贝那普利对糖尿病大鼠肾组织环氧合酶2及环氧合酶源性前列腺素表达的干预

前列腺素代谢异常与肾小球高滤过密切相关,环氧合酶(COX)是前列腺素合成的关键限速酶,有同工酶COX-1和COX-2.糖尿病肾病(DN)时肾脏COX-2的表达增多,而COX-1无明显变化[1].血管紧张素Ⅱ(AngⅡ)增多及肾脏氧化应激状态均可诱导COX-2大量产生,而应用抗氧化剂可以抑制COX-2表达[2,3].探讨AngⅡ、氧化应激和COX-2的关系以及ACEI类药物是否能通过抑制AngⅡ及氧化应激从而进一步抑制肾组织COX-2表达.可能为临床治疗DN提供新的理论依据.     

NADPH氧化酶介导血管紧张素Ⅱ诱导的腹膜间皮细胞转分化及细胞外基质积聚

目的 探讨NADPH氧化酶在血管紧张素(Ang)Ⅱ诱导的腹膜间皮细胞转分化以及细胞外基质积聚中的作用。 方法 体外培养SD大鼠原代腹膜间皮细胞，静止24 h后，随机分为以下4组：正常对照组，AngⅡ（10^-7 mol/L）组，AngⅡ+ Los(洛沙坦,10 μmol/L)组及AngⅡ+DPI（NADPH氧化酶活性抑制剂，10 μmol/L）组。应用荧光染料（DCF）及激光共聚焦显微镜检测细胞内活性氧（ROS）的产生。RT-PCR检测NADPH氧化酶亚单位p47phox以及纤溶酶原激活物抑制剂（PAI）1、α平滑肌肌动蛋白（SMA）、E钙黏蛋白（cadherin） mRNA的表达。Western印迹检测p47phox、α-SMA的蛋白表达。 结果 （1）外源性AngⅡ可显著增加大鼠腹膜间皮细胞ROS的产生，刺激15 min后，ROS 的表达较对照组上升了(3.64±0.53)倍。DPI和洛沙坦可显著抑制AngⅡ刺激后ROS的产生（P < 0.05）。（2）AngⅡ刺激腹膜间皮细胞后， NADPH氧化酶亚单位p47phox mRNA和蛋白的表达均呈上升趋势。洛沙坦和DPI可阻断由AngⅡ诱导的p47phox表达上调（P < 0.05）。（3） AngⅡ诱导α-SMA表达的上调以及 E-cadherin mRNA的下调, 洛沙坦和DPI可部分逆转AngⅡ的这种作用。（4）AngⅡ刺激8 h后可明显上调PAI-1的mRNA表达，为正常对照组的（3.06±0.77）倍。 洛沙坦和DPI可明显阻断PAI-1的表达上调（P < 0.05）。 结论 NADPH氧化酶依赖产生的ROS介导了AngⅡ诱导的腹膜间皮细胞转分化及细胞外基质积聚。阻断AngⅡ的作用及抑制NADPH氧化酶的表达和活性可作为防治腹膜纤维化的潜在治疗靶点。     

血管紧张素Ⅱ灌注诱导大鼠肾脏血管紧张素转换酶2表达改变

目的 研究血管紧张素Ⅱ(AngⅡ)灌注后大鼠肾脏血管紧张素转换酶2(ACE2)表达的改变及其意义。 方法 雄性SD大鼠随机分为AngⅡ灌注组(400 ng&#8226;kg-1&#8226;min-1)、生理盐水灌注组和健康对照组，每组6只。测定28 天内大鼠血压及尿蛋白量变化。于第28天处死动物取肾，观察组织学改变，并用免疫组化、RT-PCR及Western印迹法检测ACE2表达及分布变化；凝胶电泳迁移率分析法(EMSA)检测核因子(NF)-κB的DNA结合活性变化。 结果 (1) AngⅡ灌注组大鼠血压升高，并出现明显蛋白尿。AngⅡ灌注后第28天，部分肾小球出现轻中度系膜增生，少数有节段性硬化；部分肾小管上皮细胞变性、坏死或萎缩，间质灶性炎性细胞浸润。(2) 健康大鼠肾脏ACE2主要分布于肾小管，以近端肾小管刷状缘分布最多。AngⅡ灌注后第28天，肾皮质ACE2 mRNA及蛋白表达均明显下降(P均< 0.05)，NF-κB结合活性显著增加(P < 0.05)。相关分析表明，ACE2表达与NF-κB结合活性之间呈负相关(r = -0.64，P < 0.01)。 结论 AngⅡ灌注可致大鼠肾脏ACE2表达明显下降，后者与肾损害的程度密切相关。肾脏ACE2表达下降可能是AngⅡ引起肾损害的重要机制     

