慢性肾衰大鼠盐敏感性的中枢RAS调控机制研究
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摘要
研究背景
高血压是肾实质疾病最常见的继发疾病。容量负荷/钠潴留和肾素-血管紧张素系统(RAS)的不适当的激活是肾病患者发生高血压的主要决定因素。最近的研究显示,中枢交感神经系统(sympathetic nervous system, SNS)活动的增加参与了慢性肾功能衰竭(CRF)高血压的发生。
1CRF时高血压的发病机理
压力性利尿是体液容量和动脉压长期控制的反馈调节系统的主要部分。容量扩张引起血压升高的准确机制尚不十分清楚,高血压发生机制的自动调节理论(autoregulation theory)可以解释部分CRF患者容量扩张与血压升高的关系：容量扩张→细胞外液容量和血容量的增加→静脉回心血量增加→心输出量增加→外周组织过度灌注→自动调节血管收缩→总外周阻力增加→血压升高。
血管紧张素Ⅱ (Angiotensin Ⅱ, Ang Ⅱ)通过多种机制升高血压,包括血管收缩、钠潴留、血管重塑和增加交感神经活动；AngⅡ的1型受体(Angiotensin Ⅱ AT1-receptor, AT1R)拮抗剂特异性的阻断Ang Ⅱ与AT1R的结合,导致血管舒张、钠排泄和血压降低。RAS系统的所有成分在脑组织中均有表达；研究表明,脑内局部的RAS系统功能的异常与高血压的发生密切相关。
肾脏与交感神经的中枢核团具有直接和间接的联系,从而调节动脉压。直接的途径是通过肾神经,而间接的途径是体液和体液相关的RAAS的激活,并作用于脑内缺乏血脑屏障的RAAS作用部位。这些区域在调节血压和体液平衡方面具有重要作用。前脑室旁器官(circumventricular organs, CVOs)神经核团通过神经突出到达下丘脑核团；下丘脑室旁核(Paraventricular hypothalamic necleus, PVN)在整合肾脏传入交感神经反应方面起着重要作用,其不但可以直接通过PVN的前交感神经元调节肾脏传入交感神经活动,还具有神经激素的影响,可增加精氨酸加压素和催产素向全身循环的释放。
.2.高血压的中枢机制
越来越多的研究认为中枢神经系统控制血压调节的功能紊乱导致交感神经活动的持续增加,进而引起慢性高血压。研究从脑内传出的交感神经调节因素是认识神经源性高血压发生机制的主要靶点。
脑内控制血压调节的中枢绝大部分位于前脑的下丘脑和脑干的髓质。这些核团是高度网络化的,其在心血管控制中的主要作用是通过调节交感神经来调节血压张力。此外,这些中枢心血管通路通过介导神经激素分泌,包括精氨酸加压素刺激血管收缩和调节肾脏压力性利尿机制。下丘脑控制中心通过刺激口渴和盐欲对电解质和血液容量也有重要的调节作用。
高血压动物模型对于研究心血管调节核团对神经源性高血压发生的具体作用至关重要。和这种调节紊乱有关的主要核团包括：穹窿下器(SFO),下丘脑室旁核(PVN)、脑干的延髓头端腹外侧核(RVLM)和孤束核(NTS)。PVN在正常血压和高血压状态下对血压调节都起着枢纽角色。这个核团接受来自SFO和NTS等区域的关于心血管状态的输入信号,整合输入信号后,通过直接到达脊髓的中间外侧细胞柱(IML)和其他的交感神经调节区域包括RVLM的神经突触控制交感神经的输出信号。PVN也调节影响心血管功能的激素的产生,包括糖皮质激素和垂体加压素。
3脑内RAS系统与血压
对脑内ANGⅡ和AT1R免疫活性的分布进行比较显示,绝大多数的脑内ANGⅡ和几乎所有的脑内AT1R位于血脑屏障内神经核团,在这些部位,脑内内源性的ANGⅡ能够接触AT1R,而循环中的ANGⅡ无法到达此部位。但是在前脑存在一条AT1R的分布带,使室周器官(SFO、OVLT)和血脑屏障内的结构(PVN)联系起来。这条通路在解剖学上构成了循环(外周)和脑(中枢)ANGⅡ系统之间的生理学的联系。
ANGⅡ是RAS系统合成的增压激素,ANGⅡ的增压作用通过作用于AT1受体(AT1R)表达；ANGⅡ也可作为神经肽,通过AT1R增加下丘脑和脑干心血管调节中枢神经元的兴奋性,中枢神经系统ANGⅡ/AT1R作用的过度活跃导致神经源性高血压。
4饮食盐与高血压
大量的研究表明,随着年龄的增长,大量的饮食盐摄入是原发性高血压高发病率的唯一的最重要的可控性因素。。肾脏对钠和水的潴留是盐诱导的高血压最主要的发生机制。在血容量正常的高血压患者,其血[Na+]水平往往轻度升高(约升高1-3mmol)。然而,盐敏感的个体高盐饮食并不仅仅升高血浆的[Na+],其脑脊液的[Na+]也同时升高。
脑内感受性室周器官(包括SFO、OVLT和MnPO)能够通过钠/渗透性受体监控血浆和脑脊液中的[Na+]变化,而SFO是对渗透性刺激最重要的感受器。对SFO进行定位损毁,能够消除向脑室内注射高渗盐水(NaCl)引起的增压反应。电刺激研究证实,SFO神经元投射至下丘脑室旁核,并且向PVN传递兴奋性信号,而PVN进一步将信号投射至脊髓中间外侧细胞柱的交感神经元。电损毁PVN部位能够阻断SFO介导的增压反应。有证据表明,AngⅡ是从SFO到PVN的神经递质。
位于前脑的穹窿下器(SFO)缺乏完整的血脑屏障,因此能够感受外周或脑脊液中钠离子浓度和渗透压的微小改变。下丘脑室旁核(PVN)位于第三脑室侧面,是脑内调节体液平衡的最重要的核团；其在解剖学和功能学上与SFO的神经元联系密切。PVN含有大量的合成精氨酸加压素(AVP)的大细胞性神经元,以及少量的小细胞性神经元,其小细胞性神经元投射至脊髓的交感神经节前神经元和脑干的运动前交感神经元。因此,PVN是心血管系统和肾脏对钠离子浓度和渗透压改变产生反应的神经源性和激素性调控的中枢。阻断PVN部位的AT1R能够减弱颈内动脉注射高渗盐水引起的肾脏交感神经的兴奋。
综上所述,鉴于中枢神经系统在血压调控和高血压发生、发展中的重要地位和作用,研究慢性肾功能衰竭状态下中枢神经系统,尤其是脑部RAS系统是否发生异常改变,将进一步加深对慢性肾功能衰竭及其他慢性肾脏病高血压的发生机制的认识。
本研究将对比5/6Nx大鼠和假手术大鼠(sham大鼠)脑内心血管调控中枢的关键神经核团(SFO和PVN) RAS系统的活化水平；并观察5/6Nx和sham大鼠脑内RAS对不同浓度饮食盐的反应情况,以及伴随的外周效应(血压,肾脏损伤程度等)；在上述研究基础上,进行高盐饮食情况下大鼠脑内RAS或交感神经阻断实验,观察5/6Nx脑内RAS是否参与其血压的调节,探索5/6Nx大鼠脑内RAS与其增强的交感神经活动之间的关系,并阐述其脑内RAS与交感神经相互关系的具体机制。
材料和方法
动物模型的制备
雄性Sprague Dawley大鼠(购自南方医科大学实验动物中心)体重150-180g,行5/6肾切除术。于第10周对大鼠进行随机分组。
实验数据和标本采集
1.血压和尿样采集
在分组后给予不同刺激前,采用股动脉插管法测量麻醉状态下大鼠股动脉血压,作为各组的基础血压；在实验结束前,再次测量大鼠的股动脉血压,以刺激14天血压减去刺激前血压的差值作为股动脉血压统计数据。不同刺激的最后三天,代谢笼内单只单笼饲养,连续三天收集24h尿液,用于24h尿钠和尿蛋白定量。
2.大鼠脑标本摘取和采血
刺激结束后,大鼠3%戊巴比妥钠腹腔注射麻醉(35mg/kg)后部分大鼠摘取新鲜脑核团,用于western-blot实验检测SFO/PVN核团RAS及TH的表达,另一部分大鼠制作脑石蜡切片,用于免疫组织化学技术检测SFO/PVN核团RAS的表达和c-fos的表达。采血用于肾功能、电解质、血AngⅡ及NE检测。
第二章5/6Nx大鼠脑核团(SFO/PVN部位)RAS的表达及对饮食盐的反应研究
术后第10周对大鼠进行随机分组,大鼠按各自的饲料共饲养刺激14天。具体分组如下：
刺激14天后,对大鼠血压及血、尿生理生化指标进行检测。采用免疫组化及western blot技术对脑核团SFO/PVN部位RAS成分(AngⅡ和AT1R)表达进行检测。
第三章5/6Nx大鼠脑核团(SFO/PVN部位)RAS的细胞定位及与神经元活化的关系研究
采用第二章5/6Nx大鼠的脑石蜡切片,对脑RAS与神经元标志物或神经胶质细胞标志物进行免疫荧光双染,确定5/6Nx大鼠的脑RAS细胞定位。
采用第二章5/6Nx大鼠的脑石蜡切片,进行c-fos的免疫组化染色,观察神经元活化情况。
第四章5/6Nx大鼠脑核团(SFO/PVN部位)RAS活化与血压及交感神经的关系研究
5/6Nx术后第10周对大鼠进行随机分组,所有大鼠在高盐饲料刺激的同时,按如下分组进行阻断干预14天,具体分组如下(每组n=12只)：
①Losartan0mg IG组：纯水灌胃,体积0.5ml/kg/d
②Losartan1mg IG组：losartan灌胃剂量1mg/kg/d,体积0.5ml/kg/d
③Losartan50mg IG组：losartan灌胃剂量50mg/kg/d,体积0.5ml/kg/d
④Losartan500mg IG组：losartan灌胃剂量500mg/kg/d,体积0.5ml/kg/d
⑤Losartan0mg ICV组：人工脑脊液(aCSF)侧脑室注射,速度0.5gl/h
⑥Losartan1mg ICV组：losartan侧脑室注射,剂量1mg/kg/d,速度0.5μl/h
⑦Clonidine5.76μg ICV组：clonidine侧脑室注射,剂量5.76μg/kg/d,速度0.51μl/h
⑧Renal denervation组：行肾神经切断术,纯水灌胃,体积0.5ml/kg/d
⑨Tempol30mg/kg/d IG组：tempol灌胃剂量30mg/kg/d,体积0.5ml/kg/d
注释：灌胃组药物均已纯水作为溶剂,脑室内注射组药物以aCSF作为溶剂。I.G,灌胃给药；I.C.V,脑室内注射；aCSF,人共脑脊液。
刺激14天后,对大鼠血压及血、尿生理生化指标进行检测。采用免疫组化及western blot技术对脑核团SFO/PVN部位RAS成分(AngⅡ和AT1R)和TH表达进行检测,并用免疫组化技术检测c-fos的表达。
