雷米普利对慢性心力衰竭大鼠心肌组织中盐皮质激素受体的影响

目的探讨雷米普利对慢性心力衰竭大鼠心肌组织中盐皮质激素受体(MR)表达及心肌纤维化的影响。方法选择SD大鼠39只,选用慢性心力衰竭造模成功SD大鼠26只,随机分为慢性心力衰竭组(心衰组,13只)、雷米普利治疗组(治疗组,13只),另设假手术大鼠为对照组(13只)。雷米普利治疗4周时,用心肌Masson染色法测定大鼠心肌胶原密度,放射性免疫法测定大鼠心肌组织中血管紧张素Ⅱ(AngⅡ)、醛固酮(ALD)含量,实时荧光定量PCR、免疫印迹检测心肌MR表达。结果与对照组比较,心衰组大鼠心肌组织中AngⅡ、ALD显著升高,MR mRNA和蛋白表达显著上调,心肌胶原密度显著增加(P<0.01);与心衰组比较,治疗组大鼠心肌组织中AngⅡ、ALD显著降低.MR mRNA和蛋白表达显著下调,心肌胶原密度显著降低(P<0.01)。结论雷米普利不仅降低慢性心力衰竭大鼠心肌组织中AngⅡ、ALD含量,还可能通过下调心肌MR的表达来抑制心肌纤维化。     

Aldosterone, mineralocorticoid receptor, and heart failure

Several large clinical studies have demonstrated the important benefit of mineralocorticoid receptor (MR) antagonists in patients with heart failure, left ventricular dysfunction after myocardial infarction, hypertension or diabetic nephropathy. Aldosterone adjusts the hydro-mineral balance in the body, and thus participates decisively to the control of blood pressure. This traditional view of the action of aldosterone restricted to sodium reabsorption in epithelial tissues must be revisited. Clinical and experimental studies indicated that chronic activation of the MR in target tissues induces structural and functional changes in the heart, kidneys and blood vessels. These deleterious effects include cardiac and renal fibrosis, inflammation and vascular remodeling. It is important to underscore that these effects are due to elevated MR activation that is inadequate for the body salt requirements.Aldosterone is generally considered as the main ligand of MR. However, this is a matter of debate especially in heart. Complexity arises from the glucocorticoids with circulating concentrations much higher than those of aldosterone, and the fact that the MR has a high affinity for 11β-hydroxyglucocorticoids. Nevertheless, the beneficial effects of MR inhibition in patients with heart failure emphasize the importance of this receptor in cardiovascular tissue. Diverse experimental models and strains of transgenic mice have allowed to dissect the effects of aldosterone and the MR in the heart. Taken together experimental and clinical data clearly highlight the deleterious cardiovascular effects of MR stimulation.     

Adrenocorticotrophin-induced hypertension: effects of mineralocorticoid and glucocorticoid receptor antagonism

OBJECTIVE: To examine whether the increase of blood pressure in adrenocorticotrophin-treated rats is mediated through mineralocorticoid or glucocorticoid receptors or corticosterone 6 beta-hydroxylation inhibition. DESIGN: Rats were randomly allocated to 14 treatment groups for 10 days. The treatments included sham injection (n = 35), adrenocorticotrophin (5, 100, 500 micrograms/kg per day, subcutaneously, n = 5, 15 and 15, respectively), spironolactone (100 mg/kg per day, subcutaneously, n = 15), standard-dose or high-dose RU 486 (70 mg/kg every 3 days or 70 mg/kg per day, subcutaneously, n = 5 and 10, respectively), spironolactone + adrenocorticotrophin (100 micrograms/kg per day, n = 5, or 500 micrograms/kg per day, n = 10), standard-dose RU 486 + adrenocorticotrophin (500 micrograms/kg per day, n = 5), high-dose RU 486 + adrenocorticotrophin (100 micrograms/kg per day, n = 10), troleandomycin (40 mg/kg per day, subcutaneously, n = 5) and troleandomycin + adrenocorticotrophin (5 micrograms/kg per day, n = 5). Systolic blood pressure and metabolic parameters were measured every second day. RESULTS: Adrenocorticotrophin treatment increased systolic blood pressure dose-dependently (5 micrograms/kg per day: +14 +/- 2 mmHg; 100 micrograms/kg per day: +20 +/- 2 mmHg; 500 micrograms/kg per day: +28 +/- 2 mmHg, all P < 0.001). Adrenocorticotrophin at 100 and 500 micrograms/kg per day increased plasma sodium and decreased plasma potassium concentrations. Spironolactone did not block adrenocorticotrophin-induced systolic blood pressure changes but did block changes in plasma sodium and potassium levels. Standard-dose RU 486 did not modify the adrenocorticotrophin-induced (500 micrograms/kg per day) systolic blood pressure rise but blocked the effect of adrenocorticotrophin on body weight. High-dose RU 486 partially blocked the adrenocorticotrophin-induced (100 micrograms/kg per day) systolic blood pressure increase (adrenocorticotrophin at 100 micrograms/kg per day: 143 +/- 3 mmHg; high-dose RU 486 + adrenocorticotrophin at 100 micrograms/kg per day: 128 +/- 5 mmHg, P < 0.001) and body-weight loss. Troleandomycin did not alter the development of adrenocorticotrophin-induced hypertension. CONCLUSIONS: Spironolactone and standard-dose RU 486 did not modify adrenocorticotrophin-induced hypertension despite demonstrable antimineralocorticoid and antiglucocorticoid actions. High-dose RU 486 partially blocked adrenocorticotrophin-induced (100 micrograms/kg per day) hypertension, suggesting either a permissive effect of glucocorticoid on blood pressure or other antihypertensive actions of RU 486. Inhibition of glucocorticoid 6 beta-hydroxylation by troleandomycin did not modify adrenocorticotrophin-induced hypertension, suggesting that effects of corticosterone 6 beta-hydroxylation in adrenocorticotrophin-induced hypertension are negligible.     