血管紧张素Ⅱ诱导足细胞c-Abl的表达改变

目的 探讨血管紧张素Ⅱ( AngⅡ)诱导下大鼠肾脏及条件永生性小鼠足细胞c-Abl的表达变化.方法 采用AngⅡ泵(400ng·kg-1·min-1)植入SD大鼠皮下的方法建立AngⅡ输注模型,24只大鼠成模后被随机分为AngⅡ输注2周组、AngⅡ输注4周组、AngⅡ输注+替米沙坦(ARB,3 mg·kg-1·min-1)干预2周组及4周组,同时设生理盐水输注组和健康对照组,每组6只.分别于成模后2周末、4周末处死大鼠取肾.电镜下观察肾脏足细胞超微结构的改变;免疫荧光法检测肾脏c-Abl表达;实时PCR及Western印迹检测c-Abl mRNA及蛋白水平的改变.对于体外培养条件永生性小鼠足细胞,免疫荧光法检测足细胞肾脏c-Abl的表达,实时PCR及Western印迹法检测足细胞在AngⅡ不同作用浓度(10-9 mol/L～10-6 mol/L)及不同作用时间点(0h、3h、6h、12 h、24 h)c-Abl mRNA和蛋白水平的变化.结果 (1)免疫荧光检测结果显示肾脏足细胞有c-Abl表达;实时PCR及Western印迹结果显示AngⅡ输注2周组和4周组大鼠肾脏c-Abl表达增加(P＜0.05),ARB干预组大鼠肾脏c-Abl表达较同期AngⅡ输注组均有降低(P＜0.05).(2)体外培养的条件永生性小鼠足细胞胞质及胞核有c-Abl表达.AngⅡ可诱导培养的小鼠足细胞c-Abl mRNA及蛋白表达增加(P＜0.05),且呈时间和剂量依赖性.结论 c-Abl在肾足细胞及体外培养足细胞均有表达,AngⅡ可诱导c-Abl表达上调.c-Abl可能参与了AngⅡ诱导的足细胞损伤.     

血管紧张素Ⅱ通过ROS-EGFR-JNK-AP-1信号通路诱导肾小球系膜细胞增殖

目的 探讨血管紧张素Ⅱ（AngⅡ）是否通过活性氧-表皮生长因子受体-c-Jun氨基末端激酶-活化蛋白1（ROS-EGFR-JNK-AP-1）信号通路诱导肾小球系膜细胞增殖。 方法 体外培养人肾小球系膜细胞，用AngⅡ（100 nmol/L）、葡萄糖氧化酶（GO）（1 U/L）、血管紧张素受体拮抗剂洛沙坦（10 μmol/L）、乙酰半胱氨酸（NAC，10 μmol/L）、NADPH氧化酶抑制剂apocynin（10 μmol/L）、硫酸二亚苯基碘（DPI，10 μmol/L）、EGFR阻断剂AG1478（10 μmol/L）处理细胞。以未处理细胞为阴性对照。应用3H-胸腺嘧啶（3H-TdR）掺入法和细胞计数测定系膜细胞增殖；荧光探针2,7-二氯二氢荧光素乙酰乙酸（DCFDA）检测细胞内ROS 的产生；Western 印迹检测EGFR 和JNK 活化。 结果 AngⅡ（100 nmol/L） 可显著促进肾小球系膜细胞ROS 产生，AngⅡ 刺激60 min，系膜细胞产生ROS是对照组2.26 倍。AngⅡ可呈时间和剂量依赖性诱导系膜细胞EGFR 磷酸化，AngⅡ刺激5 min，EGFR 活化明显增加，至30 min 达到高峰，随AngⅡ刺激剂量增加，EGFR活化亦显著增强，AngⅡ（100 nmol/L） 刺激30 min，EGFR 磷酸化是对照组的3.96 倍。洛沙坦、NAC以及apocynin和 DPI 显著抑制AngⅡ诱导的EGFR 磷酸化，同时AG1478 几乎完全阻断AngⅡ诱导的系膜细胞增殖；洛沙坦、NAC、apocynin、DPI 和AG1478 显著抑制AngⅡ诱导的JNK 活化。 结论 ROS-EGFR-JNK-AP-1信号通路参与AngⅡ诱导的肾小球系膜细胞增殖。NADPH 氧化酶抑制剂和EGFR 受体拮抗剂能显著抑制AngⅡ诱导的系膜细胞增殖，可能是一种新的治疗途径。     

