腺苷1型受体在慢性心肾综合征水钠潴留中作用机制初探

发布时间：2018-06-05 04:36

本文选题：心肾综合征 + DOCA-Salt小鼠　；参考：《北京协和医学院》2015年博士论文

【摘要】：一、研究背景：心肾综合征(Cardiorenal Syndrome, CRS)是同时累及心脏和肾脏的一大组疾病的统称,包括心脏(或肾脏)的急性或慢性功能不全导致肾脏(或心脏)的急性或慢性功能不全,或者共同因素同时导致心脏和肾脏的结构和功能损害。心功能和肾功能之间有着紧密联系,心肾同时受累,大大增加了疾病的诊治难度和死亡率1,2。根据2010年ADKI (Acute Dialysis Quality Initiative)共识,心肾综合征共分为5型3。CRS的病理生理机制尚未完全阐明,各型CRS在病生理机制上存在共同特征,可分为血流动力学机制与非血流动力学机制4。其中血流动力学机制的核心是水钠潴留和容量负荷增加,而非血流动力学机制包括肾素-血管紧张素-醛固酮系统(Renin-Angiotensin-Aldosterone System, RAAS)和交感神经系统激活、氧化应激、炎症反应等。目前CRS动物模型多采用心肾手术构建、以急性变化为主,没有很好的模拟病理生理机制的慢性CRS动物模型,限制了对其病生理机制的探究。高醛固酮高盐饮食(DOCA-Salt)小鼠模型模拟了盐敏感高血压的病生理状态,前期我们观察到肾脏结构和功能的损害。本研究试图探索该模型是否同时存在心脏损害,从而为探究CRS机制提供平台。腺苷1型受体(Adenosine A1 Receptor, A1AR)通过调节肾脏管球反馈、促进心脏释放心房利钠肽(Atrial Natriuretic Peptide, ANP)调控水盐负荷；在非血流动力学机制方面,A1AR对RAAS起到直接抑制作用、对炎症和凋亡起到调节作用。但目前没有研究系统地探索在DOCA-Salt小鼠模型上A1AR对于心肾损害的意义。前期我们观察到A1AR主要通过血流动力学因素保护DOCA-Salt小鼠肾脏结构和功能,本研究试图通过血流动力学机制和非血流动力学机制两方面探索A1AR对DOCA-Salt小鼠心脏损害是否同样具有保护作用。二、研究目的：1.观察DOCA-Salt鼠心脏损害,探索DOCA-Salt模型作为慢性CRS模型的可能性；2.探索A1AR在DOCA-Salt小鼠水钠潴留中的作用；3.探索A1AR在DOCA-Salt小鼠心肌肥大、心肌组织微血管密度、炎症和纤维化中的作用。三、研究方法及结果：1. DOCA-Salt小鼠心脏损害在C57BL/6J野生型小鼠上,行左肾切除术,术中于左肾窝埋置醋酸脱氧皮质酮(Deoxycorticosterone Acetate, DOCA)缓释药片,术后予高盐饮食,建立DOCA-Salt小鼠模型。采用尾套法测量血压、心率,观察到DOCA-Salt小鼠心率随实验周数显著下降(0周685.8±±25.4 vs.8周526.6±103.9次/分,P0.01),平均动脉压显著上升(0周74.6±4.4 vs.8周88.5±6.5 mmHg, P0.01)。分别于第4周和第8周处死小鼠,并留取心脏标本,称量全心重量,观察到第8周时心脏重量指数显著增加(7.05±0.83 vs.5.16±0.20mg/g, P=0.007)。常规HE染色观察心肌组织镜下改变并测量左心室厚度无显著变化。Van Gieson胶原染色观察心肌组织纤维化程度,发现从第4周起出现局灶性间质纤维化；运用Realtime PCR方法分析转化生长因子-β1 (Transforming Growth Factor-β1, TGF-β1)、胶原纤维1 (Collagenl)在实验组和对照组间无差异。CD34免疫组化染色观察并分析心肌微血管密度,发现第4周时微血管密度显著增加([1.01±0.003]%vs.[0.59±0.001]%,P=0.030),第8周时微血管密度较对照组无显著变化。CD68免疫组化染色观察并分析巨噬细胞浸润程度,发现DOCA-Salt小鼠巨噬细胞浸润程度与对照组无差异；运用Realtime PCR方法分析趋化因子2(Chemokine C-C motif ligand 2, CCL2) mRNA表达水平也无差异。2. DOCA-Salt小鼠水盐负荷在第0、4、8周用代谢笼留24h尿,观察到DOCA-Salt小鼠24h尿量(0周1173±464 vs.4周3606±2359 vs.8周7635±3017μl,两两比较P0.05)及24h尿钠排出量(0周133.3±39.5 vs.4周798.4±719.4 vs.8周1788±854.0μmol/d,两两比较P0.05)随实验周数显著增加,尿渗透压(0周1801±642 vs.4周910±213mOsm/kg H2O,P0.05;0周1801±642 vs.8周860±233mOsm/kg H2O, P0.05)显著下降。Realtime PCR方法观察到心肌组织Corin和ANP mRNA表达在第4周显著增加,分别为31.9倍(P=0.005)和180.7倍(P=0.030),第8周时与对照组无差异。Realtime PCR方法观察到DOCA-Salt小鼠心肌组织腺苷合成酶CD73 mRNA表达从第4周起显著上调(4周18.79倍,P=0.001；8周8.28倍,P=0.036), A1AR mRNA第8周开始出现显著上调(12.12倍,P=0.013),但Western Blot方法没有观察到A1AR蛋白表达水平的显著变化。3.A1AR在DOCA-Salt小鼠心脏损害中的作用建立A1AR-/- DOCA-Salt小鼠模型,观察到A1AR-/- DOCA-Salt小鼠心率随实验周数下降(4周636±64.2 vs.0周696.7±35.2,P0.05),平均动脉压无显著变化。24h尿量相对于野生型实验组显著增加(7003±3742 vs.3606±2359μl, P0.05),24h尿钠排出量较对照增加(:1246.5±859.8 vs.146.6±71.2μmol/d, P0.05),尿渗透压较对照下降(702±165 vs.1394±397 mOsm/kg, P0.05)。心肌组织ANPmRNA表达显著上调(12.65倍,P=0.026),Corin mRNA表达在实验组与对照组间无差异。A1AR-/- DOCA-Salt小鼠第4周心脏重量指数及左心室厚度无显著变化；心肌组织微血管密度无显著升高；心肌巨噬细胞浸润程度、CCL2 mRNA表达水平在实验组与对照组间无差异；仅1只(共6只)小鼠心脏出现局灶性间质纤维化,TGF-β1和Collagenl mRNA表达水平在实验组与对照组间无差异。四、研究结论：本实验条件下1. DOCA-Salt小鼠血压升高,心率下降,早期(4周)心脏微血管密度显著增加、出现局灶性间质纤维化,晚期(8周)心脏重量指数显著增加。结合前期研究观察到肾脏损害,DOCA-Salt小鼠存在心肾共同损害,可作为慢性CRS模型。2. DOCA-Salt小鼠血压升高,提示存在水钠潴留,其水盐负荷受到相关因素调节：心脏CD73-A1AR和Corin-ANP通路激活,ANP限制水和钠的重吸收；24h尿量和24h尿钠排出量显著增加；前期研究观察到肾脏CD73-A1AR通路激活,提示管球反馈持续激活。A1AR-/- DOCA-Salt小鼠管球反馈丧失,24h尿量显著增加,血压较基线和对照组均无升高,提示A1AR加重水钠潴留。3.A1AR-/- DOCA-Salt小鼠早期(4周)出现局灶性间质纤维化,心脏重量指数和微血管密度无显著变化。未观察到A1AR在DOCA-Salt小鼠模型中对心肌肥大、炎症和纤维化的明确保护作用。
