RNA阵列技术检测自发性高血压大鼠甲羟戊酸途径酶的基因表达

目的：探讨甲羟戊酸(MVA)途径的9个酶在不同周龄自发性高血压大鼠(SHR)发病过程中的基因表达变化特点。方法:提取2、4、6、8、10、12不同周龄雄性SHR以及正常血压大鼠(WKY)的心室肌、血管平滑肌、肝脏和肾脏4种组织的总RNA，共294个样品，利用高通量RNA阵列技术(RNA array)检测甲羟戊酸途径的9个酶的基因在不同周龄SHR和WKY大鼠中mRNA表达谱的改变。 结果:（1）SHR大鼠从第6周开始收缩压明显高于WKY(P<0.01)。 （2）12周龄SHR大鼠体内血清胆固醇浓度明显低于WKY大鼠，组织中的胆固醇浓度没有明显差异(P<0.01)。（3）在SHR大鼠心脏、血管、肝脏、肾脏组织中：MVA中间产物的合成酶如法呢醇焦磷酸合成酶(FDS)、异戊烯化焦磷酸化异构酶(IDI)、法呢醇转移酶α亚基(FT1)和β亚基(FT2)的表达明显高于WKY(P<0.01)。（4）SHR肾脏组织中羟甲基戊二酰辅酶A还原酶（HMGR）、甲羟戊酸激酶（MVK）、焦磷酸甲羟戊酸脱羧酶（MVD）、鲨烯合成酶（SQS）和鲨烯环氧化酶（SQ） 表达较为一致，早期(2-4 周)SHR大鼠的基因表达明显高于WKY，随着周龄增加SHR的表达进一步增高，且与WKY相比都有明显差异(P<0.01) 。（5）在心脏、血管和肝脏中，HMGR、MVK、MVD、SQS和SQ的基因表达无明显规律。结论:SHR大鼠随着周龄的增长甲羟戊酸途径中各酶基因表达的改变，以非胆固醇产物类合成酶(如IDI、FDS和FT1、FT2)表达增加为特点，这一改变是否与高血压有关尚待进一步验证。     

自发性高血压大鼠甲状旁腺的异常表现

观察自发性高血压大鼠（ＳＨＲ）的甲状旁腺。发现在其腺体中除大量正常的主细胞外，还具有一种特殊的新型细胞。这些细胞的形态不规则，边界不清。ＨＥ染色后胞浆比主细胞着色深，核的形态不规则且染色更暗。此种细胞在甲状旁腺中成团状分布，与主细胞形成鲜明的对比。另外，经实验证实在ＳＨＲ的血浆中存在一种特殊的高血压因子（ＨＦ），可以使正常大鼠的血压产生延迟性升高，其高峰在４５ｍｉｎ出现，此特点与血中已知的增压物质的即刻效应和延期出现的效应有本质的不同。因此，推论这种具有独特的理化性质和生理功能高血压因子的来源与甲状旁腺有密切关系。     

人类心房利钠肽基因在自发性高血压大鼠体内表达及其对血压的影响

目的研究人类心房利钠肽（hANP）基因在自发性高血压大鼠（SHRs）体内表达、持续时间和对血压的影响。方法 12只8周龄雄性SHRs,随机分为两组,于左腿股四头肌注射pcDNA3.1-hANP质粒的为实验组,在同一部位注射pcDNA3.1空质粒的为对照组,每周测量大鼠尾动脉收缩压,及利用放射免疫方法监测血中hANP水平。结果转基因后第1周起与对照组比较,实验组血压开始下降,两组动物一直相差（13±3.1）mmHg（P〈0.05）,其作用可持续10周;且放射免疫方法监测实验组血中hANP水平较高;RT-PCR和Western印迹杂交技术检测显示实验组hANP基因在肌肉组织中高效表达,对照组则未见表达。结论肌肉注射法将hANP基因导入SHRs,hANP基因可在肌肉组织中高效表达,且hANP可释放入血,降低SHRs的血压,显示了hANP基因对高血压患者治疗的可能性。     