血管紧张素Ⅱ对足细胞nephrin 磷酸化水平的影响

目的 通过建立血管紧张素Ⅱ( AngⅡ)输注大鼠模型及体外足细胞培养,观察AngⅡ刺激对足细胞nephrin磷酸化水平的影响.方法 30只SPF级Wistar大鼠皮下埋置渗透性微泵,随机分为AngⅡ组(AngⅡ400 ng· kg-1·min-1,n=12)、替米沙坦(Tel)组(AngⅡ+Tel 3 mg· kg-1·d-1,n=12)和正常对照组(生理盐水代替AngⅡ,n=6),于实验0、7、14、21、28 d测量大鼠尾动脉收缩压,收集24 h尿液,检测尿白蛋白.分别在14、28 d处死动物,收集血液标本,检测血肌酐;收集肾脏标本,透射电镜观察肾小球足细胞形态,放免法检测血浆及肾组织AngⅡ水平;Western印迹检测nephrin表达及其磷酸化水平.体外培养小鼠永生化足细胞,AngⅡ (10-6 mol/L)刺激不同时间,并行洛沙坦(10-5 mol/L)干预,Western印迹法检测足细胞nephrin 表达及其磷酸化水平.异硫氰酸荧光素( FITC)-鬼笔毒环肽(phalloidin)染色标记足细胞F-actin,用外周F-actin环评分系统(CFS)半定量分析足细胞骨架运动.结果 (1)与正常对照组同时间点相比,AngⅡ组大鼠自7d起尾动脉收缩压开始升高(P＜0.05),随着作用时间的延长,血压持续升高.AngⅡ输注7d大鼠开始出现蛋白尿,并持续增加.各组大鼠血肌酐、尿肌酐及内生肌酐清除率无明显变化.AngⅡ输注大鼠血浆及肾组织AngⅡ浓度明显增高(均P＜0.05).(2)与正常对照组相比,AngⅡ输注组大鼠肾小球足细胞nephrin表达明显减少,其磷酸化水平亦显著下降(P＜0.05).(3)与正常对照组相比,AngⅡ刺激早期(3～6 h),足细胞nephrin磷酸化表达即明显下调(P＜0.05),刺激12～24 h维持低水平表达.(4)AngⅡ刺激后,F-actin排列紊乱,逐渐向细胞外周分布形成F-actin环,CFS评分显著高于正常对照组(P＜0.05).结论 正常状态下足细胞nephrin维持一定水平磷酸化状态,AngⅡ可以诱导足细胞nephrin磷酸化水平下调.nephrin磷酸化水平改变可能是AngⅡ诱导足细胞骨架重排、足突融合的重要分子机制.     

愈肾颗粒对血管紧张素Ⅱ刺激大鼠肾系膜细胞TGF-β表达的影响

目的:观察血管紧张素Ⅱ(Ang Ⅱ)对大鼠肾小球系膜细胞转化生长因子-β1(TGF-β1) mRNA表达及中药愈肾颗粒的影响.方法:采用体外培养法培养大鼠肾小球系膜细胞,应用血清药理学方法制取中药复方愈肾颗粒血清、中药对照药物肾炎舒及西药对照药物泼尼松药物血清,利用Ang Ⅱ作为刺激因子,应用反转录多聚酶链反应技术(RT-PCR)观察各组间TGF-β1 mRNA表达的情况.结果:(1)Ang Ⅱ能明显促进系膜细胞的TGF-β1 的表达;(2)愈肾颗粒低剂量、高剂量组的TGF-β1 mRNA相对光密度比值明显低于病理组(P<0.01),愈肾颗粒对TGF-β1的mRNA表达有下调作用;(3)对照药物肾炎舒可抑制TGF-β1的表达,但是对照药泼尼松对TGF-β1的表达,与病理组比较无统计学差异.结论:Ang Ⅱ能促进系膜细胞TGF-β1的mRNA表达.愈肾颗粒能明显抑制肾小球系膜细胞TGF-β1基因表达.     