统计学方法：
数据以mean±SE表示,所有统计由统计软件SPSS13.0完成。假手术和肾衰组实验前基础数据比较采用独立样本T检验；刺激14天后,相同饮食盐的假手术和肾衰组之间的比较采用独立样本T检验；假手术或肾衰组在不同饮食盐刺激14天后的组内比较,符合方差齐性检验的数据,采用one-way ANOVA,当P<0.05时,用Post Hoc Tests的LSD法进行两两比较；方差不齐时采用Welch检验；当P<0.05时,两两比较采用Dunnett'T3法。阻断剂干预组之间比较采用one-way ANOVA,方法同前。P<0.05为差异有统计学意。
结果
1.造模术后10周大鼠血压及肾功能
在术后第十周进行不同饮食盐刺激前,5/6Nx大鼠血压已显著高于sham组(P<0.05),5/6Nx组血肌酐和24小时尿蛋白排泄量也显著升高(P<0.05),证明5/6Nx大鼠造模成功。
2.不同浓度饮食盐刺激14天大鼠的生理及血、尿生化指标
在正常盐情况下,5/6Nx大鼠血压水平已显著高于假手术大鼠(P<0.05)。高盐饮食能够显著升高5/6Nx大鼠的血压、血NE水平、血钠和24h尿蛋白(P<0.05),而低盐饮食则能显著降低5/6Nx大鼠的血压(P<0.05)。饮食盐的改变对sham大鼠血压、血钠水平及24h尿蛋白无影响。
Sham和5/6Nx大鼠24h尿量和24h尿钠排泄量随着饮食盐的增加而增加(P<0.05),而血AngⅡ和NE随着饮食盐的增加而受到抑制(P<0.05)
3.不同浓度饮食盐刺激14天大鼠股动脉血压的改变
经过14天不同的饮食盐刺激,假手术大鼠血压无明显改变。与5/6Nx正常盐组比较,高盐饮食显著升高5/6Nx大鼠血压(P<0.05),而低盐饮食则能降低5/6Nx大鼠血压(P<0.05)。
4.免疫组化检测不同浓度饮食盐刺激14天大鼠脑SFO/PVN核团AngⅡ的表达
相同饮食盐时,5/6Nx大鼠SFO核团和PVN核团AngⅡ的表达均高于假手术大鼠(P<0.05)；高盐饮食进一步增强了5/6Nx大鼠AngⅡ在SFO和PVN的表达(P<0.05),而对sham大鼠AngⅡ表达无影响。与正常盐组比较,低盐饮食能够降低sham和5/6Nx大鼠的AngⅡ的表达(P<0.05)。
5.免疫组化和western blot检测不同浓度饮食盐刺激14天大鼠脑SFO/PVN核团ATlR的表达
相同盐饮食时,5/6Nx大鼠SFO和PVN核团AT1R的表达高于假手术大鼠(P<0.05)；高盐饮食进一步增强了5/6Nx大鼠AT1R在SFO和PVN的表达(P<0.05),而对sham大鼠ATlR表达无影响。与正常盐组比较,低盐饮食能够降低sham和5/6Nx大鼠的AT1R的表达(P<0.05)。
6.5/6Nx大鼠SFO/PVN核团AngⅡ和AT1R的细胞定位研究
免疫荧光双染显示,在SFO和PVN核团,AngⅡ和AT1R均定为在神经元细胞上,神经胶质细胞未见表达AngⅡ和AT1R。
7.不同饮食盐刺激5/6Nx大鼠脑SFO和PVN核团神经元的活化
相同盐饮食时,5/6Nx大鼠SFO和PVN核团c-fos的表达高于假手术大鼠(P<0.05)；高盐饮食进一步增强了5/6Nx大鼠AT1R在SFO和PVN的表达(P<0.05),而对sham大鼠c-fos表达无影响。与正常盐组比较,低盐饮食能够降低sham和5/6Nx大鼠的c-fos的表达(P<0.05)。
8不同阻断处理14天对高盐饮食的5/6Nx大鼠生理指标的影响
14天的不同阻断处理对大鼠的体重、血肌酐和血AngⅡ浓度无影响。与0mglosartan灌胃组比较,脑室内注射losartan (lmg)能够显著降低血压(P<0.05),降低血钠水平(P<0.05),极显著的抑制血浆NE水平(P<0.01),并增加24h尿量和24h尿钠排出量(P<0.05),而24h尿蛋白排出量显著降低(P<0.05),且对循环RAS系统无影响,而相同剂量的losartan通过灌胃给药则对上述指标无任何影响,证明脑室内注射losartan (lmg)是通过作用于中枢神经系统产生降压作用；因循环RAS系统未受影响,其对血NE水平的抑制和尿量、尿钠排泄量的增加则反映了对肾脏交感神经的抑制；脑室内注射交感神经抑制剂clonidine或进行肾神经切断术产生与losartan脑室内注射类似的抑制效果(P<0.05);50mg losartan灌胃已能够降低血压、血钠、血浆NE水平以及尿蛋白排泄量(P<0.05),并增加24h尿量和24h尿钠排出量(P<0.05),并随着剂量的增加(500mg losartan灌胃)而增强(P<0.05)；抗氧化剂tempol灌胃对高盐饲养的5/6Nx大鼠的血压、血钠、血浆NE水平以及尿蛋白排泄量均有抑制作用(P<0.05),并能够增加24h尿量和24h尿钠排出量(P<0.05),提示氧化应激机制参与了高盐饲养的5/6Nx大鼠高血压的发生,并于交感神经有关。
9不同阻断处理14天对高盐饮食的5/6Nx大鼠股动脉血压的改变
与Omg losartan灌胃组比较,脑室内注射losartan (1mg)能够显著降低血压(P<0.05),而相同剂量的losartan灌胃对血压无影响,随着losartan灌胃剂量的增加,50mg时血压已显著降低(P<0.05),500mg则进一步增强降压幅度(P<0.05),达到与losartan (1mg) ICV相似的效果；脑室内注射clonidine、肾神经切断术和外周给予tempol均能够使血压显著降低(P<0.05)。
10不同阻断处理14天对高盐饮食的5/6Nx大鼠SFO/PVN核团RAS表达的影响
免疫组化检测显示,与Omg losartan灌胃组比较,脑室内注射losartan (1mg)增强了SFO/PVN核团AngⅡ的表达(P<0.05),但显著抑制了SFO/PVN核团AT1R的表达(P<0.05)；而灌胃给予0-50mg的氯沙坦对AngⅡ和AT1R的表达均无影响,500mg氯沙坦灌胃上调了AngⅡ的表达(P<0.05)和抑制AT1R的表达(P<0.05),灌胃给予抗氧化剂tempol亦未能影响脑内AngⅡ和AT1R的表达；交感神经阻断实验发现,切断肾神经能显著降低SFO/PVN核团AngⅡ和AT1R的表达(P<0.05),中枢给予交感神经抑制剂clonidine无此效果,提示肾神经传入信号对脑内AngⅡ表达具有刺激作用。
Western blot检测SFO/PVN核团ATlR的表达进一步印证了免疫组化的结果。
11不同阻断处理14天对高盐饮食的5/6Nx大鼠SFO/PVN核团TH表达的影响
内源性儿茶酚胺(去甲肾上腺素和肾上腺素)介导了交感神经对血压的调控,酪氨酸羟化酶(TH)是儿茶酚胺系统的限速酶,其表达水平反映了交感神经的活动状态。Western blot检测显示,与Omg losartan灌胃组比较,脑室内注射losartan (1mg)显著抑制了SFO/PVN核团TH的表达(P<0.05),而相同剂量的losartan灌胃对TH表达无影响,表明脑内TH的表达受到局部RAS系统的调控；大剂量losartan (500mg)灌胃也能抑制TH的表达(P<0.05),灌胃给予抗氧化剂tempol未影响脑内TH的表达;交感神经阻断实验发现,切断肾神经能显著降低SFO/PVN核团TH的表达(P<0.05),中枢给予交感神经抑制剂clonidine无此效果,提示5/6Nx大鼠脑内TH的高度表达受到病态的肾脏传入信号的影响。
12不同阻断处理14天对高盐饮食的5/6Nx大鼠SFO/PVN核团c-fos表达的影响
c-fos作为神经元活化的标志,其大量表达提示神经元受到短期或持续的刺激而处于活动状态。不同阻断处理的5/6Nx大鼠SFO/PVN核团c-fos表达情况见图7和表8。与Omg losartan灌胃组比较,脑室内注射losartan (1mg)显著抑制了SFO/PVN核团c-fos的表达(P<0.05),而相同剂量的losartan灌胃对c-fos表达无影响,表明losartan能够抑制高盐饮食的5/6Nx大鼠SFO/PVN核团内神经元的活化；大剂量losartan (500mg)灌胃也能抑制-fos的表达(P<0.05),灌胃给予抗氧化剂Itempol未影响脑内c-fos的表达；交感神经阻断实验发现,切断肾神经能显著降低SFO/PVN核团c-fos的表达(P<0.05),中枢给予交感神经抑制剂clonidine无此效果,提示5/6Nx大鼠肾脏传入信号激活了SFO/PVN核团的神经元。
论文总结
1.5/6Nx大鼠下丘脑心血管调控中枢(交感中枢)存在RAS的活化；
2.5/6Nx大鼠脑内活化的RAS系统通过刺激交感中枢引起血压升高；
3.高盐饮食能够加重5/6Nx大鼠肾损伤,并通过肾脏传入神经进一步激活脑内RAS系统,进而激活交感中枢,导致高血压恶化；
4.在5/6Nx大鼠的肾脏和下丘脑心血管调控中枢(交感中枢)之间存在正反馈环路,加速了肾损伤的进展。
Chapter one Background
Renal parenchymal disease is the most common secondary cause of hypertension, The pathogenesis of hypertension in patients with renal disease has been classically considered to be due to the combined result of volume overload/sodium retention and inappropriate activation of the rennin-angiotensin-aldosterone system (RAAS). Recent evidence now strongly suggests that increased sympathetic nervous system (SNS) activity is also involved.