自发性高血压大鼠FXYD5基因表达的时空分布

目的:研究大鼠FXYD5(FXYD domain-containing ion transport regulator 5)基因表达在自发性高血压大鼠（SHR）及正常血压大鼠（Wistar-Kyoto, WKY）的时间与空间分布差异。方法:取4周、8周、13周及21周龄SHR的肾脏、肝脏、肠系膜二、三级动脉分支、大脑和心脏组织，以同周龄WKY作对照，采用Northern blotting方法分析FXYD5mRNA表达丰度。结果:Northern blotting的结果显示：13周、21周龄SHR的肾脏组织FXYD5基因表达丰度较同周龄WKY低；在肺组织，除8周龄该基因的表达水平在SHR组略低于对照组外，其它周龄两组没有差别。结论:FXYD5基因在SHR中有着特异性的组织分布特点，并且在不同周龄的SHR， FXYD5基因在肾脏的表达水平不是恒定不变的，在时间上呈一钟形变化。提示FXYD5基因可能是一个高血压相关的基因，可能与高血压的发病有关。     

Low-Intensity physical activity beneficially alters the ultrastructural renal morphology of spontaneously hypertensive rats

INTRODUCTION AND OBJECTIVE:

Kidney disorders can cause essential hypertension, which can subsequently cause renal disease. High blood pressure is also common among those with chronic kidney disease; moreover, it is a well-known risk factor for a more rapid progression to kidney failure. Because hypertension and kidney function are closely linked, the present study aimed to observe the beneficial effects of low-intensity physical activity on structural and ultrastructural renal morphology and blood pressure in normotensive and spontaneously hypertensive rats.

METHOD:

Male Wistar-Kyoto rats and spontaneously hypertensive rats were randomly allocated into four groups: sedentary or exercised Wistar-Kyoto and sedentary or exercised spontaneously hypertensive rats. The exercise lasted 20 weeks and consisted of treadmill training for 1 hour/day, 5 days/week.

RESULTS:

The exercised, spontaneously hypertensive rats showed a significant blood pressure reduction of 26%. The body masses of the Wistar-Kyoto and spontaneously hypertensive strains were significantly different. There were improvements in some of the renal structures of the animals treated with physical activity: (i) the interdigitations of the proximal and distal convoluted tubules; (ii) the basal membrane of the proximal and distal convoluted tubules; and (iii) in the basal membrane, slit diaphragm and pedicels of the glomerular filtration barrier. The spontaneously hypertensive rats also showed a decreased expression of connexin-43.

CONCLUSION:

Physical exercise could be a therapeutic tool for improving kidney ultrastructure and, consequently, renal function in hypertensive individuals.     

Inhibition of renal sympathetic nerve traffic from cardiac receptors in normotensive and spontaneously hypertensive rats

The reflex inhibition of the sympathetic outflow to the kidney was examined during volume load with horse plasma in 6 normotensive rats (NCR) and 6 spontaneously hypertensive rats (SHR). The rats were anesthetized with chloralose and urethane. The arterial baroreceptors were denervated. The renal nervous inhibition was mediated via the vagal nerves and was mainly due to activation of receptors in the left side of the heart. The average thresholds in mean left atrial pressure for renal nervous inhibition was 5.4 mmHg for NCR and 9.2 mmHg for SHR indicating a clear resetting of the reflex arch in the hypertensive animal: The reason is probably a decreased distensibility of the wall of the left atrium due to a chronic elevation of left atrial pressure. This resetting of the atrial receptors in the hypertensive animals is probably of importance to allow an adequate filling pressure of the hypertrophied left ventricle and might also be of importance for the reflex neural control of renal function in these animals.     

人类心房利钠肽基因在自发性高血压大鼠体内表达及对血压和肾脏的组织学影响

目的研究人类心房利钠肽（hANP）基因在自发性高血压大鼠（SHR）体内表达、持续时间和对血压及肾脏的组织学影响。方法 12只8周龄雄性SHR,随机分为两组,于左腿股四头肌注射pc DNA3.1-h ANP质粒的为实验组,在同一部位注射pc DNA3.1空质粒的为对照组,每周测量大鼠尾动脉收缩压,及利用放射免疫方法监测血中h ANP水平;10周后处死动物,RT-PCR和Western印迹杂交技术检测h ANP基因表达情况;HE染色和Masson染色观察对肾脏的组织形态学的影响。结果转基因后第1周起与对照组比较,实验组血压开始下降,两组动物一直相差（12±3.1）mm Hg（P〈0.05）,其作用可持续10周;且放射免疫方法监测实验组血中hANP水平较高;RT-PCR和Western印迹杂交技术检测显示实验组hANP基因在肌肉组织中高效表达,对照组则未见表达;组织形态学分析结果显示实验组肾脏组织细胞形态基本正常,胶原沉积明显减少。结论肌肉注射法将hANP基因导入SHR,hANP基因可在肌肉组织中高效表达,且hANP可释放如血,降低SHR的血压并对高血压靶器官肾脏的损害起到了治疗及预防作用。显示了hANP基因对高血压患者治疗的可能性。     