12-脂氧化酶对系膜细胞血管紧张素Ⅱ1型受体表达的影响

目的 探讨12-脂氧化酶(12-LO)对系膜细胞血管紧张素(Ang)Ⅱ1型受体(AT1R)表达的影响。方法 用AngⅡ刺激正常和12-LO基因敲除小鼠肾系膜细胞后，观察p38 MAPK活性和细胞外基质(ECM)蛋白的变化。用12-LO的作用产物12(S)-HETE刺激的系膜细胞、转染12-LO基因的系膜细胞和采用显微切割法从正常和12-LO基因敲除小鼠肾脏提取的肾小球来观察AT1R的表达。采用RT-PCR和Western印迹分别检测目标基因mRNA和蛋白的表达。结果 AngⅡ的刺激可诱导正常系膜细胞p38 MAPK活性和ECM蛋白表达增高。然而，AngⅡ的刺激不能诱导12-LO基因敲除小鼠系膜细胞p38 MAPK活性和ECM蛋白表达升高。剂量依赖性和时间依赖性实验结果表明12(S)-HETE刺激可引起系膜细胞AT1蛋白水平增高，且AT1R mRNA水平升高有统计学意义(P < 0.01)。敲除肾小球内12-LO基因可有效地降低AT1R mRNA的表达(P < 0.01)，转染12-LO基因至系膜细胞使AT1R蛋白和mRNA的表达明显增多(P < 0.01)。结论 12-LO可上调系膜细胞AT1R的表达。     

伊贝沙坦对大鼠单侧输尿管结扎致肾损伤的保护作用

血管紧张素Ⅱ受体拮抗剂(ARB)通过阻断血管紧张素Ⅱ(AngⅡ)与其受体1(AT1)特异性结合，发挥其降压、减少蛋白尿、保护肾功能的作用。许多献报道，ARB治能疗肾小球疾病＾[1]，对糖尿病大鼠的肾脏具有保护作用＾[2]，但对肾小管间质疾病的影响很少见有报道。本拟通过伊贝沙坦对大鼠单侧输尿管结扎(UUO)致肾间质损伤的作用，了解其对肾脏的保护作用及机制。     

芬戈莫德对血管紧张素Ⅱ介导的慢性肾脏病的影响

目的通过建立血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)灌注的大鼠肾损害模型,使用药物芬戈莫德(Fingolimod,FTY720)进行干预,来评估该药物对大鼠肾功能的影响,并探讨FTY720对AngⅡ灌注大鼠肾脏炎症改变的作用及可能涉及的机制,从而为肾脏疾病的治疗提供新的策略。方法 36只SPF级雄性SD大鼠随机分为3组:(1)模型组12只,即AngⅡ灌注组,皮下植入含AngⅡ的渗透性微量泵,持续灌注28 d(AngⅡ:400 ng·kg~(-1)·min~(-1));(2)干预组12只,即AngⅡ+FTY720干预组,在模型组的基础上,FTY720以0.5 g·kg~(-1)·d~(-1)灌胃28 d;(3)对照组12只,以等量的生理盐水灌胃28 d。每周称体质量,留24 h尿液,并于第28天处死各组大鼠,经心脏采血,并留取肾脏标本。放射免疫法检测各组大鼠血液中AngⅡ及肾匀浆组织中AngⅡ的浓度。对尿液及血液样本进行尿蛋白与血生化分析。光镜下观察各组大鼠肾组织病理学改变并行Jablonski评分。免疫印迹法检测炎症因子TNF-α、IL-6在肾脏中的表达。结果 (1)AngⅡ可导致模型组大鼠血肌酐、尿素氮水平升高,尿蛋白排泄明显增加,炎症因子TNF-α、IL-6在肾脏组织中的表达水平升高;(2)干预组的肾脏病理损伤较模型组减轻,且Jablonski评分明显低于模型组,血肌酐、尿素氮水平明显降低,尿蛋白明显减少,肾脏组织中炎症因子TNF-α、IL-6水平明显降低,差异具有统计学意义(P0.05)。结论 FTY720可减轻AngⅡ诱导的大鼠肾脏的病理损伤,可能是通过减少炎症因子的表达发挥了减轻尿蛋白,改善肾功能的作用。     