1Pathogenesis of Hypertension in Renal Failure
Pressure natriuresis is a central component of the feedback system for the long-term control of fluid volume and arterial pressure. However, in the presence of a defect in renal sodium excretory function, blood pressure is elevated to offset the abnormal pressure natriuresis relationship. Salt retention and the subsequent volume expansion also contribute to the phenomena of autoregulation, whereby increased tissue blood flow stimulates widespread vascular constriction, further increasing blood pressure. Therefore, volume retention and sodium balance is important in the hypertension associated with renal disease and has been well documented in end-stage renal disease (ESRD) patients.
There is overwhelming evidence for a role of the RAAS in the development of hypertension and progression of kidney disease in renal failure. This is most apparent when considering the success of angiotensin-converting enzyme (ACE) inhibitors or angiotensin (Ang) II receptor antagonists in controlling blood pressure in renal insufficiency and the fact that these drugs also provide blood pressure-independent renoprotection.
Afferent signals from the kidney are detected by chemoreceptors, which respond to ischaemic metabolites, uraemic toxins and the overall ionic composition of the renal interstitium, and mechanoreceptors, which monitor hydrostatic pressure changes in the kidney. The associated stimulation of renal afferents evokes a reflex increase in sympathetic activity and blood pressure. Whether mechanoreceptor-mediated responses or activation of renal chemoreceptors, the kidney has both direct and indirect connections to central nuclei of the SNS that are involved in arterial pressure regulation. The hypothalamus, in particular, plays a number of important roles in the integrated renal afferent sympathetic response. Not only does it have the ability to regulate renal efferent sympathetic nerve activity directly via presympathetic PVN neurons, but it also has a neurohormonal influence, augmenting the release of arginine vasopressin and oxytocin into the systemic circulation.
2Brain Control of Blood Pressure
The brain plays a critical role in the regulation of blood pressure (BP) through the incorporation of a complex signaling network that processes information from the circulation regarding changes in vascular tone. In turn, this network responds by modulating sympathetic nerve activity (SNA) and by secreting hormones into the circulation help to adjust the degree of vascular resistance in order to maintain a homeostatic BP set point that is known to be governed by renal pressure-natriuresis mechanisms. However, an accumulating body of work has suggested that dysfunctions in the central nervous system (CNS) regulatory mechanisms of BP control can lead to sustained increase in SNA, which can lead to chronic hypertension.
The BP regulatory brain centers are situated, for the most part, in the hypothalamus of the forebrain and in the medulla of the brainstem. These nuclei are highly networked and their major function in cardiovascular control involves the regulation of SNA to modulate BP tone. In addition, these central cardiovascular pathways mediate neurohormone secretion, including arginine-vasopressin (AVP) that stimulates vasoconstriction and modulates the renal pressure-natriuresis mechanism. Hypothalamic control centers are also important in the regulation of electrolyte and plasma fluid volume via stimulation of thirst and increasing salt appetite.
Animal models of hypertension have been vital in detailing the contribution of various cardiovascular regulatory brain nuclei to neurogenic hypertension. Key nuclei that have been involved in this disorder include; the organum subfornicale (SFO), the paraventricular nucleus (PVN) of the hypothalamus, the rostral ventrolateral medulla (RVLM) and the nucleus of the solitary tract (NTS) of the brainstem. The PVN plays a pivotal role in regulating blood pressure in both the normotensive and hypertensive states. This nucleus receives neural input concerning cardiovascular status from regions such as the SFO and NTS, integrates the input and controls sympathetic outflow by sending projections directly to the intermediolateral cell column (IML) of the spinal cord, and by sending projections to other sympathetic regulatory regions including the RVLM. The PVN also regulates the production of hormones that influence cardiovascular function including glucocorticoids and vasopressin.
3Brain Renin-Angiotensin System and Blood Pressure
The RAS is one of the most important mechanisms for regulating blood pressure, and the major bioactive hormone of this system is Ang II. The pressor actions of Ang II are mediated by its binding to AT1R. Since then, AT1R and all the components for the RAS have been found in various brain regions that are involved in the regulation of SNA as well as the regulation of fluid and electrolyte balance. Furthermore, the physiological significance of a brain RAS is underscored by observations of increased sensitivity to Ang II, and of increased ATIR expression in the cardiovascular regulatory brain areas of animal models of neurogenic hypertension. In addition, genetic or pharmacological interventions of brain RAS components have been shown to attenuate neurogenic hypertension.
In addition, increased RAS activity in the brain increases AVP secretion, which is found to exacerbate hypertension in rats. At the other extreme, a deficit of RAS function in key regions in the brain, such as the magnocellular region of the PVN or the SON, results in impaired production and secretion of AVP, the main cause of centrally mediated diabetes insipidus.
Each component of the RAS has been identified in the brain. Evidence of their role in central BP control and function in hypertension has been determined, both pharmacologically and by genetic manipulation. Data from multiple laboratories show that brain RAS components are compartmentalized within specific brain nuclei and even within specific cells, which would result in increased efficiency of RAS function. These findings help assert that a functional and competent RAS is active in the brain.
4Dietary salt and hypertension
There is broad agreement that excess dietary salt (NaCl) is the single most important controllable factor responsible for the rise in BP with advancing age in our culture and, thus, for the high incidence of essential hypertension. According to widely accepted dogma, salt and, consequently, water retention by the kidneys is a major factor in the pathogenesis of salt-induced hypertension. Slightly elevated plasma [Na+](by about1-3mM) with apparent normovolemia is often observed in hypertensive humans. The high-salt intake in salt-sensitive subjects elevates not only plasma [Na+] but cerebrospinal fluid (CSF)[Na+] as well.
A variety of lesion and stimulation studies have indicated the importance of areas of the anteroventral region of the third ventricle (AV3V), specifically the MnPO and SFO, in cardiovascular and fluid-balance regulation. Additionally, there is substantial neuroanatomical and electrophysiological evidence indicating that the circumventricular organs, such as the organum vasculosum of lamina terminalis and SFO project to the MnPO and PVN. The organum vasculosum of lamina terminalis, SFO, AV3V, and MnPO were identified by the viral neuronal label, and they were found to be a part of the descending sympathetic pathway linked to cardiovascular control. In addition to the anatomical evidence, the stimulation studies showed that electrical stimulation of the SFO produces action potentials in efferent neurons of the PVN. These circumventricular organs may play an important role in the sympathoexcitatory pathway by providing information regarding the circulating humoral milieu, since their neuronal cell bodies are positioned at the site of weak blood-brain barrier and can thereby interact with molecules in the circulation, such as ANG II. There is evidence that ANG II acts as a neurotransmitter from the SFO to the PVN.
A brain region crucial for the regulation of body fluid homeostasis is the paraventricular nucleus of the hypothalamus (PVN). Located lateral to the third ventricle, it is anatomically and functionally connected to neurons residing in forebrain sensory circumventricular organs, parts of the brain lacking a functional blood-brain barrier known for their ability to sense small changes in sodium concentration/osmolality. It includes large vasopressin-producing magnocellular neurons and smaller parvocellular neurons that project to spinal preganglionic sympathetic neurons and premotor sympathetic neurons in the brainstem. The PVN is thus a plausible site for coordination of neurogenic and hormonal actions on the cardiovascular system and the kidney in response to changes in sodium concentration/osmolality.
Hypertension is a common presentation of CRF. The present study was conducted to test the hypothesis that dysregulation of RAS occur in brain of CRF rats, which contribute to the sympathetic overactivity and development of hypertension in CRF, and then we will clarify the relationship between brain RAS and sympathetic activity in CRF rats.
Materials and methods
Preparation of Animal Model
All animal procedures were approved by the Animal Experiment and Care Committee of the Sourthern Medical University. Sixty adult male Sprague-Dawley rats (initial weight,150-180g, Sourthern Medical University Animal Experiment Center) were maintained under shandardized conditions. The animals were subjected to two-step,5/6nephrectomy (5/6Nx, by performing a right nephrectomy with surgical resection of the two-thords of the left kidney) or to sham operation (controls). Ten weeks after the operation, the5/6Nx rats were randomized into subgroups.
Experimental data and specimen collection
1. Blood pressure and urine collection
Three days before the end of the study period,24-hour urine samples were collected for three consecutive days, and the blood pressure was determined in conscious rats by the indirect tail-cuff method also. Systolic blood pressure before and14days after for rats on different stimulate were monitored via a pressure transducer placed in the femoral artery, the change in systolic blood pressure is used as the statistic.
2. Blood collect and brain perfusion
At the completion of each protocol, rats were anesthetized with pentobarbital sodium (40mg/kg, i.p.), Trunk blood was collected in chilled vacuum tubes, and plasma samples were separated and stored at-80℃until assayed. Part of the rat brain tissue were collected for western blot studies, and the others were used for immunohistochemical studies.
Chapter two Affects of salt diet on central renin-angiotensin system of5/6nephrectomy rat model
After14days stimulation, urine and Blood were collected for biochemical indicators test; the brain tissue were used for brain RAS detect by immunohistochemistry or western blot.
Chapter three Cellular localization of brain RAS in5/6Nx rats and the relationship between the RAS and the neuron activation
The rat brain paraffin section from Chapter two were used for double lable brain RAS with NSE or GFAP by immunofluorescence, or for c-fos quantitative by immunohistochemistry.