自发性高血压大鼠肾脏L-精氨酸/一氧化氮系统与红细胞L-精氨酸转运

目的:研究自发性高血压大鼠(SHR)肾脏(L-精氨酸／一氧化氮)L-Arg／NO系统的改变及其与红细胞L-Arg转运的关系。方法:检测16周龄SHR、卡托普利治疗4周的16周龄SHR(CAP)和16周龄WKY大鼠肾脏L-Arg转运、NOS活性、NO₂^-和cGMP含量,红细胞L-Arg转运。结果: SHR肾脏高低亲和L-Arg转运体的Vmax均低于WKY组(P＜0.01,P＜0.05),Km值则无明显差异。NOS活性、NO₂^-、cGMP含量分别较WKY组低35.4%、36.2%和85.2%(P＜0.05或P＜0.01)。CAP组高亲和L-Arg转运体的Vmax、NOS活性均高于SHR组(+90%,P＜0.01;+58.6%,P＜0.05)。NOS活性与高亲和L-Arg转运体的Vmax呈正相关,r=0.585,P＜0.05。红细胞L-Arg转运的改 变与肾脏相似, SHR组的Vmax低于WKY组(－30%,P＜0.01),CAP组高于SHR组(+26.5%,P＜0.01),Km值组间比较无明显差异。红细胞L-Arg转运的Vmax与肾脏高或低亲和L-Arg转运体的Vmax均呈正相关,r=0.8434,P＜0.01(高亲和)和r=0.5255,P＜0.05(低亲和)。 结论:SHR肾脏L-Arg/NO系统活动抑制,卡托普利治疗明显解除此抑制状态。肾脏L-Arg转运的改变与红细胞L-Arg转运的改变基本一致。     

Influence of neonatal sympathectomy on proximal renal resistance artery function in spontaneously hypertensive rats

Renal transplantation experiments have shown that the kidney contributes to chronic sympathectomy-induced arterial pressure reduction in spontaneously hypertensive rats (SHR). The underlying mechanisms are currently unclear but may include alterations in the function of small renal arteries. Neonatal SHR were sympathectomized by intraperitoneal guanethidine injections and removal of adrenal medullary tissue. Controls were sham- or hydralazine-treated. At 12 weeks of age, distal interlobar artery segments were investigated using small-vessel wire myography. Vessels from sympathectomized animals showed increased sensitivity to noradrenaline (NE). Vasopressin- and endothelin-1-induced vasoconstriction was similar in all groups (as reflected by the pD₂, i.e. –logEC₅₀, where EC₅₀ is the molar concentration of agonist eliciting a half-maximal response). Maximum vasopressin-induced tension was similar in all groups while endothelin-1-induced maximum tension was significantly higher in sympathectomized than in sham-treated SHR. The sensitivity of NE-induced vasoconstriction to extracellular Ca²⁺ did not differ between groups while sensitivity to L-type Ca²⁺ channel activation was significantly higher in both sympathectomized and hydralazine-treated animals than in sham-treated animals. Endothelium-dependent and independent vasodilation were similar in all groups. Sequential blockade of NO-synthase and cyclooxygenase had similar effects in all groups. In conclusion, neonatal sympathectomy does not induce any changes in the function of isolated proximal renal resistance arteries from SHR that could explain the blood pressure lowering effect of a kidney graft from sympathectomized SHR.     