Mycophenolate mofetil ameliorates nephropathy in the obese Zucker rat

BACKGROUND: The obese Zucker rat has metabolic condition resembling type II diabetes, including hyperlipidemia, obesity, insulin resistance, and hyperglycemia. With advancing age, the obese Zucker rat develops glomerulosclerosis, proteinuria, and renal failure. Since immune cells play a central role in the development of chronic renal injury, we evaluated the potential benefit of mycophenolate mofetil (MMF), alone and in combination with angiotensin receptor type 1 blockade (ARB) in the obese Zucker rat. METHODS: Thirteen-week-old male obese Zucker rats (fa/fa) were randomly assigned to four experimental groups (five rats each) that received the following treatments for 3 months: (1) losartan (100 mg/L in the drinking water), (2) MMF (20 mg/kg/day), (3) MMF and losartan, and (4) placebo. Lean Zucker rats (N = 5) were included as normal controls. Renal function, biochemical parameters, renal histology, and immunohistology were evaluated. RESULTS: The placebo-treated obese Zucker rats exhibited proteinuria and significant glomerular and tubulointerstitial injury in association with renal immune cell infiltration. Proteinuria, histologic damage, and renal immune cell infiltration were all reduced by MMF treatment alone or in combination with ARB. The improvement of proteinuria and structural damage was more pronounced in the group that received the combination of MMF and losartan. CONCLUSION: MMF treatment alone, and especially in combination with ARB, improves nephropathy in the obese Zucker rat.     

Renal cyclooxygenase-2 in obese Zucker (fatty) rats

BACKGROUND: Cyclooxygenase (COX) isoforms, COX-1 and COX-2, are involved in production of prostanoids in the kidney. Increases in renal COX-2 expression have been implicated in the pathophysiology of progressive renal injury, including type 1 diabetes. Thromboxane A(2) (TxA(2)) has been suggested as the key mediator of these effects resulting in up-regulation of prosclerotic cytokines and extracellular matrix proteins. Unlike type 1 diabetes, renal COX has not been studied in models of type 2 diabetes. METHODS: Renal cortical COX protein expression, and urinary excretion of stable metabolites of prostaglandin E(2) (PGE(2)) and TxA(2), in association with metabolic parameters, were determined in 4-and 12-week-old Zucker fatty rats (fa/fa rat) (ZDF4 and ZDF12), a model of type 2 diabetes, and in age-matched littermates with no metabolic defect (Zucker lean) (ZL4 and ZL12). RESULTS: Western blotting revealed increased COX-2 expression in ZDF4 as compared to ZL4 (245 +/- 130%) (P < 0.05). This increase in COX-2 was even more apparent in 12-week-old ZDF rats (650 +/- 120%) (P < 0.01). All groups of rats demonstrated COX-2-positive cells in typical cortical localizations [macula densa, thick ascending loop of Henle (TALH)]. In contrast to COX-2, COX-1 expression was 30% lower in ZDF12. These changes in COX expression were associated with enhanced urinary excretion of prostanoids, in parallel with the development of metabolic abnormalities. Moreover, increases in prostanoid excretion in ZDF12 were in part reduced by wortmannin (100 mug/kg), used as inhibitor of insulin signaling. CONCLUSION: Renal cortical COX-2 protein expression and function were increased in ZDF rats, as compared to controls, whereas COX-1 exhibited opposite regulation. The changes in COX-2 paralleled metabolic abnormalities, and were at least in part a four consequence of hyperinsulinemia. These abnormalities may play a role in renal pathophysiology in this model of type 2 diabetes.     