[Abstract]:One, study background: Cardiorenal Syndrome (CRS) is a general name for a large group of diseases involving the heart and kidney, including acute or chronic dysfunction of the heart (or kidney), causing acute or chronic dysfunction of the kidney (or heart), or co factors that cause structural and functional impairment of the heart and kidney simultaneously. There is a close relationship between function and renal function, and the heart and kidney are simultaneously involved, which greatly increases the difficulty and mortality of the diagnosis and treatment of the disease 1,2. according to the consensus of the 2010 ADKI (Acute Dialysis Quality Initiative). The pathophysiological mechanism of the heart and kidney syndrome divided into 5 types of 3.CRS has not been fully explained, and the various types of CRS have common characteristics in the physiological mechanism of the disease, The hemodynamic mechanism and non hemodynamic mechanism 4., the core of the hemodynamic mechanism is sodium retention and increased volume load, while non hemodynamic mechanisms include the renin angiotensin aldosterone system (Renin-Angiotensin-Aldosterone System, RAAS) and sympathetic nervous system activation, oxidative stress, and inflammatory response At present, most of the CRS animal models are constructed by cardiac and renal surgery. The chronic CRS animal model with acute changes and no good simulation of pathophysiology has limited the physiological mechanism of its disease. The model of high aldosterone high salt diet (DOCA-Salt) mice model simulated the physiological state of salt sensitive hypertension, and we observed the kidney in the early stage. Damage of structure and function. This study attempts to explore whether the model has cardiac damage at the same time, and thus provides a platform for exploring the CRS mechanism. Adenosine A1 Receptor (A1AR) regulates the cardiac release of natriuretic peptide (Atrial Natriuretic Peptide, ANP) by regulating the feedback of the renal tubular bulb (A1AR) to regulate the water and salt load in the heart, and in non blood flow. In the dynamic mechanism, A1AR plays a direct inhibitory effect on RAAS and regulates inflammation and apoptosis. However, there is no systematic exploration of the significance of A1AR on the damage to the heart and kidney in the DOCA-Salt mouse model. We observed that A1AR mainly protects the structure and function of the kidney of DOCA-Salt mice by hemodynamic factors. The study tries to explore whether A1AR has the same protective effect on the heart damage of DOCA-Salt mice through two aspects of hemodynamic mechanism and non hemodynamic mechanism. Two, the purpose of this study is: 1. to observe the heart damage of DOCA-Salt rats and