Chapter four The relationship between brain RAS and blood pressure or SNA in5/6Nx rats
Ten week after5/6Nx, the rats were fed a high-salt (4%) diet, randomized into9groups (n=12in each group) and treated as follows for14days, respectively:①Losartan Omg/kg/d by intragastric gavage (IG):Rats were administrated with daily intragastric injection of endotoxin-free phosphate-buffered saline (PBS)(pH7.4);②Losartan lmg/kg/d IG:Rats were administrated with daily intragastric injection of losartan (lmg/kg per day, Sigma Chemical, St Louis, MO, USA);③Losartan50mg/kg/d IG:Rats were administrated with daily intragastric injection of losartan (50mg/kg per day);④Losartan500mg/kg/d IG:Rats were administrated with daily intragastric injection of losartan (500mg/kg per day);⑤Losartan Omg/kg/d by intracerebroventricular injection (ICV):Rats were administrated with daily intracerebroventricular injection of artificial cerebrospinal fluid (aCSF);⑥Losartan1mg/kg/d ICV:Rats were administrated with daily intracerebroventricular injection of losartan(1mg/kg per day);⑦Clonidine5.76g/kg/d ICV:Rats were administrated with daily intracerebroventricular injection of clonidine (5.76g/kg per day, Sigma Chemical, St Louis, MO, USA);⑨Renal denervation (RDX);⑨Tempol30mg/kg/d IG:Rats were administrated with daily intragastric injection of tempol (30mg/kg per day, Sigma Chemical, St Louis, MO, USA).
After14days stimulation, urine and Blood were collected for biochemical indicators test; the brain tissue were used for brain RAS and TH and c-fos detect by immunohistochemistry or western blot.
Statistical Analyses
All data are presented as mean±SE. Continuous variables between groups were compared using one-way ANOVA, followed by LSD method when P<0.05. Nonparametric test is used when heterogeneity of variance. Statistical analyses were conducted with SPSS13.0for Windows (SPSS, Chicago, IL). Significance was defined as P<0.05.
Results
1. Characteristics of rats post-5/6nephrectomy versus sham-operated rats on10weeks
The base line blood pressure (systolic blood pressure) was determined in conscious rats by the indirect tail-cuff method. Systolic pressure and serum creatinine and24-hour urinary protein excretion were higher in5/6Nx rats versus sham rats, prove5/6Nx rats modeling success.
2. Changes in general characteristics after14days different salt diet stimulation in sham-operated and5/6Nx rats
Blood pressure, serum sodium levels and24h urine protein had no significant difference in sham-operated rats with14days different salt diet (P>0.05). Compared with sham-operated rats,5/6Nx rats had a significantly higher blood pressure with the same normal salt diet (P<0.05). Compared with normal salt5/6Nx group, the blood pressure and serum sodium levels and24h urine protein in high salt5/6Nx group was significantly increased (P<0.05); The low-salt diet can significantly lower the blood pressure of the rats with5/6Nx (P<0.05).
The24h urinary volume and24h urinary sodium excretion were increased with increasing dietary salt (P<0.05), but the blood Ang II has been suppressed with increasing dietary salt in both sham and5/6Nx rats (P<0.05).
3. Changes in systolic blood pressure before and14days after for rats on low, normal and high salt diets
SBP of femoral blood pressure had no significant difference in sham-operated rats with14days different salt diet (P>0.05). Compared with normal salt5/6Nx group, the SBP of femoral blood pressure in high salt5/6Nx group was significantly increased (P<0.05); The low-salt diet can significantly lower the blood pressure of the rats with5/6Nx (P<0.05).
4. Levels of Ang Ⅱ in SFO and PVN after14days different salt diet stimulation in sham-operated and5/6Nx rats
Compared with sham-operated rats,5/6Nx rats had a significantly higher Ang II in SFO and PVN with the same normal salt diet (P<0.05). Compared with normal salt5/6Nx group, the Ang II in SFO and PVN in high salt5/6Nx group was significantly increased (P<0.05); The low-salt diet can significantly lower the Ang Ⅱ in SFO and PVN in sham and5/6Nx rats (P<0.05).
5. Levels of AT1R in SFO and PVN after14days different salt diet stimulation in sham-operated and5/6Nx rats
Compared with sham-operated rats,5/6Nx rats had a significantly higher AT1R in SFO and PVN with the same normal salt diet (P<0.05). Compared with normal salt5/6Nx group, the AT1R in SFO and PVN in high salt5/6Nx group was significantly increased (P<0.05); The low-salt diet can significantly lower the AT1R in SFO and PVN in sham and5/6Nx rats (P<0.05).
6. Cellular localization of Ang Ⅱ or AT1R in SFO and PVN in5/6Nx rats
Ang II or AT1R immunoreactivity is colocalized on neurons within SFO and PVN.
7. Neurons activation in SFO and PVN of5/6Nx rats with different salt diet
Compared with sham-operated rats,5/6Nx rats had a significantly higher c-fos in SFO and PVN with the same normal salt diet (P<0.05). Compared with normal salt5/6Nx group, the c-fos in SFO and PVN in high salt5/6Nx group was significantly increased (P<0.05); The low-salt diet can significantly lower the c-fos in SFO and PVN in sham and5/6Nx rats (P<0.05).
8. General characteristics respond to14days of blockade with various approaches in5/6Nx rats
Systolic blood pressure and urinary albumin excretion markedly decreased in salt-loaded5/6Nx rats with losartan ICV at lmg/kg/d (P<0.05). Losartan IG at higher doses (50-500mg/kg/d), clonidine ICV, renal denervation, or tempol IG also significantly attenuated systolic blood pressure and urinary albumin excretion in high-salt-fed5/6Nx animals (P<0.05). NE level markedly decreased in salt-loaded5/6Nx rats combined with losartan ICV at lmg/kg/d, clonidine ICV or renal denervation (P<0.05). Losartan IG at higher doses (50-500mg/kg/d) and tempol IG also significantly attenuated renal NE levels in high-salt-fed5/6Nx animals (P<0.05).
9. Changes in systolic blood pressure before and14days after losartan or clonidine blockade in sham-operated and5/6Nx rats
Systolic blood pressure markedly decreased in salt-loaded5/6Nx rats with losartan ICV at lmg/kg/d (P<0.05). Losartan IG at higher doses (50-500mg/kg/d), clonidine ICV, renal denervation, or tempol IG also significantly attenuated systolic blood pressure in high-salt-fed5/6Nx animals (P<0.05).
10. Levels of RAS expression in SFO and PVN nucleus after14days of blockade with various approaches in5/6Nx rats
We previously showed that high-salt diet enhance RAS expression in SFO and PVN nucleus in5/6Nx rats. To verify the role of central RAS in the development of hypertension and the relationship between central RAS and SNS, the5/6Nx rats treated with high-salt diet were blockaded by different approaches to central or periphery RAS and SNS. Immunohistochemistry was used to map the expression of Ang II. The operation of renal denervation eliminate the increase of Ang II expression induced by high-salt diet (P<0.05vs5/6Nx rats with IG Omg of losartan), conversely, lmg of losartan given by ICV or500mg losartan given by IG further increased Ang II expression in both SFO and PVN nucleus (P<0.05vs5/6Nx rats with IG Omg of losartan), whereas the losartan (lmg and50mg) or tempol given by IG and clonidine given by ICV had no effect.
Immunohistochemistry was also used to map the expression of ATIR. Changes of AT1R expression response to various blockades are different to Ang II. Low dose of losartan (lmg) given by ICV prominently suppressed the expression of ATIR in SFO and PVN nucleus (P<0.05vs5/6Nx rats with IG Omg of losartan), while the same dose or even much larger dose of losartan (50mg) given by IG didn't have any effect on expression of ATIR, the effect of500mg of losartan given by IG was comparable with that of1mg of losartan given by ICV (P<0.05vs5/6Nx rats with IG Omg of losartan). The operation of renal denervation had restrained the high expression of AT1R in SFO and PVN nucleus (P<0.05vs5/6Nx rats with IG Omg of losartan). Clonidine or tempol had no effect on AT1R expression.
Western blot was used to further determine the ATIR changes, and obtained similar results to immunohistochemistry.The high expression of AT1R in SFO and PVN nucleus induced by high-salt diet were suppressed by ICV1mg of losartan or IG500mg of losartan or renal denervation (P<0.05vs5/6Nx rats with IG Omg of losartan), whereas the losartan (1mg and50mg) or tempol given by IG and clonidine given by ICV had no effect.
11. Levels of tyrosine hydroxylase (TH) expression in SFO and PVN nucleus after14days of blockade with various approaches in5/6Nx rats
To verify the relationship between central RAS and SNS, tyrosine TH levels in SFO and PVN nucleus in5/6Nx rats were detected by western blot. ICV lmg of losartan significantly decreased the expression of TH in SFO and PVN nucleus (P<0.05vs5/6Nx rats with IG Omg of losartan), high dose of losartan (500mg) given by IG or renal denervation had similar effect on TH (P<0.05vs5/6Nx rats with IG Omg of losartan), whereas the losartan (lmg and50mg) or tempol given by IG and clonidine given by ICV had no effect.
12. Levels of c-fos expression in SFO and PVN nucleus after14days of blockade with various approaches in5/6Nx rats
To clarify the effect of various blockades on neurons activation in SFO and PVN of5/6Nx rats treated with high-salt diet, the c-fos was quantified by immunohistochemistry. We found that high level of c-fos was restored by Low dose of losartan (1mg) given by ICV (P<0.05vs5/6Nx rats with IG Omg of losartan), in contrast, the same dose of losartan and even more large of losartan (50mg) given by IG didn't change the c-fos expression in SFO and PVN nucleus, while the large dose of losartan (500mg) had comparable effect with that of lmg of losartan given by ICV (P<0.05vs5/6Nx rats with IG Omg of losartan). Moreover, we found that c-fos was significantly restored by renal denervation (P<0.05vs5/6Nx rats with IG Omg of losartan), but not by clonidine or tempol.
Paper summarizes
1. RAS was activated in brain cardiovascular centre (sympathetic integration site) in5/6Nx rats;
2. Overactive brain RAS may raises blood pressure by stimulating the central sympathetic nervous system in5/6Nx rats;
3. High-salt diet can aggravate kidney injury and further activation brain RAS by afferent nerves from the kidneys, thereby activating central sympathetic nervous system and worsen the hypertension;
4. There exists a positive feedback loop between the kidney and brain cardiovascular centre (sympathetic integration site), which can accelerated the progress of kidney injury.