The metabolic and microcirculatory impact of orthotopic liver transplantation on the obese Zucker rat

BACKGROUND: The purpose of this study was to investigate the metabolic alterations in the recipient and microcirculatory changes to the graft in the first 3 months after orthotopic liver transplantation (OLT) of nonsteatotic liver grafts from lean rats into obese Zucker rats. METHODS: Body weight and plasma lipids were measured for 3 months post-OLT. Graft perfusion (hepatic microcirculatory perfusion [HMP]) and vascular structure were measured in vivo at 3 months. Liver biopsy specimens were obtained throughout for morphologic analysis. Sham-operation obese and lean Zucker rats acted as controls. RESULTS: Plasma cholesterol levels were elevated from 2 months after OLT, whereas plasma triglyceride levels were reduced (P<0.05). Plasma high-density lipoprotein cholesterol concentrations increased from the first month after OLT (P<0.05). HMP in OLT animals (137+/-3 perfusion units [PU]) (P<0.05) was intermediate between lean (221+/-11 PU) and obese controls (113+/-5 PU). Hepatic cord width in the OLT group was similar to that in lean controls. Mean liver-to-body weight ratios in OLT animals (4.12%+/-0.39%) were significantly higher than in lean controls (3.25%+/-0.1%). The number of viable hepatocytes per high-power field in the OLT animals was lower than in the lean animals but higher than in obese controls (P<0.05). The transplanted livers showed moderate to marked microvesicular fatty change (MIFC) and glycogen deposition at 3 months after OLT. CONCLUSIONS: Transplantation of a nonsteatotic liver into an obese Zucker rat initially has a positive effect on lipid metabolism. However, 3 months after OLT, the donor liver became steatotic with MIFC changes and reduced perfusion. The authors' results emphasize the importance of the recipient's metabolic status in the maintenance of liver graft function after OLT.     

Expression of a novel PDGF isoform, PDGF-C, in normal and diseased rat kidney

Platelet-derived growth factor-C (PDGF-C) is a new member of the PDGF family. Its expression in normal and diseased kidney is unknown. Rabbit antisera were generated against human full-length, core domain, and mouse PDGF-C, and their specificity was confirmed by Western blot analyses. Renal PDGF-C expression was analyzed by immunohistochemistry in normal rats (n = 8), mesangioproliferative anti-Thy 1.1 nephritis (n = 4 each at days 1, 4, 6, and 85), passive Heymann nephritis (PHN, n = 4), puromycin nephrosis (PAN, n = 2), Milan normotensive rats (MN, n = 2), and obese Zucker rats (n = 3). PDGF-C expression was also studied in anti-Thy 1.1 rats treated with PDGF-B aptamer antagonists (n = 5) or irrelevant control aptamers (n = 5). PDGF-C was constitutively expressed in arterial smooth muscle cells and collecting duct epithelial cells. Mesangial PDGF-C was markedly upregulated in anti-Thy 1.1 nephritis in parallel with the peak mesangial cell proliferation. Furthermore, PDGF-CC acted as a potent growth factor for mesangial cells in vitro. Inhibition of PDGF-B via specific aptamers reduced the injury in anti-Thy 1.1 nephritis but did not affect the glomerular PDGF-C overexpression or the mitogenicity of PDGF-CC in vitro. In PHN, PAN, and obese Zucker rats, glomeruli remained negative for PDGF-C despite severe glomerular injury. PDGF-C localized to podocytes at sites of focal and segmental sclerosis in MN. Interstitial PDGF-C expression was increased at sites of fibrosing injury in obese Zucker rats. The use of the different antisera resulted in virtually identical findings. It is concluded that PDGF-C is a novel mesangial cell mitogen that is constitutively expressed in the kidney and specifically upregulated in mesangial, visceral epithelial, and interstitial cells after predominant injury to these cells. PDGF-C may therefore be involved in the pathogenesis of renal scarring.     