to explore the possibility of DOCA-Salt model as a chronic CRS model; 2. explore A1AR in the retention of water and sodium retention in DOCA-Salt mice. The role of A1AR to explore the role of A1AR in myocardial hypertrophy, microvascular density, inflammation and fibrosis in DOCA-Salt mice. Three, research methods and results: 1. DOCA-Salt mouse heart damage in C57BL/6J wild type mice, left nephrectomy, implantation of Deoxycorticosterone Acetate, DOC in the left renal fossa (DOC). A) sustained release tablets were given high salt diet and established DOCA-Salt mice model. The blood pressure and heart rate were measured by tail sleeve method. The heart rate of DOCA-Salt mice decreased significantly with the number of weeks (0 weeks 685.8 + + 25.4 vs.8 weeks, 526.6 + 103.9 / min, P0.01), and the average arterial pressure increased significantly (0 weeks 74.6 + 4.4 vs.8 weeks, 88.5 + 6.5 mmHg, P0.01), respectively. Respectively, at week fourth weeks, respectively. Eighth weeks and eighth weeks, the mice were killed and heart specimens were left. The cardiac weight was weighed and the heart weight index increased significantly at eighth weeks (7.05 + 0.83 vs.5.16 + 0.20mg/g, P=0.007). Routine HE staining was used to observe the changes of the myocardial tissue under the microscope and the left ventricular thickness was not significantly changed by.Van Gieson collagen staining to observe the degree of fibrosis of the myocardium. The focal interstitial fibrosis was observed from fourth weeks, and the Realtime PCR method was used to analyze the TGF - beta 1 (Transforming Growth Factor- beta 1, TGF- beta 1), and the collagen fiber 1 (Collagenl) was observed and analyzed between the experimental group and the control group. The microvascular density of the cardiac muscle was observed and the microvascular density was significant at fourth weeks. Increased ([1.01 + 0.003]%vs.[0.59 + 0.001]%, P=0.030), microvascular density in eighth weeks had no significant changes compared with the control group..CD68 immunohistochemical staining was observed and the infiltration degree of macrophages was analyzed. The infiltration degree of macrophage in DOCA-Salt mice was not different from that of the control group; the Realtime PCR method was used to analyze chemokine factor 2 (Chemokine C-C motif lig). The expression level of and 2, CCL2) was also no difference in the water and salt load of.2. DOCA-Salt mice. 24h urine was kept in the metabolic cage at week 0,4,8. The 24h urine volume was observed in DOCA-Salt mice (1173 + 464 vs.4 weeks, 1173 + 464 vs.4 weeks, 3606 + 2359 vs.8, 7635 + 3017 Mu L, 22 comparison) and sodium excretion (0 weeks 133.3 + 39.5). 