ACE inhibitors and mineralocorticoid receptor blockade in patients with congestive heart failure

The two major outcome trials on the combination of angiotensin-converting enzyme (ACE) inhibitors and mineralocorticoid receptor (MR) antagonists in heart failure are RALES (Randomized Aldactone Evaluation Study) and EPHESUS (Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study). There have also been studies in essential hypertension, and in diabetic hypertensive patients, on the cardiac and renal effects of ACE inhibitors and MR antagonists, individually and in combination. In the clinical studies on heart failure, in outcome trials and the smaller studies using surrogate end points, a combination of ACE inhibition and MR blockade is superior to ACE inhibition alone, and in the hypertension studies to either agent alone. Some insight into their distinct sites of protective action may be gained from studies on experimental animal preparations. The principal caveat in the use of combination therapy is the possibility of hyperkalemia, which should be minimal in patients with creatine clearance greater than 30 mL/min and with the low doses of MR antagonist shown to be effective in outcome trials.     

醛固酮合成酶和盐皮质激素受体基因在充血性心力衰竭心肌的表达

目的　研究醛固酮合成酶 (Aldosterone synthase,CYP11B2 )及盐皮质激素受体 (Mineralocorticoid receptor,MR)基因在心功能不全心肌中的表达。方法　选取 2 5例行瓣膜置换术的慢性心力衰竭 (CHF)患者左心室乳头肌组织 10 0 mg,同时取 6例健康心肌组织作为对照 ,提取组织总 RNA,并逆转录为 c DNA,以特定的寡核苷酸为引物进行聚合酶链反应 ,β- actin作为内参照 ,分别观察 CYP11B2及 MR在不同心功能状态心肌中的表达。结果　在 6例正常心肌组织中 CYP11B2基因的表达为阴性 ,2 5例 CHF患者中有 10例表达阳性 ,分别为心功能 级～ 级 2例 , 级～ 级 8例 ;轻度心力衰竭患者 (心功能 ～ 级 )与重度心力衰竭患者 (心功能 ～ 级 )表达阳性率分别为 15 .4 %和 6 6 .7% ,差异有显著性 (P=0 .0 15 )。正常心肌、心功能 ～ 级和心功能 ～ 级心肌 MR/ β- actin分别为 1.2 8± 0 .13,0 .92± 0 .12 ,0 .80± 0 .2 1,与正常对照组相比 ,CHF时 MR基因表达明显下降 (P<0 .0 0 1) ,而心功能 ～ 级与心功能 ～ 级之间 ,无显著性差异 (P=0 .0 83)。慢性心力衰竭患者中 ,CYP11B2基因表达阳性与阴性组之间 MR/ β- actin表达分别为 0 .78± 0 .2 0和 0 .94± 0 .13,二者间具有显著性差异 (P=0 .0 18)。CYP11B2     

The role of mineralocorticoid receptor antagonists (MRAs) in very old patients with heart failure