高血压是肾实质疾病最常见的继发疾病。容量负荷/钠潴留和肾素-血管紧张素系统(RAS)的不适当的激活是肾病患者发生高血压的主要决定因素。最近的研究显示,中枢交感神经系统(sympathetic nervous system, SNS)活动的增加参与了慢性肾功能衰竭(CRF)高血压的发生。
1CRF时高血压的发病机理
压力性利尿是体液容量和动脉压长期控制的反馈调节系统的主要部分。容量扩张引起血压升高的准确机制尚不十分清楚,高血压发生机制的自动调节理论(autoregulation theory)可以解释部分CRF患者容量扩张与血压升高的关系：容量扩张→细胞外液容量和血容量的增加→静脉回心血量增加→心输出量增加→外周组织过度灌注→自动调节血管收缩→总外周阻力增加→血压升高。
血管紧张素Ⅱ (Angiotensin Ⅱ, Ang Ⅱ)通过多种机制升高血压,包括血管收缩、钠潴留、血管重塑和增加交感神经活动；AngⅡ的1型受体(Angiotensin Ⅱ AT1-receptor, AT1R)拮抗剂特异性的阻断Ang Ⅱ与AT1R的结合,导致血管舒张、钠排泄和血压降低。RAS系统的所有成分在脑组织中均有表达；研究表明,脑内局部的RAS系统功能的异常与高血压的发生密切相关。
肾脏与交感神经的中枢核团具有直接和间接的联系,从而调节动脉压。直接的途径是通过肾神经,而间接的途径是体液和体液相关的RAAS的激活,并作用于脑内缺乏血脑屏障的RAAS作用部位。这些区域在调节血压和体液平衡方面具有重要作用。前脑室旁器官(circumventricular organs, CVOs)神经核团通过神经突出到达下丘脑核团；下丘脑室旁核(Paraventricular hypothalamic necleus, PVN)在整合肾脏传入交感神经反应方面起着重要作用,其不但可以直接通过PVN的前交感神经元调节肾脏传入交感神经活动,还具有神经激素的影响,可增加精氨酸加压素和催产素向全身循环的释放。
.2.高血压的中枢机制
越来越多的研究认为中枢神经系统控制血压调节的功能紊乱导致交感神经活动的持续增加,进而引起慢性高血压。研究从脑内传出的交感神经调节因素是认识神经源性高血压发生机制的主要靶点。
脑内控制血压调节的中枢绝大部分位于前脑的下丘脑和脑干的髓质。这些核团是高度网络化的,其在心血管控制中的主要作用是通过调节交感神经来调节血压张力。此外,这些中枢心血管通路通过介导神经激素分泌,包括精氨酸加压素刺激血管收缩和调节肾脏压力性利尿机制。下丘脑控制中心通过刺激口渴和盐欲对电解质和血液容量也有重要的调节作用。
高血压动物模型对于研究心血管调节核团对神经源性高血压发生的具体作用至关重要。和这种调节紊乱有关的主要核团包括：穹窿下器(SFO),下丘脑室旁核(PVN)、脑干的延髓头端腹外侧核(RVLM)和孤束核(NTS)。PVN在正常血压和高血压状态下对血压调节都起着枢纽角色。这个核团接受来自SFO和NTS等区域的关于心血管状态的输入信号,整合输入信号后,通过直接到达脊髓的中间外侧细胞柱(IML)和其他的交感神经调节区域包括RVLM的神经突触控制交感神经的输出信号。PVN也调节影响心血管功能的激素的产生,包括糖皮质激素和垂体加压素。
3脑内RAS系统与血压
对脑内ANGⅡ和AT1R免疫活性的分布进行比较显示,绝大多数的脑内ANGⅡ和几乎所有的脑内AT1R位于血脑屏障内神经核团,在这些部位,脑内内源性的ANGⅡ能够接触AT1R,而循环中的ANGⅡ无法到达此部位。但是在前脑存在一条AT1R的分布带,使室周器官(SFO、OVLT)和血脑屏障内的结构(PVN)联系起来。这条通路在解剖学上构成了循环(外周)和脑(中枢)ANGⅡ系统之间的生理学的联系。
ANGⅡ是RAS系统合成的增压激素,ANGⅡ的增压作用通过作用于AT1受体(AT1R)表达；ANGⅡ也可作为神经肽,通过AT1R增加下丘脑和脑干心血管调节中枢神经元的兴奋性,中枢神经系统ANGⅡ/AT1R作用的过度活跃导致神经源性高血压。
4饮食盐与高血压
大量的研究表明,随着年龄的增长,大量的饮食盐摄入是原发性高血压高发病率的唯一的最重要的可控性因素。。肾脏对钠和水的潴留是盐诱导的高血压最主要的发生机制。在血容量正常的高血压患者,其血[Na+]水平往往轻度升高(约升高1-3mmol)。然而,盐敏感的个体高盐饮食并不仅仅升高血浆的[Na+],其脑脊液的[Na+]也同时升高。
脑内感受性室周器官(包括SFO、OVLT和MnPO)能够通过钠/渗透性受体监控血浆和脑脊液中的[Na+]变化,而SFO是对渗透性刺激最重要的感受器。对SFO进行定位损毁,能够消除向脑室内注射高渗盐水(NaCl)引起的增压反应。电刺激研究证实,SFO神经元投射至下丘脑室旁核,并且向PVN传递兴奋性信号,而PVN进一步将信号投射至脊髓中间外侧细胞柱的交感神经元。电损毁PVN部位能够阻断SFO介导的增压反应。有证据表明,AngⅡ是从SFO到PVN的神经递质。
位于前脑的穹窿下器(SFO)缺乏完整的血脑屏障,因此能够感受外周或脑脊液中钠离子浓度和渗透压的微小改变。下丘脑室旁核(PVN)位于第三脑室侧面,是脑内调节体液平衡的最重要的核团；其在解剖学和功能学上与SFO的神经元联系密切。PVN含有大量的合成精氨酸加压素(AVP)的大细胞性神经元,以及少量的小细胞性神经元,其小细胞性神经元投射至脊髓的交感神经节前神经元和脑干的运动前交感神经元。因此,PVN是心血管系统和肾脏对钠离子浓度和渗透压改变产生反应的神经源性和激素性调控的中枢。阻断PVN部位的AT1R能够减弱颈内动脉注射高渗盐水引起的肾脏交感神经的兴奋。
综上所述,鉴于中枢神经系统在血压调控和高血压发生、发展中的重要地位和作用,研究慢性肾功能衰竭状态下中枢神经系统,尤其是脑部RAS系统是否发生异常改变,将进一步加深对慢性肾功能衰竭及其他慢性肾脏病高血压的发生机制的认识。
本研究将对比5/6Nx大鼠和假手术大鼠(sham大鼠)脑内心血管调控中枢的关键神经核团(SFO和PVN) RAS系统的活化水平；并观察5/6Nx和sham大鼠脑内RAS对不同浓度饮食盐的反应情况,以及伴随的外周效应(血压,肾脏损伤程度等)；在上述研究基础上,进行高盐饮食情况下大鼠脑内RAS或交感神经阻断实验,观察5/6Nx脑内RAS是否参与其血压的调节,探索5/6Nx大鼠脑内RAS与其增强的交感神经活动之间的关系,并阐述其脑内RAS与交感神经相互关系的具体机制。
材料和方法
动物模型的制备
雄性Sprague Dawley大鼠(购自南方医科大学实验动物中心)体重150-180g,行5/6肾切除术。于第10周对大鼠进行随机分组。
实验数据和标本采集
1.血压和尿样采集
在分组后给予不同刺激前,采用股动脉插管法测量麻醉状态下大鼠股动脉血压,作为各组的基础血压；在实验结束前,再次测量大鼠的股动脉血压,以刺激14天血压减去刺激前血压的差值作为股动脉血压统计数据。不同刺激的最后三天,代谢笼内单只单笼饲养,连续三天收集24h尿液,用于24h尿钠和尿蛋白定量。
2.大鼠脑标本摘取和采血
刺激结束后,大鼠3%戊巴比妥钠腹腔注射麻醉(35mg/kg)后部分大鼠摘取新鲜脑核团,用于western-blot实验检测SFO/PVN核团RAS及TH的表达,另一部分大鼠制作脑石蜡切片,用于免疫组织化学技术检测SFO/PVN核团RAS的表达和c-fos的表达。采血用于肾功能、电解质、血AngⅡ及NE检测。
第二章5/6Nx大鼠脑核团(SFO/PVN部位)RAS的表达及对饮食盐的反应研究
术后第10周对大鼠进行随机分组,大鼠按各自的饲料共饲养刺激14天。具体分组如下：
刺激14天后,对大鼠血压及血、尿生理生化指标进行检测。采用免疫组化及western blot技术对脑核团SFO/PVN部位RAS成分(AngⅡ和AT1R)表达进行检测。
第三章5/6Nx大鼠脑核团(SFO/PVN部位)RAS的细胞定位及与神经元活化的关系研究
采用第二章5/6Nx大鼠的脑石蜡切片,对脑RAS与神经元标志物或神经胶质细胞标志物进行免疫荧光双染,确定5/6Nx大鼠的脑RAS细胞定位。
采用第二章5/6Nx大鼠的脑石蜡切片,进行c-fos的免疫组化染色,观察神经元活化情况。
第四章5/6Nx大鼠脑核团(SFO/PVN部位)RAS活化与血压及交感神经的关系研究
5/6Nx术后第10周对大鼠进行随机分组,所有大鼠在高盐饲料刺激的同时,按如下分组进行阻断干预14天,具体分组如下(每组n=12只)：
①Losartan0mg IG组：纯水灌胃,体积0.5ml/kg/d
②Losartan1mg IG组：losartan灌胃剂量1mg/kg/d,体积0.5ml/kg/d
③Losartan50mg IG组：losartan灌胃剂量50mg/kg/d,体积0.5ml/kg/d
④Losartan500mg IG组：losartan灌胃剂量500mg/kg/d,体积0.5ml/kg/d
⑤Losartan0mg ICV组：人工脑脊液(aCSF)侧脑室注射,速度0.5gl/h
⑥Losartan1mg ICV组：losartan侧脑室注射,剂量1mg/kg/d,速度0.5μl/h
⑦Clonidine5.76μg ICV组：clonidine侧脑室注射,剂量5.76μg/kg/d,速度0.51μl/h
⑧Renal denervation组：行肾神经切断术,纯水灌胃,体积0.5ml/kg/d
⑨Tempol30mg/kg/d IG组：tempol灌胃剂量30mg/kg/d,体积0.5ml/kg/d
注释：灌胃组药物均已纯水作为溶剂,脑室内注射组药物以aCSF作为溶剂。I.G,灌胃给药；I.C.V,脑室内注射；aCSF,人共脑脊液。
刺激14天后,对大鼠血压及血、尿生理生化指标进行检测。采用免疫组化及western blot技术对脑核团SFO/PVN部位RAS成分(AngⅡ和AT1R)和TH表达进行检测,并用免疫组化技术检测c-fos的表达。
统计学方法：