Effect of angiotensinⅡ type 1 receptor blocker on the 12-lipoxygenase activity and P-cadherin expression in type 2 diabetic rat glomeruli

Objective To investigate the effect of angiotensinⅡ(AngⅡ) type 1 receptor blocker (ARB) on 12-lipoxygenase (12-LO) activity and P-cadherin expression in type 2 diabetic rat glomeruli. Methods Podocytes were stimulated by 10-7 mol/L AngⅡ for 24 hours. 12(S)-HETE (1 mg•kg-1•d-1) and AngⅡ (400 ng•kg-1•min-1) were infused to rats by osmotic mini-pump for 1 week and 2 weeks respectively. Rats fed with high fat diet received low dose streptozotocin (STZ) to make type 2 diabetes and divided into 2 groups: low dose STZ (DN group), low dose STZ＋ARB treatment (Losartan group). Rats fed with regular chow were used as control group. All the rats were sacrificed after 6 weeks. Urine, blood, kidney cortical tissue and isolated glomeruli by sieving method were collected at the end of study respectively. ELISA, RT-PCR and Western blotting for related target were performed respectively. Results AngⅡincreased 12(S)-HETE levels in podocytes and glomeruli (all P＜0.01). AngⅡ levels in the glomeruli were significantly increased by 12(S)-HETE stimulation (P＜0.01). Blood glucose, kidney/body weight and 24 hour urinary protein were increased in DN group compared with that in control group (all P＜0.01). However, urine protein, Kidney/body weight were decreased in Losartan group compared with DN group (all P＜0.05). Increment of 12(S)-HETE content and decrement of P-cadherin expression were observed in DN glomeruli compared with that in control group(all P＜0.01). These abnormalities were prevented by administration of the losartan (all P＜0.05). Conclusions AngⅡ can down-regulate glomerular P-cadherin expression via activation of 12-LO.ARB can ameliorate the progression of DN via up-regulation of glomerular P-cadherin through inhibition of 12-LO activation in type 2 DN rats.     

Effect of hereditary obesity on renal expressions of NO synthase, caveolin-1, AKt, guanylate cyclase, and calmodulin

BACKGROUND: Obesity has emerged as a major cause of diabetes, cardiovascular disease, and renal insufficiency worldwide. Obese Zucker rats exhibit hyperphagia, obesity, insulin resistance, hyperlipidemia, and glomerulosclerosis and are frequently used as a model to study hereditary form of metabolic syndrome. Nitric oxide plays a major role in preservation of renal function and structure. The present study was designed to test the hypothesis that renal disease in this model may be associated with down-regulation of endothelial (eNOS) and neuromal NO synthases (nNOS) in the kidney. The study further sought to explore expressions of caveolin-1, phospho AKt, and calmodulin, which regulate activities of constituitive NOS isoforms, as well as soluble guanylate cyclase (sGC), which is involved in NO signaling. METHODS: Twenty-two-week-old male obese and lean Zucker rats were studied. Body weight, serum lipids, urine albumin excretion, and renal tissue abundance of the above proteins were determined. RESULTS: Serum glucose and arterial pressure were unchanged, whereas urinary NO metabolite (NO(chi)) excretion and renal tissue nitrotyrosine abundance were markedly reduced (denoting depressed NO production) in the obese versus lean Zucker rats. This was accompanied by significant glomerulosclerosis, tubulointerstitial damage, renal immune cell infiltration, marked down-regulations of renal tissue eNOS and nNOS, mild reduction of caveolin-1, and unchanged calmodulin, phospho-AKt, and sGC. CONCLUSION: Hereditary obesity can result in down-regulations of kidney eNOS and nNOS, marked reduction of NO production, and glomerulosclerosis prior to the onset of frank diabetes and hypertension.     