Mineralocorticoid receptor antagonists (MRAs) have been effective in reducing total mortality in patients with heart failure (HF) and a reduced left ventricular ejection fraction. Due to the finding that aldosterone levels decrease with age, one might question the effectiveness of MRAs in very old patients (≥80?years of age), those at the greatest risk for developing HF with a preserved left ventricular ejection fraction (PEF). However, while aldosterone levels decrease with age, there is also a decrease in the enzyme 11 beta HSD2 levels with age, thereby allowing cortisol to stimulate the mineralocorticoid receptor (MR), which in younger patients with higher levels of 11 beta HSD 2 levels is converted to cortisone which cannot activate the MR. There is also an increase in the expression of the MR in the vascular wall with age. Thus, there is reason to believe that MRAs might be effective in reducing cardiovascular mortality and the incidence of hospitalizations for HF in very old patients with HFPEF. There is also reason to believe that MRAs might favorably affect many of the comorbid conditions associated with HFPEF in very old patients. The safety and efficacy of this hypothesis is currently under investigation in the NHLBI sponsored TOPCAT trial.     

Antiproteinuric effects of mineralocorticoid receptor blockade in patients with chronic renal disease

We have recently shown that mineralocorticoid receptor blockade may represent optimal therapy for patients with early diabetic nephropathy who show aldosterone breakthrough during angiotensin-converting enzyme (ACE) inhibitor treatment, and who no longer show the maximal antiproteinuric effects of ACE inhibition. In this study, we explored the effects of the mineralocorticoid receptor antagonist spironolactone on urinary protein excretion in patients with chronic renal disease with proteinuria persistently more than 0.5 g/d, despite maintained blood pressure (BP) control, and including the use of an ACE inhibitor (trandolapril) for at least 10 months. After a 12-week study period of spironolactone treatment (25 mg/d), BP did not change but urinary protein excretion was significantly reduced. The extent of the reduction was on average significantly greater in diabetic patients than in nondiabetics. In patients with diabetic nephropathy, although urinary type IV collagen did not decrease after conventional treatment, it was significantly reduced by spironolactone. None of the patients developed serious hyperkalemia, and no other adverse events were observed. All patients in this study had relatively well preserved renal function. In conclusion, the present study demonstrates that in patients with chronic renal disease with proteinuria persistently more than 0.5 g/d, despite BP control and the use of an ACE inhibitor, adding spironolactone to the conventional treatment produces beneficial effects on urinary protein excretion, particularly in patients with diabetes. Our study suggests that attenuation of mineralocorticoid receptor-mediated effects may become a new goal for patients who escape the antiproteinuric effects of the conventional treatment. Additional, larger, prospective, randomized double-blind studies will be needed for general acceptance of this strategy.     

Protective effects of mineralocorticoid receptor blockade against neuropathy in experimental diabetic rats

Aims: Mineralocorticoid receptor (MR) blockade is an effective treatment for hypertension and diabetic nephropathy. There are no data on the effects of MR blockade on diabetic peripheral neuropathy (DPN). The aim of this study was to determine whether MRs are present in the peripheral nerves and to investigate the effectiveness of MR blockade on DPN in streptozotocin (STZ)‐induced diabetic rats. Methods: Expression of MR protein and messenger RNA (mRNA) was examined in the peripheral nerves using Western blot analysis and RT‐PCR. We next studied the effects of the selective MR antagonist eplerenone and the angiotensin II receptor blocker candesartan on motor and sensory nerve conduction velocity (NCV), morphometric changes and cyclooxygenase‐2 (COX‐2) gene and NF‐κB protein expression in the peripheral nerves of STZ‐induced diabetic rats. Results: Expression of MR protein and mRNA in peripheral nerves was equal to that in the kidney. Motor NCV was significantly improved by 8 weeks of treatment with either eplerenone (39.1 ± 1.2 m/s) or candesartan (46.4 ± 6.8 m/s) compared with control diabetic rats (33.7 ± 2.0 m/s) (p < 0.05). Sensory NCV was also improved by treatment with candesartan or eplerenone in diabetic rats. Eplerenone and candesartan caused significant improvement in mean myelin fibre area and mean myelin area compared with control diabetic rats (p < 0.05). COX‐2 mRNA and NF‐κB protein were significantly elevated in the peripheral nerves of diabetic rats compared with control rats, and treatment with eplerenone or candesartan reduced these changes in gene expression (p < 0.05). Conclusion: MR blockade may have neuroprotective effects on DPN.     