22 compared with P0.05), the urine osmotic pressure (0 weeks, 1801 + 642 vs.4 weeks 910 + 213mOsm/kg H2O, P0.05, 0 weeks 1801 + 642 vs.8 weeks, 860 + 233mOsm/kg H2O, P0.05) decreased significantly by.Realtime PCR method and observed that the myocardial tissue Corin and expressions were increased in fourth weeks, respectively, and 31.9 times and 180.7 times respectively, At eighth weeks, there was no difference between the control group and the control group.Realtime PCR method to observe the expression of adenosine synthetase CD73 mRNA in DOCA-Salt mice from fourth weeks (4 weeks, 18.79 times, P=0.001, 8 weeks 8.28 times, P=0.036), and A1AR mRNA eighth weeks began to rise significantly (12.12 times, P= 0.013), but Western Blot method did not observe the expression of protein The effect of.3.A1AR on the heart damage of DOCA-Salt mice was established by.3.A1AR. The heart rate of A1AR-/- DOCA-Salt mice decreased with the number of weeks (4 weeks 636 + 64.2 vs.0 weeks 696.7 + 35.2, P0.05), and the average arterial pressure had no significant change in the mean arterial pressure, which was significantly increased (7003 + 3742 VS) compared with the wild type experimental group. .3606 + 2359 L, P0.05), 24h urine sodium excretion increased (1246.5 + 859.8 vs.146.6 + 71.2 u mol/d, P0.05), and urinary osmotic pressure decreased (702 + 165 vs.1394 + 397 mOsm/kg, P0.05). There was no significant change in cardiac weight index and left ventricular thickness in fourth weeks, no significant increase in myocardial microvascular density, and no difference in the level of macrophage infiltration and CCL2 mRNA expression between the experimental group and the control group; only 1 (a total of 6) mice had focal interstitial fibrosis, and the expression level of TGF- beta 1 and Collagenl mRNA was in the heart. There was no difference between the experimental group and the control group. Four, the study concluded that the blood pressure of 1. DOCA-Salt mice increased, the heart rate decreased, the cardiac microvascular density increased significantly at the early (4 weeks), the focal interstitial fibrosis appeared, and the heart weight index of the late (8 weeks) increased significantly. The kidney damage was observed in the early stage of the study, and the heart of the DOCA-Salt mice existed. Common renal damage can be used as a chronic CRS model in.2. DOCA-Salt mice, suggesting the presence of sodium retention. The water and salt load is regulated by related factors: the activation of the heart CD73-A1AR and Corin-ANP pathway, the restriction of the reabsorption of water and sodium by ANP; the increase of 24h urine volume and the excretion of 24h urine sodium; the early study observed the CD73-A1AR pathway in the kidney. Activation, suggesting that tube feedback continued to activate.A1AR-/- DOCA-Salt mice feedback loss, 24h urine volume increased significantly, blood pressure was not higher than the baseline and control group, suggesting that A1AR increased sodium retention of.3.A1AR-/- DOCA-Salt mice early (4 weeks) focal interstitial fibrosis, cardiac weight index and microvascular density did not change significantly. The protective effect of A1AR on myocardial hypertrophy, inflammation and fibrosis in DOCA-Salt mice was observed.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R541.6;R692

【共引文献】