aneurysms. J Am Coll Surg 1995,181:371-382
5. Powell J, Greenhalgh RM:Cellular, enzymatic, and genetic factors in the pathogenesis of abdominal aortic aneurysms. J Vasc Surg 1989,9:297-304
6. Uwe Schonbeck, Galina K. Sukhova, Norbert Gerdes, and Peter Libby. TH2 Predominant Immune Responses Prevail in Human Abdominal Aortic Aneurysm. Am. J. Pathol., Aug 2002; 161:499-506.
7. K Shimizu, M Shichiri, P Libby, RT Lee, and RN Mitchell. Th2-predominant inflammation and blockade of IFN-gamma signaling induce aneurysms in allografted aortas. J Clin Invest, Jul 2004; 114(2):300-8.
8. Kwang Kon Koh, Sang Min Park, and Michael J. Quon. Leptin and Cardiovascular Disease:Response to Therapeutic Interventions. Circulation, Jun 2008; 117:3238-3249.
9. M. Otero, R. Lago, R. Gomez, C. Dieguez, F. Lago, J. Gomez-Reino, and O. Gualillo. Towards a pro-inflammatory and immunomodulatory emerging role of leptin. Rheumatology, Aug 2006; 45:944-950.
10. Crowther M, Goodall S, Jones JL, Bell PR, Thompson MM. Localization of matrix metalloproteinase 2 within the aneurysmal and normal aortic wall. Br J Surg. 2000;87:1391-1400.
11. Elmore JR, Keister BF, Franklin DP, Youkey JR, Carey DJ. Expression of matrix metalloproteinases and TIMPs in human abdominal aortic aneurysms. Ann Vase Surg. 1998;12:221-228.
12. Thompson RW, Holmes DR, Mertens RA, Liao S, Botney MD, Mecham RP, Welgus HG, Parks WC. Production and localization of 92-kilodalton gelatinase in abdominal aortic aneurysms:an elastolytic metalloproteinase expressed by aneurysm-infiltrating macrophages. J Clin Invest.1995;96:318-326.
13. Curci JA, Liao SX, Huffman MD, Shapiro SD, Thompson RW. Expression and localization of macrophage elastase (matrix metalloproteinase-12) in abdominal aortic aneurysms. J Clin Invest.1998; 102:1900-1910.
14. Tamarina NA, McMillan WD, Shively VP, Pearce WH. Expression of matrix metalloproteinases and their inhibitors in aneurysms and normal aorta. Surgery. 1997; 122:264-271.
15. Mao D, Lee JK, Van Vickle SJ, Thompson RW. Expression of collagenase-3 (MMP-13) in human abdominal aortic aneurysms and vascular smooth muscle cells in culture. Biochem Biophys Res Commun.1999; 261:904-910.
16. Nollendorfs A, Greiner TC, Nagase H, Baxter BT. The expression and localization of membrane type-1 matrix metalloproteinase in human abdominal aortic aneurysms. J Vase Surg.2001;34:316-322.
17. Pyo R, Lee JK, Shipley JM, Curci JA, Mao D, Ziporin SJ, Ennis TL, Shapiro SD, Senior RM, Thompson RW. Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms. J Clin Invest.2000;105:1641-1649.
18. Longo GM, Xiong W, Greiner TC, Zhao Y, Fiotti N, Baxter BT. Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms. J Clin Invest.2002; 110:625-632.
19. Carmeliet P, Moons L, Lijnen R, Baes M, Lemaitre V, Tipping P, Drew A, Eeckhout Y, Shapiro S, Lupu F, Collen D. Urokinase-generated plasmin activates matrix metalloproteinases during aneurysm formation. Nat Genet. 1997; 17:439-444.
20. Wang YX, Martin-McNulty B, Freay AD, Sukovich DA, Halks-Miller M, Li WW, Vergona R, Sullivan ME, Morser J, Dole WP, Deng GG. Angiotensin II increases urokinase-type plasminogen activator expression and induces aneurysm in the abdominal aorta of apolipoprotein E-deficient mice. Am J Pathol. 2001;159:1455-1464.
21. Deng GG, Martin-McNulty B, Sukovich DA, Freay A, Halks-Miller M, Thinnes T, Loskutoff DJ, Carmeliet P, Dole WP, Wang YX. Urokinase-type plasminogen activator plays a critical role in angiotensin II-induced abdominal aortic aneurysm. Circ Res.2003;92:510-517.
22. Sasaguri T, Arima N, Tanimoto A, Shimajiri S, Hamada T, Sasaguri Y:A role for interleukin 4 in production of matrix metalloproteinase 1 by human aortic smooth muscle cells. Atherosclerosis 1998,138:247-253
23. Chizzolini C, Rezzonico R, De Luca C, Burger D, Dayer JM:Th2 cell membrane factors in association with IL-4 enhance matrix metalloproteinase-1 (MMP-1) while decreasing MMP-9 production by granulocyte-macrophage colony-stimulating factor-differentiated human monocytes. J Immunol 2000,164:5952-5960
24. Borghaei RC, Rawlings JR PL, Mochan E:Interleukin-4 suppression of interleukin-1-induced transcription of collagenase (MMP-1) andstromelysin 1 (MMP-3) in human synovial fibroblasts. Arthritis Rheum 1998,41:1398-1406
25. Nemoto O, Yamada H, Kikuchi T, Shinmei M, Obata K, Sato H, Seiki M, Shimmei M:Suppression of matrix metalloproteinase-3 synthesis by interleukin-4 in human articular chondrocytes. J Rheumatol 1997,24:1774-1779
26. Frostegard J, Ulfgren AK, Nyberg P, Hedin U, Swedenborg J, Andersson U, Hansson GK:Cytokine expression in advanced human atherosclerotic plaques: dominance of pro-inflammatory (Th1) and macrophage-stimulating cytokines. Atherosclerosis 1999,145:33-43
27. Soraya Taleb, Olivier Herbin, Hafid Ait-Oufella, Wim Verreth, Pierre Gourdy, Veronique Barateau, Regine Merval, Bruno Esposito, Karine Clement, Paul Holvoet, Alain Tedgui, and Ziad Mallat. Defective Leptin/Leptin Receptor Signaling Improves Regulatory T Cell Immune Response and Protects Mice From Atherosclerosis. Arterioscler Thromb Vasc Biol, Dec 2007; 27:2691-2698.
28. Peter F. Bodary, Shufang Gu, Yuechun Shen, Alyssa H. Hasty, Joshua M. Buckler, and Daniel T. Eitzman. Recombinant Leptin Promotes Atherosclerosis and Thrombosis in Apolipoprotein E-Deficient Mice. Arterioscler Thromb Vasc Biol, Aug 2005; 25:1634.