大剂量糖皮质激素上调血管内皮细胞MR表达的意义

目的 观察UPS和Dex对人脐静脉内皮细胞盐皮质激素受体(MR)的表达情况,以探讨糖皮质激素作用的新机制.方法 利用内毒素脂多糖LPS“致伤”血管内皮细胞,用地塞米松进行“治疗”,然后通过RT-PCR和免疫组化的方法检测血管内皮细胞盐皮质激素受体(MR)的表达情况.结果 内皮细胞本身能明显表达MRmRNA,运用不同浓度Dex和100 ng/mlLPS刺激后,可使人脐静脉内皮细胞内MRmRNA表达明显下降,应用大剂量(10-6mol/L)的Dex刺激细胞3h,MRmRNA的表达无明显变化,6h开始有升高,12 h达高峰,24 h回升近刺激前水平.免疫组化的结果与RT-PCR一致.结论 大剂量的DEX( 10-6mol/l)可上调MR的表达,GC引起GR和MR表达的不同变化可能是机体对损伤应激的一种调节机制,表明糖皮质激素作用存在新的机制.     

Interdomain interactions in the mineralocorticoid receptor

The potential for interaction between the N-terminal domain and the C-terminal region (hinge and ligand-binding domain) of the mineralocorticoid receptor (MR) was examined using the mammalian-2-hybrid assay. The MR C-terminal region was fused to the GAL4 DNA-binding domain (GAL4-MRC). To examine if the AF-2 is involved in the interaction, as has been reported for other steroid hormone receptors, it was inactivated by point mutation (E962A). The N-terminal domain was fused to the VP16 transactivation domain (VP16-MRNT). In the mammalian-2-hybrid assay both GAL4-MRC and GAL4-MRC(E962A) interact with VP16-MRNT in an aldosterone-dependent manner. The GAL4-MRC(E962A) construct was used in subsequent experiments to examine the AF-2-independent N/C-interaction. The MR antagonist spironolactone inhibits the aldosterone-mediated association of the two domains. GAL4-MRC(E962A) interacts weakly with the GR or AR N-terminal domains in the presence of aldosterone. No dimerization between GAL4-MRC(E962A) and VP16-MRC is observed. Interestingly, cortisol produces a much weaker N/C-interaction than aldosterone, and it is possible that the N/C-interaction may contribute to observed functional differences in the MR bound to the two ligands.     

心力衰竭时中枢盐皮质激素受体通过促炎因子发挥交感兴奋作用

目的探讨RU28318阻断中枢盐皮质激素受体（MR）的作用,验证慢性充血性心力衰竭时,中枢内MR的交感兴奋作用是通过提高外周血促炎因子（PIC）实现的。方法采用结扎冠状动脉左前降支致心肌梗死的方法制备心衰大鼠模型,假手术对照只穿线不结扎。大鼠分两部分处理：一部分给药组大鼠6周内每天口服MR阻滞剂奥孕酸钾（RU28318）,每天30mg／kg,溶于饮用水中,对照组给普通饮用水;另一部分使用抗PIG药物己酮可可碱（pentoxifylline,fyIx）,每天30mg／kg,溶人饮用水中,以确定单纯促炎因子减少能否足以引起RU28318所致的结果。6周后,保证各组至少有12只动物存活。测定各组大鼠血流动力学指标,评价左心功能;电生理记录肾交感神经放电（RSNA）;ELISA技术测定血浆去甲肾上腺素（NE）,PIC包括肿瘤坏死因子-α（TNF-α）、白介素-α（IL-1α）含量,以及脑组织TNF—d和脑脊液中前列腺素E2（PGE2）水平。通过免疫组织化学染色和Westernblot评价环氧酶-2（cox-2）的表达。结果对心衰大鼠外周使用抑制PIC合成的药物PTX产生的各项结果和用RU28318处理的心衰大鼠很相似：治疗后的心衰大鼠右室体重比（RV／BW）降低,左室舒张末压（LVEDP）、肺体重比（1ung／BW）下降,说明右室重构和肺淤血都有所减轻;但心功能没有明显改善（左室最大上升／下降速率LV＋dp／dtmax变化没有统计学意义）;血浆PIC中TNF—d、IL-1和IL-6水平均降低;肾交感神经放电减弱,同时血中NE减少;下丘脑室旁核（PVN）COX-2染色减弱,蛋白含量降低;同时脑脊液中PGE2减少。结论慢性心衰时,利用RU28318阻断MR产生的交感活动抑制是通过降低血中促弗I天l千耗椎阳的．