人类心房利钠肽基因在自发性高血压大鼠体内表达及其对血压、心脏、血管的影响

目的研究人类心房利钠肽（hANP）基因在自发性高血压大鼠（SHR）体内表达、持续时间和对血压及心脏、血管的影响。方法12只8周龄雄性SHR,随机分为两组,于左腿股四头肌注射pcDNA3．1-hANP质粒的为实验组,在同一部位注射pcDNA3．1空质粒的为对照组,每周测量大鼠尾动脉收缩压,及利用放射免疫方法监测血中hANP水平;10周后处死动物。RT—PCR和Western印迹杂交技术检测hANP基因表达情况;分别称量体重、全心脏和左心室重量,大体评价对心肌重构的影响;苏木素-伊红（HE）染色和胶原组织学（VG）染色观察对心脏、血管组织形态学的影响。结果转基因后第1周起与对照组比较,实验组血压开始下降,两组动物一直相差（12±3．1）mmHg（P〈0．05）,其作用可持续10周;且放射免疫方法监测实验组血中hANP水平较高;RT—PCR和Western印迹杂交技术检测显示实验组hANP基因在肌肉组织中高效表达,对照组则未见表达;实验组心脏重量／体重比和左心室重量／心脏重量比明显低于对照组。组织形态学分析结果显示实验组心肌、血管组织细胞形态基本正常,胶原沉积明显减少。结论肌肉注射法将hANP基因导入SHR,hANP基因可在肌肉组织中高效表达,且hANP可释放如血,降低SHR的血压并对心脏、血管高血压靶器官的损害起到了预防作用。显示了hANP基因对高血压患者治疗的可能性。     

Induction of cardiac angiotensin I-converting enzyme with dietary NaCl-loading in genetically hypertensive and normotensive rats

We have recently shown that the angiotensin I converting enzyme (ACE) gene is linked to NaCl-loaded blood pressure in the stroke-prone spontaneously hypertensive rat (SHRSP), and that high-NaCl loading selectively stimulates ACE in the aorta of SHRSP but not in normotensive Wistar-Kyoto (WKY) rats. We therefore investigated the relationship between cardiac ACE and the development of hypertension and left ventricular hypertrophy in response to normal- and high-NaCl diet in these rats. ACE mRNA and ACE activity were measured in left ventricular tissue after completion of hemodynamic characterization of the animals. While SHRSP rats increased blood pressure (P<0.0001) and heart rate (P<0.005) in response to high NaCl, blood pressure remained unchanged in WKY. Similarly, relative left ventricular weight increased only in SHRSP after high NaCl (P<0.002). A significant two- to threefold increase of cardiac ACE mRNA and fourfold stimulation of ACE enzyme activity in response to high NaCl was found in both WKY and SHRSP rats (P<0.005). The induction of ACE gene expression was significantly more pronounced in SHRSP compared to WKY (P<0.02), whereas no significant strain differences in left ventricular ACE activity were found after either normal- or high-NaCl diet. Thus, arterial blood pressure and left ventricular weight remained unchanged in the WKY rats despite the activation of left ventricular ACE activity after high-NaCl exposure. These results demonstrate that left ventricular ACE activity is equally upregulated in response to high-NaCl in the normotensive and hypertensive strain, independently from the development of hypertension. We conclude that the pretranslational induction of left ventricular ACE with high-NaCl loading may be important both for the regulation of cardiac angiotensins and kinins and for local therapeutic ACE inhibition in the heart during high-salt status.Abbreviations ACE Angiotensin I-converting enzyme - Ang Angiotensin - LVH Left ventricular hypertrophy - PCR Polymerase chain reaction - SHRSP Stroke-prone spontaneously hypertensive rat - WKY Wistar-Kyoto     

Altered cellular kinetics in the growth plate of the femoral head of spontaneously hypertensive rats

Purpose

Pathologic changes in the growth plate remain unknown in Legg-Calvé-Perthes (LCP) disease. Spontaneously hypertensive rats have proven to be a good model for studying LCP disease. This study investigated the histopathologic changes and the expression of vascular endothelial growth factor in the growth plate of spontaneously hypertensive rats (SHR).

Materials and Methods

Sixty SHR rats were divided into two groups: those showing osteonecrosis (SHR+n group: 32), and those showing normal ossification (SHR-n group: 28). Thirty Wister Kyoto rats served as a control. For histomorphological measurement, the length of each zone of the growth plate was measured. Cell kinetics was measured by 5-bromo-2''-deoxyuridin (BrdU) immunohistochemistry and transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assays. Vascular endothelial growth factor (VEGF) immunohistochemistry was used to identify of expression of VEGF.

Results

The lengths of growth plates of the SHR+n group were significantly shorter in the initial growth period than those of the other groups. The lowest proliferative rate and the highest apoptosis rate were observed in the SHR+n group at the initial growth period. The expression of VEGF in the growth plate of the SHR group was lower than the control group, and it was lower in the SHR+n group than in the SHR-n group.