数据以mean±SE表示,所有统计由统计软件SPSS13.0完成。假手术和肾衰组实验前基础数据比较采用独立样本T检验；刺激14天后,相同饮食盐的假手术和肾衰组之间的比较采用独立样本T检验；假手术或肾衰组在不同饮食盐刺激14天后的组内比较,符合方差齐性检验的数据,采用one-way ANOVA,当P<0.05时,用Post Hoc Tests的LSD法进行两两比较；方差不齐时采用Welch检验；当P<0.05时,两两比较采用Dunnett'T3法。阻断剂干预组之间比较采用one-way ANOVA,方法同前。P<0.05为差异有统计学意。
结果
1.造模术后10周大鼠血压及肾功能
在术后第十周进行不同饮食盐刺激前,5/6Nx大鼠血压已显著高于sham组(P<0.05),5/6Nx组血肌酐和24小时尿蛋白排泄量也显著升高(P<0.05),证明5/6Nx大鼠造模成功。
2.不同浓度饮食盐刺激14天大鼠的生理及血、尿生化指标
在正常盐情况下,5/6Nx大鼠血压水平已显著高于假手术大鼠(P<0.05)。高盐饮食能够显著升高5/6Nx大鼠的血压、血NE水平、血钠和24h尿蛋白(P<0.05),而低盐饮食则能显著降低5/6Nx大鼠的血压(P<0.05)。饮食盐的改变对sham大鼠血压、血钠水平及24h尿蛋白无影响。
Sham和5/6Nx大鼠24h尿量和24h尿钠排泄量随着饮食盐的增加而增加(P<0.05),而血AngⅡ和NE随着饮食盐的增加而受到抑制(P<0.05)
3.不同浓度饮食盐刺激14天大鼠股动脉血压的改变
经过14天不同的饮食盐刺激,假手术大鼠血压无明显改变。与5/6Nx正常盐组比较,高盐饮食显著升高5/6Nx大鼠血压(P<0.05),而低盐饮食则能降低5/6Nx大鼠血压(P<0.05)。
4.免疫组化检测不同浓度饮食盐刺激14天大鼠脑SFO/PVN核团AngⅡ的表达
相同饮食盐时,5/6Nx大鼠SFO核团和PVN核团AngⅡ的表达均高于假手术大鼠(P<0.05)；高盐饮食进一步增强了5/6Nx大鼠AngⅡ在SFO和PVN的表达(P<0.05),而对sham大鼠AngⅡ表达无影响。与正常盐组比较,低盐饮食能够降低sham和5/6Nx大鼠的AngⅡ的表达(P<0.05)。
5.免疫组化和western blot检测不同浓度饮食盐刺激14天大鼠脑SFO/PVN核团ATlR的表达
相同盐饮食时,5/6Nx大鼠SFO和PVN核团AT1R的表达高于假手术大鼠(P<0.05)；高盐饮食进一步增强了5/6Nx大鼠AT1R在SFO和PVN的表达(P<0.05),而对sham大鼠ATlR表达无影响。与正常盐组比较,低盐饮食能够降低sham和5/6Nx大鼠的AT1R的表达(P<0.05)。
6.5/6Nx大鼠SFO/PVN核团AngⅡ和AT1R的细胞定位研究
免疫荧光双染显示,在SFO和PVN核团,AngⅡ和AT1R均定为在神经元细胞上,神经胶质细胞未见表达AngⅡ和AT1R。
7.不同饮食盐刺激5/6Nx大鼠脑SFO和PVN核团神经元的活化
相同盐饮食时,5/6Nx大鼠SFO和PVN核团c-fos的表达高于假手术大鼠(P<0.05)；高盐饮食进一步增强了5/6Nx大鼠AT1R在SFO和PVN的表达(P<0.05),而对sham大鼠c-fos表达无影响。与正常盐组比较,低盐饮食能够降低sham和5/6Nx大鼠的c-fos的表达(P<0.05)。
8不同阻断处理14天对高盐饮食的5/6Nx大鼠生理指标的影响
14天的不同阻断处理对大鼠的体重、血肌酐和血AngⅡ浓度无影响。与0mglosartan灌胃组比较,脑室内注射losartan (lmg)能够显著降低血压(P<0.05),降低血钠水平(P<0.05),极显著的抑制血浆NE水平(P<0.01),并增加24h尿量和24h尿钠排出量(P<0.05),而24h尿蛋白排出量显著降低(P<0.05),且对循环RAS系统无影响,而相同剂量的losartan通过灌胃给药则对上述指标无任何影响,证明脑室内注射losartan (lmg)是通过作用于中枢神经系统产生降压作用；因循环RAS系统未受影响,其对血NE水平的抑制和尿量、尿钠排泄量的增加则反映了对肾脏交感神经的抑制；脑室内注射交感神经抑制剂clonidine或进行肾神经切断术产生与losartan脑室内注射类似的抑制效果(P<0.05);50mg losartan灌胃已能够降低血压、血钠、血浆NE水平以及尿蛋白排泄量(P<0.05),并增加24h尿量和24h尿钠排出量(P<0.05),并随着剂量的增加(500mg losartan灌胃)而增强(P<0.05)；抗氧化剂tempol灌胃对高盐饲养的5/6Nx大鼠的血压、血钠、血浆NE水平以及尿蛋白排泄量均有抑制作用(P<0.05),并能够增加24h尿量和24h尿钠排出量(P<0.05),提示氧化应激机制参与了高盐饲养的5/6Nx大鼠高血压的发生,并于交感神经有关。
9不同阻断处理14天对高盐饮食的5/6Nx大鼠股动脉血压的改变
与Omg losartan灌胃组比较,脑室内注射losartan (1mg)能够显著降低血压(P<0.05),而相同剂量的losartan灌胃对血压无影响,随着losartan灌胃剂量的增加,50mg时血压已显著降低(P<0.05),500mg则进一步增强降压幅度(P<0.05),达到与losartan (1mg) ICV相似的效果；脑室内注射clonidine、肾神经切断术和外周给予tempol均能够使血压显著降低(P<0.05)。
10不同阻断处理14天对高盐饮食的5/6Nx大鼠SFO/PVN核团RAS表达的影响
免疫组化检测显示,与Omg losartan灌胃组比较,脑室内注射losartan (1mg)增强了SFO/PVN核团AngⅡ的表达(P<0.05),但显著抑制了SFO/PVN核团AT1R的表达(P<0.05)；而灌胃给予0-50mg的氯沙坦对AngⅡ和AT1R的表达均无影响,500mg氯沙坦灌胃上调了AngⅡ的表达(P<0.05)和抑制AT1R的表达(P<0.05),灌胃给予抗氧化剂tempol亦未能影响脑内AngⅡ和AT1R的表达；交感神经阻断实验发现,切断肾神经能显著降低SFO/PVN核团AngⅡ和AT1R的表达(P<0.05),中枢给予交感神经抑制剂clonidine无此效果,提示肾神经传入信号对脑内AngⅡ表达具有刺激作用。
Western blot检测SFO/PVN核团ATlR的表达进一步印证了免疫组化的结果。
11不同阻断处理14天对高盐饮食的5/6Nx大鼠SFO/PVN核团TH表达的影响
内源性儿茶酚胺(去甲肾上腺素和肾上腺素)介导了交感神经对血压的调控,酪氨酸羟化酶(TH)是儿茶酚胺系统的限速酶,其表达水平反映了交感神经的活动状态。Western blot检测显示,与Omg losartan灌胃组比较,脑室内注射losartan (1mg)显著抑制了SFO/PVN核团TH的表达(P<0.05),而相同剂量的losartan灌胃对TH表达无影响,表明脑内TH的表达受到局部RAS系统的调控；大剂量losartan (500mg)灌胃也能抑制TH的表达(P<0.05),灌胃给予抗氧化剂tempol未影响脑内TH的表达;交感神经阻断实验发现,切断肾神经能显著降低SFO/PVN核团TH的表达(P<0.05),中枢给予交感神经抑制剂clonidine无此效果,提示5/6Nx大鼠脑内TH的高度表达受到病态的肾脏传入信号的影响。
12不同阻断处理14天对高盐饮食的5/6Nx大鼠SFO/PVN核团c-fos表达的影响
c-fos作为神经元活化的标志,其大量表达提示神经元受到短期或持续的刺激而处于活动状态。不同阻断处理的5/6Nx大鼠SFO/PVN核团c-fos表达情况见图7和表8。与Omg losartan灌胃组比较,脑室内注射losartan (1mg)显著抑制了SFO/PVN核团c-fos的表达(P<0.05),而相同剂量的losartan灌胃对c-fos表达无影响,表明losartan能够抑制高盐饮食的5/6Nx大鼠SFO/PVN核团内神经元的活化；大剂量losartan (500mg)灌胃也能抑制-fos的表达(P<0.05),灌胃给予抗氧化剂Itempol未影响脑内c-fos的表达；交感神经阻断实验发现,切断肾神经能显著降低SFO/PVN核团c-fos的表达(P<0.05),中枢给予交感神经抑制剂clonidine无此效果,提示5/6Nx大鼠肾脏传入信号激活了SFO/PVN核团的神经元。
论文总结
1.5/6Nx大鼠下丘脑心血管调控中枢(交感中枢)存在RAS的活化；
2.5/6Nx大鼠脑内活化的RAS系统通过刺激交感中枢引起血压升高；
3.高盐饮食能够加重5/6Nx大鼠肾损伤,并通过肾脏传入神经进一步激活脑内RAS系统,进而激活交感中枢,导致高血压恶化；
4.在5/6Nx大鼠的肾脏和下丘脑心血管调控中枢(交感中枢)之间存在正反馈环路,加速了肾损伤的进展。
Chapter one Background
Renal parenchymal disease is the most common secondary cause of hypertension, The pathogenesis of hypertension in patients with renal disease has been classically considered to be due to the combined result of volume overload/sodium retention and inappropriate activation of the rennin-angiotensin-aldosterone system (RAAS). Recent evidence now strongly suggests that increased sympathetic nervous system (SNS) activity is also involved.