厄贝沙坦对糖尿病大鼠肾脏骨桥蛋白表达及肾纤维化的影响

目的 探讨不同剂量厄贝沙坦对糖尿病大鼠肾组织骨桥蛋白( OPN)表达及小管间质纤维化的影响.方法 将63只8周龄雄性Wistar大鼠按随机数字表法分为正常对照组(Ctrl组,n=7)、糖尿病组(DM组,n=14)、30 mg/kg肼屈嗪干预组(DM+ Hyd组,n=12)、25mg·kg-1· d-1厄贝沙坦干预组(DM+Irb25组,n=10)、50 mg·kg-1·d-1厄贝沙坦干预组(DM+Irb50组,n=9)和200 mg · kg-1·d-1厄贝沙坦干预组(DM+Irb200组,n=11).糖尿病模型造模成功后4周,灌胃法给予相应剂量的肼屈嗪和厄贝沙坦.第12周末,观察各组大鼠24h尿白蛋白排泄率(UAER)、内生肌酐清除率(Ccr);PAS及Masson染色观察各组大鼠肾脏病理形态和胶原纤维沉积;ELISA法测定各组大鼠肾组织AngⅡ含量;实时定量PCR检测大鼠肾组织转化生长因子(TGF)β1、OPN mRNA的表达.结果 药物干预各组大鼠UAER、Ccr较DM组显著减少(均P＜ 0.05).DM组大鼠肾小球肥大,系膜基质增生,肾小球及小管间质有大量胶原纤维沉积;药物干预后,肾小球及肾小管上述病变减轻.DM组大鼠肾组织AngⅡ水平及TGF-β1、OPN mRNA表达均显著升高(均P＜0.05),给予肼屈嗪及厄贝沙坦干预后,AngⅡ、TGF-β1、OPN mRNA表达显著降低(均P＜0.05),且随着厄贝沙坦剂量的递增而递减.相关分析结果显示,DM组大鼠肾组织AngⅡ水平与OPN mRNA的表达呈正相关(r=0.74,P＜0.01).结论 厄贝沙坦通过减少肾脏局部AngⅡ水平,进而减少肾脏TGF-β1、OPN mRNA的表达,最终减轻小管间质纤维化,发挥肾脏保护作用,且这种保护作用具有剂量依赖性.     

梗阻性肾病大鼠肾组织periostin的表达及意义

目的 研究梗阻性肾病大鼠肾组织periostin表达的变化及其与肾间质纤维化的相关性.方法 18只SD雄性大鼠按随机数字法分成3组:假手术组、模型组和贝那普利组,每组6只.用单侧输尿管结扎法建立梗阻性肾病大鼠模型.RT-PCR检测各组大鼠肾组织periostin和转化生长因子(TGF) β1的mRNA表达;ELISA检测各组大鼠肾组织periostin、血管紧张素Ⅱ( AngⅡ)、TGF- β1的蛋白水平;HE及Masson染色观察肾间质变化;免疫组化检测肾组织Ⅰ型胶原蛋白.结果 与假手术组比较,模型组大鼠肾组织periostin、TGF-β1、AngⅡ蛋白表达显著升高(均P＜0.05).与模型组比较,贝那普利组上述蛋白表达显著降低,差异有统计学意义(均P＜0.05).与假手术组比较,模型组大鼠肾组织periostin和TGF- β1的mRNA表达显著上调,差异有统计学意义(均P＜0.05),而贝那普利组periostin和TGF- β1的mRNA表达则显著下调(均P＜0.05).肾组织periostin蛋白表达与AngⅡ、TGF-β1和Ⅰ型胶原的蛋白表达以及肾间质纤维化积分均呈正相关(r值分别为0.652、0.781、0.776和0.825,均P＜0.05).结论 梗阻性肾病大鼠肾组织periostin呈高表达,其可能参与了梗阻性肾病肾间质纤维化进程.     

Impact of dietary fatty acid supplementation on renal injury in obese Zucker rats

We previously reported that renal injury in hyperlipidemic, obese Zucker rats was associated with a relative deficiency of tissue polyunsaturated fatty acids (PUFA). In the present study 10-week-old obese Zucker rats were pair fed regular chow or chow containing either 20% sunflower oil rich in n-6 PUFA, fish oil rich in n-3 PUFA, coconut oil medium-chain saturated fatty acid, or beef tallow long-chain saturated fatty acid. At 34 weeks of age there were comparable reductions in albuminuria, mesangial matrix expansion, and glomerulosclerosis in the fish oil and sunflower oil groups. While both fish oil and sunflower oil reduced serum triglycerides, and improved the composition of triglyceride-enriched lipoproteins, only fish oil decreased serum cholesterol. The effect of the dietary fatty acid supplementation on fatty acid profiles were similar in isolated glomeruli and cortical tissue. In general, the amelioration in injury in the fish oil and sunflower oil fed rats was most closely linked to glomerular levels of PUFA, either n-6 or n-3. These data suggest that hyperlipidemia and abnormalities in tissue FA are closely linked, and that dietary supplementation with PUFA may ameliorate chronic, progressive renal injury.