Conclusion

The growth plate of the SHR+n group was found to be affected by disease process of ischemic necrosis of the femoral head, and this might explain the relative overgrowth of the greater trochanter in the later stages of LCP disease.     

Alterations in tissue concentration and gene expression of adrenomedullin in spontaneously hypertensive rats

In order to elucidate the role of adrenomedullin in hypertension, we have compared concentrations of immunoreactive rat adrenomedullin and adrenomedullin messenger RNA levels in tissues of 8-week-old spontaneously hypertensive rats (SHR) with those of age-matched Wistar-Kyoto rats (WKY). The adrenomedullin immunoreactivity concentrations in adrenal gland and cardiac atrium were significantly higher in SHR than in WKY. The adrenomedullin content of cardiac ventricle was also significantly higher in SHR than in WKY. The rat adrenomedullin messenger RNA levels in adrenal gland and heart of SHR were also higher than those of WKY. These results suggest that adrenomedullin participates in the mechanism to counteract the blood pressure elevation in SHR.     

Changes in brainstem and spinal adrenoceptor binding with ageing in spontaneously hypertensive and Wistar-Kyoto rats

Adrenoceptor binding in membranes prepared from the brainstem and thoracic spinal cord of male spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats aged 6 and 36-40 weeks has been compared. Binding of [3H]prazosin (alpha 1-receptors), [3H]rauwolscine (alpha 2-receptors) and [3H]dihydroalprenolol (DHA: beta-receptors) fell with age in both regions in both strains. Strain, independent of age, was also a significant determinant of binding for all three ligands in brainstem and for [3H]DHA in spinal cord. The changes in receptor binding may reflect differences in noradrenergic activity with age and between SHR and WKY rats.     

ACE2在高血压大鼠左心室重构中的作用及缬沙坦干预的作用

目的观察血管紧张素转换酶2(ACE2)在自发性高血压大鼠(SHR)左心室中的表达以及缬沙坦干预后对ACE2表达水平的影响。方法 24只12周龄雄性SHR随机分为SHR组、缬沙坦组,12只同龄雄性血压正常的Wistar大鼠作为正常对照组。10周后处死,测定心脏重量指数(HWI)、左心室重量指数(LVWI);酶联免疫法测定血浆中AngⅡ的浓度;碱水解法测定心肌中羟脯氨酸的含量;反转录-聚合酶链反应检测心肌中ACE2的表达。结果与正常对照组比较,SHR组LVWI、血浆中AngⅡ的浓度以及心肌羟脯氨酸的含量均增高(P<0.05),心肌组织中ACE2的表达显著降低(P<0.05);与SHR组比较,缬沙坦组LVWI、血浆中AngⅡ的浓度以及心肌羟脯氨酸的含量均降低(P<0.05),心肌组织中ACE2表达显著增高(P<0.05)。结论缬沙坦可逆转高血压左心室重构,机制可能与增加心肌中ACE2的表达有关。     

自发性高血压大鼠心肌组织microRNA-133a与TGF-β1蛋白表达的改变及意义

 目的：观察microRNA-133a（miR-133a）与转化生长因子-β1（transforming growth factor β1,TGF-β1）蛋白在自发性高血压大鼠（spontaneously hypertensive rats,SHR）心肌组织中的表达改变和关系。方法：取12只18周龄雄性自发性高血压大鼠为SHR组,12只18周龄雄性Wistar-Kyoto （WKY）大鼠为对照组,通过无创血压测量分析系统测大鼠尾动脉血压,Masson染色检测心肌胶原容积分数（collagen volume fraction,CVF）和血管周围胶原面积比率（perivascular collagen area ratio, PVCA）,实时荧光定量PCR检测miR-133a表达水平,免疫组化和Western blotting法检测心肌TGF-β1蛋白表达。结果：与对照组比较,SHR组的收缩压和舒张压明显升高（P<0.01）,心肌CVF和PVCA明显升高（P<0.01）,TGF-β1蛋白表达水平明显升高（P＜0.01）,miR-133a表达水平明显降低（P<0.01）,SHR组心肌miR-133a表达水平为对照组的（23.9±4.6）%;SHR组心肌组织miR-133a与TGF-β1蛋白表达水平呈负相关（r=-0.791, P<0.01）。结论：SHR心肌组织miR-133a表达下调,伴随TGF-β1蛋白表达升高和胶原合成增加。miR-133a与TGF-β1可能参与SHR大鼠的心肌纤维化。