1Pathogenesis of Hypertension in Renal Failure
Pressure natriuresis is a central component of the feedback system for the long-term control of fluid volume and arterial pressure. However, in the presence of a defect in renal sodium excretory function, blood pressure is elevated to offset the abnormal pressure natriuresis relationship. Salt retention and the subsequent volume expansion also contribute to the phenomena of autoregulation, whereby increased tissue blood flow stimulates widespread vascular constriction, further increasing blood pressure. Therefore, volume retention and sodium balance is important in the hypertension associated with renal disease and has been well documented in end-stage renal disease (ESRD) patients.
There is overwhelming evidence for a role of the RAAS in the development of hypertension and progression of kidney disease in renal failure. This is most apparent when considering the success of angiotensin-converting enzyme (ACE) inhibitors or angiotensin (Ang) II receptor antagonists in controlling blood pressure in renal insufficiency and the fact that these drugs also provide blood pressure-independent renoprotection.
Afferent signals from the kidney are detected by chemoreceptors, which respond to ischaemic metabolites, uraemic toxins and the overall ionic composition of the renal interstitium, and mechanoreceptors, which monitor hydrostatic pressure changes in the kidney. The associated stimulation of renal afferents evokes a reflex increase in sympathetic activity and blood pressure. Whether mechanoreceptor-mediated responses or activation of renal chemoreceptors, the kidney has both direct and indirect connections to central nuclei of the SNS that are involved in arterial pressure regulation. The hypothalamus, in particular, plays a number of important roles in the integrated renal afferent sympathetic response. Not only does it have the ability to regulate renal efferent sympathetic nerve activity directly via presympathetic PVN neurons, but it also has a neurohormonal influence, augmenting the release of arginine vasopressin and oxytocin into the systemic circulation.
2Brain Control of Blood Pressure
The brain plays a critical role in the regulation of blood pressure (BP) through the incorporation of a complex signaling network that processes information from the circulation regarding changes in vascular tone. In turn, this network responds by modulating sympathetic nerve activity (SNA) and by secreting hormones into the circulation help to adjust the degree of vascular resistance in order to maintain a homeostatic BP set point that is known to be governed by renal pressure-natriuresis mechanisms. However, an accumulating body of work has suggested that dysfunctions in the central nervous system (CNS) regulatory mechanisms of BP control can lead to sustained increase in SNA, which can lead to chronic hypertension.
The BP regulatory brain centers are situated, for the most part, in the hypothalamus of the forebrain and in the medulla of the brainstem. These nuclei are highly networked and their major function in cardiovascular control involves the regulation of SNA to modulate BP tone. In addition, these central cardiovascular pathways mediate neurohormone secretion, including arginine-vasopressin (AVP) that stimulates vasoconstriction and modulates the renal pressure-natriuresis mechanism. Hypothalamic control centers are also important in the regulation of electrolyte and plasma fluid volume via stimulation of thirst and increasing salt appetite.
Animal models of hypertension have been vital in detailing the contribution of various cardiovascular regulatory brain nuclei to neurogenic hypertension. Key nuclei that have been involved in this disorder include; the organum subfornicale (SFO), the paraventricular nucleus (PVN) of the hypothalamus, the rostral ventrolateral medulla (RVLM) and the nucleus of the solitary tract (NTS) of the brainstem. The PVN plays a pivotal role in regulating blood pressure in both the normotensive and hypertensive states. This nucleus receives neural input concerning cardiovascular status from regions such as the SFO and NTS, integrates the input and controls sympathetic outflow by sending projections directly to the intermediolateral cell column (IML) of the spinal cord, and by sending projections to other sympathetic regulatory regions including the RVLM. The PVN also regulates the production of hormones that influence cardiovascular function including glucocorticoids and vasopressin.
3Brain Renin-Angiotensin System and Blood Pressure
The RAS is one of the most important mechanisms for regulating blood pressure, and the major bioactive hormone of this system is Ang II. The pressor actions of Ang II are mediated by its binding to AT1R. Since then, AT1R and all the components for the RAS have been found in various brain regions that are involved in the regulation of SNA as well as the regulation of fluid and electrolyte balance. Furthermore, the physiological significance of a brain RAS is underscored by observations of increased sensitivity to Ang II, and of increased ATIR expression in the cardiovascular regulatory brain areas of animal models of neurogenic hypertension. In addition, genetic or pharmacological interventions of brain RAS components have been shown to attenuate neurogenic hypertension.
In addition, increased RAS activity in the brain increases AVP secretion, which is found to exacerbate hypertension in rats. At the other extreme, a deficit of RAS function in key regions in the brain, such as the magnocellular region of the PVN or the SON, results in impaired production and secretion of AVP, the main cause of centrally mediated diabetes insipidus.
Each component of the RAS has been identified in the brain. Evidence of their role in central BP control and function in hypertension has been determined, both pharmacologically and by genetic manipulation. Data from multiple laboratories show that brain RAS components are compartmentalized within specific brain nuclei and even within specific cells, which would result in increased efficiency of RAS function. These findings help assert that a functional and competent RAS is active in the brain.
4Dietary salt and hypertension
There is broad agreement that excess dietary salt (NaCl) is the single most important controllable factor responsible for the rise in BP with advancing age in our culture and, thus, for the high incidence of essential hypertension. According to widely accepted dogma, salt and, consequently, water retention by the kidneys is a major factor in the pathogenesis of salt-induced hypertension. Slightly elevated plasma [Na+](by about1-3mM) with apparent normovolemia is often observed in hypertensive humans. The high-salt intake in salt-sensitive subjects elevates not only plasma [Na+] but cerebrospinal fluid (CSF)[Na+] as well.
A variety of lesion and stimulation studies have indicated the importance of areas of the anteroventral region of the third ventricle (AV3V), specifically the MnPO and SFO, in cardiovascular and fluid-balance regulation. Additionally, there is substantial neuroanatomical and electrophysiological evidence indicating that the circumventricular organs, such as the organum vasculosum of lamina terminalis and SFO project to the MnPO and PVN. The organum vasculosum of lamina terminalis, SFO, AV3V, and MnPO were identified by the viral neuronal label, and they were found to be a part of the descending sympathetic pathway linked to cardiovascular control. In addition to the anatomical evidence, the stimulation studies showed that electrical stimulation of the SFO produces action potentials in efferent neurons of the PVN. These circumventricular organs may play an important role in the sympathoexcitatory pathway by providing information regarding the circulating humoral milieu, since their neuronal cell bodies are positioned at the site of weak blood-brain barrier and can thereby interact with molecules in the circulation, such as ANG II. There is evidence that ANG II acts as a neurotransmitter from the SFO to the PVN.
A brain region crucial for the regulation of body fluid homeostasis is the paraventricular nucleus of the hypothalamus (PVN). Located lateral to the third ventricle, it is anatomically and functionally connected to neurons residing in forebrain sensory circumventricular organs, parts of the brain lacking a functional blood-brain barrier known for their ability to sense small changes in sodium concentration/osmolality. It includes large vasopressin-producing magnocellular neurons and smaller parvocellular neurons that project to spinal preganglionic sympathetic neurons and premotor sympathetic neurons in the brainstem. The PVN is thus a plausible site for coordination of neurogenic and hormonal actions on the cardiovascular system and the kidney in response to changes in sodium concentration/osmolality.
Hypertension is a common presentation of CRF. The present study was conducted to test the hypothesis that dysregulation of RAS occur in brain of CRF rats, which contribute to the sympathetic overactivity and development of hypertension in CRF, and then we will clarify the relationship between brain RAS and sympathetic activity in CRF rats.
Materials and methods
Preparation of Animal Model
All animal procedures were approved by the Animal Experiment and Care Committee of the Sourthern Medical University. Sixty adult male Sprague-Dawley rats (initial weight,150-180g, Sourthern Medical University Animal Experiment Center) were maintained under shandardized conditions. The animals were subjected to two-step,5/6nephrectomy (5/6Nx, by performing a right nephrectomy with surgical resection of the two-thords of the left kidney) or to sham operation (controls). Ten weeks after the operation, the5/6Nx rats were randomized into subgroups.
Experimental data and specimen collection
1. Blood pressure and urine collection
Three days before the end of the study period,24-hour urine samples were collected for three consecutive days, and the blood pressure was determined in conscious rats by the indirect tail-cuff method also. Systolic blood pressure before and14days after for rats on different stimulate were monitored via a pressure transducer placed in the femoral artery, the change in systolic blood pressure is used as the statistic.
2. Blood collect and brain perfusion
At the completion of each protocol, rats were anesthetized with pentobarbital sodium (40mg/kg, i.p.), Trunk blood was collected in chilled vacuum tubes, and plasma samples were separated and stored at-80℃until assayed. Part of the rat brain tissue were collected for western blot studies, and the others were used for immunohistochemical studies.
Chapter two Affects of salt diet on central renin-angiotensin system of5/6nephrectomy rat model
After14days stimulation, urine and Blood were collected for biochemical indicators test; the brain tissue were used for brain RAS detect by immunohistochemistry or western blot.
Chapter three Cellular localization of brain RAS in5/6Nx rats and the relationship between the RAS and the neuron activation
The rat brain paraffin section from Chapter two were used for double lable brain RAS with NSE or GFAP by immunofluorescence, or for c-fos quantitative by immunohistochemistry.
Chapter four The relationship between brain RAS and blood pressure or SNA in5/6Nx rats
Ten week after5/6Nx, the rats were fed a high-salt (4%) diet, randomized into9groups (n=12in each group) and treated as follows for14days, respectively:①Losartan Omg/kg/d by intragastric gavage (IG):Rats were administrated with daily intragastric injection of endotoxin-free phosphate-buffered saline (PBS)(pH7.4);②Losartan lmg/kg/d IG:Rats were administrated with daily intragastric injection of losartan (lmg/kg per day, Sigma Chemical, St Louis, MO, USA);③Losartan50mg/kg/d IG:Rats were administrated with daily intragastric injection of losartan (50mg/kg per day);④Losartan500mg/kg/d IG:Rats were administrated with daily intragastric injection of losartan (500mg/kg per day);⑤Losartan Omg/kg/d by intracerebroventricular injection (ICV):Rats were administrated with daily intracerebroventricular injection of artificial cerebrospinal fluid (aCSF);⑥Losartan1mg/kg/d ICV:Rats were administrated with daily intracerebroventricular injection of losartan(1mg/kg per day);⑦Clonidine5.76g/kg/d ICV:Rats were administrated with daily intracerebroventricular injection of clonidine (5.76g/kg per day, Sigma Chemical, St Louis, MO, USA);⑨Renal denervation (RDX);⑨Tempol30mg/kg/d IG:Rats were administrated with daily intragastric injection of tempol (30mg/kg per day, Sigma Chemical, St Louis, MO, USA).
After14days stimulation, urine and Blood were collected for biochemical indicators test; the brain tissue were used for brain RAS and TH and c-fos detect by immunohistochemistry or western blot.
Statistical Analyses
All data are presented as mean±SE. Continuous variables between groups were compared using one-way ANOVA, followed by LSD method when P<0.05. Nonparametric test is used when heterogeneity of variance. Statistical analyses were conducted with SPSS13.0for Windows (SPSS, Chicago, IL). Significance was defined as P<0.05.
Results
1. Characteristics of rats post-5/6nephrectomy versus sham-operated rats on10weeks
The base line blood pressure (systolic blood pressure) was determined in conscious rats by the indirect tail-cuff method. Systolic pressure and serum creatinine and24-hour urinary protein excretion were higher in5/6Nx rats versus sham rats, prove5/6Nx rats modeling success.
2. Changes in general characteristics after14days different salt diet stimulation in sham-operated and5/6Nx rats
Blood pressure, serum sodium levels and24h urine protein had no significant difference in sham-operated rats with14days different salt diet (P>0.05). Compared with sham-operated rats,5/6Nx rats had a significantly higher blood pressure with the same normal salt diet (P<0.05). Compared with normal salt5/6Nx group, the blood pressure and serum sodium levels and24h urine protein in high salt5/6Nx group was significantly increased (P<0.05); The low-salt diet can significantly lower the blood pressure of the rats with5/6Nx (P<0.05).
The24h urinary volume and24h urinary sodium excretion were increased with increasing dietary salt (P<0.05), but the blood Ang II has been suppressed with increasing dietary salt in both sham and5/6Nx rats (P<0.05).
3. Changes in systolic blood pressure before and14days after for rats on low, normal and high salt diets
SBP of femoral blood pressure had no significant difference in sham-operated rats with14days different salt diet (P>0.05). Compared with normal salt5/6Nx group, the SBP of femoral blood pressure in high salt5/6Nx group was significantly increased (P<0.05); The low-salt diet can significantly lower the blood pressure of the rats with5/6Nx (P<0.05).
4. Levels of Ang Ⅱ in SFO and PVN after14days different salt diet stimulation in sham-operated and5/6Nx rats
Compared with sham-operated rats,5/6Nx rats had a significantly higher Ang II in SFO and PVN with the same normal salt diet (P<0.05). Compared with normal salt5/6Nx group, the Ang II in SFO and PVN in high salt5/6Nx group was significantly increased (P<0.05); The low-salt diet can significantly lower the Ang Ⅱ in SFO and PVN in sham and5/6Nx rats (P<0.05).
5. Levels of AT1R in SFO and PVN after14days different salt diet stimulation in sham-operated and5/6Nx rats
Compared with sham-operated rats,5/6Nx rats had a significantly higher AT1R in SFO and PVN with the same normal salt diet (P<0.05). Compared with normal salt5/6Nx group, the AT1R in SFO and PVN in high salt5/6Nx group was significantly increased (P<0.05); The low-salt diet can significantly lower the AT1R in SFO and PVN in sham and5/6Nx rats (P<0.05).
6. Cellular localization of Ang Ⅱ or AT1R in SFO and PVN in5/6Nx rats
Ang II or AT1R immunoreactivity is colocalized on neurons within SFO and PVN.
7. Neurons activation in SFO and PVN of5/6Nx rats with different salt diet
Compared with sham-operated rats,5/6Nx rats had a significantly higher c-fos in SFO and PVN with the same normal salt diet (P<0.05). Compared with normal salt5/6Nx group, the c-fos in SFO and PVN in high salt5/6Nx group was significantly increased (P<0.05); The low-salt diet can significantly lower the c-fos in SFO and PVN in sham and5/6Nx rats (P<0.05).
8. General characteristics respond to14days of blockade with various approaches in5/6Nx rats
Systolic blood pressure and urinary albumin excretion markedly decreased in salt-loaded5/6Nx rats with losartan ICV at lmg/kg/d (P<0.05). Losartan IG at higher doses (50-500mg/kg/d), clonidine ICV, renal denervation, or tempol IG also significantly attenuated systolic blood pressure and urinary albumin excretion in high-salt-fed5/6Nx animals (P<0.05). NE level markedly decreased in salt-loaded5/6Nx rats combined with losartan ICV at lmg/kg/d, clonidine ICV or renal denervation (P<0.05). Losartan IG at higher doses (50-500mg/kg/d) and tempol IG also significantly attenuated renal NE levels in high-salt-fed5/6Nx animals (P<0.05).
9. Changes in systolic blood pressure before and14days after losartan or clonidine blockade in sham-operated and5/6Nx rats
Systolic blood pressure markedly decreased in salt-loaded5/6Nx rats with losartan ICV at lmg/kg/d (P<0.05). Losartan IG at higher doses (50-500mg/kg/d), clonidine ICV, renal denervation, or tempol IG also significantly attenuated systolic blood pressure in high-salt-fed5/6Nx animals (P<0.05).
10. Levels of RAS expression in SFO and PVN nucleus after14days of blockade with various approaches in5/6Nx rats
We previously showed that high-salt diet enhance RAS expression in SFO and PVN nucleus in5/6Nx rats. To verify the role of central RAS in the development of hypertension and the relationship between central RAS and SNS, the5/6Nx rats treated with high-salt diet were blockaded by different approaches to central or periphery RAS and SNS. Immunohistochemistry was used to map the expression of Ang II. The operation of renal denervation eliminate the increase of Ang II expression induced by high-salt diet (P<0.05vs5/6Nx rats with IG Omg of losartan), conversely, lmg of losartan given by ICV or500mg losartan given by IG further increased Ang II expression in both SFO and PVN nucleus (P<0.05vs5/6Nx rats with IG Omg of losartan), whereas the losartan (lmg and50mg) or tempol given by IG and clonidine given by ICV had no effect.
Immunohistochemistry was also used to map the expression of ATIR. Changes of AT1R expression response to various blockades are different to Ang II. Low dose of losartan (lmg) given by ICV prominently suppressed the expression of ATIR in SFO and PVN nucleus (P<0.05vs5/6Nx rats with IG Omg of losartan), while the same dose or even much larger dose of losartan (50mg) given by IG didn't have any effect on expression of ATIR, the effect of500mg of losartan given by IG was comparable with that of1mg of losartan given by ICV (P<0.05vs5/6Nx rats with IG Omg of losartan). The operation of renal denervation had restrained the high expression of AT1R in SFO and PVN nucleus (P<0.05vs5/6Nx rats with IG Omg of losartan). Clonidine or tempol had no effect on AT1R expression.
Western blot was used to further determine the ATIR changes, and obtained similar results to immunohistochemistry.The high expression of AT1R in SFO and PVN nucleus induced by high-salt diet were suppressed by ICV1mg of losartan or IG500mg of losartan or renal denervation (P<0.05vs5/6Nx rats with IG Omg of losartan), whereas the losartan (1mg and50mg) or tempol given by IG and clonidine given by ICV had no effect.
11. Levels of tyrosine hydroxylase (TH) expression in SFO and PVN nucleus after14days of blockade with various approaches in5/6Nx rats
To verify the relationship between central RAS and SNS, tyrosine TH levels in SFO and PVN nucleus in5/6Nx rats were detected by western blot. ICV lmg of losartan significantly decreased the expression of TH in SFO and PVN nucleus (P<0.05vs5/6Nx rats with IG Omg of losartan), high dose of losartan (500mg) given by IG or renal denervation had similar effect on TH (P<0.05vs5/6Nx rats with IG Omg of losartan), whereas the losartan (lmg and50mg) or tempol given by IG and clonidine given by ICV had no effect.
12. Levels of c-fos expression in SFO and PVN nucleus after14days of blockade with various approaches in5/6Nx rats
To clarify the effect of various blockades on neurons activation in SFO and PVN of5/6Nx rats treated with high-salt diet, the c-fos was quantified by immunohistochemistry. We found that high level of c-fos was restored by Low dose of losartan (1mg) given by ICV (P<0.05vs5/6Nx rats with IG Omg of losartan), in contrast, the same dose of losartan and even more large of losartan (50mg) given by IG didn't change the c-fos expression in SFO and PVN nucleus, while the large dose of losartan (500mg) had comparable effect with that of lmg of losartan given by ICV (P<0.05vs5/6Nx rats with IG Omg of losartan). Moreover, we found that c-fos was significantly restored by renal denervation (P<0.05vs5/6Nx rats with IG Omg of losartan), but not by clonidine or tempol.
Paper summarizes
1. RAS was activated in brain cardiovascular centre (sympathetic integration site) in5/6Nx rats;
2. Overactive brain RAS may raises blood pressure by stimulating the central sympathetic nervous system in5/6Nx rats;
3. High-salt diet can aggravate kidney injury and further activation brain RAS by afferent nerves from the kidneys, thereby activating central sympathetic nervous system and worsen the hypertension;
4. There exists a positive feedback loop between the kidney and brain cardiovascular centre (sympathetic integration site), which can accelerated the progress of kidney injury.
引文
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