【作者】 谭红梅；

【导师】 祝善俊；

【作者基本信息】 第三军医大学 ， 内科学， 2008， 博士

【摘要】 研究背景保护肾脏功能对延缓慢性心力衰竭(CHF)患者的病情进程具有重要意义。流行病学证据表明,在CHF患者中,即使轻度肾功能减退也是导致预后不良的重要危险因素。近年来,CHF患者心脏和肾脏间的相互作用已成为学术界热切关注的焦点。已有研究表明,CHF患者往往伴有以肾小球滤过功能受损为主要特征的肾功能异常,同时肾脏交感神经压力反射在CHF患者中也是受损的。因此,研究早期肾功能减退在CHF病程进展中的作用,具有非常重要的意义。目前,血清肌酐(CREA)、尿素氮(UREA)是临床上用于检测肾功能最常用的生化指标,但其浓度受肌肉、饮食、众多疾病状态等诸多因素的影响而发生改变,且只有在肾功能严重下降时,其水平才高于正常值,因此不能作为诊断CHF患者早期肾功能减退的检测指标。血清胱抑素C(CysC)、内皮素-1(ET-1)、尿液微量白蛋白(MA)、β2-微球蛋白(β2-MG)、N-乙酰-β-氨基葡萄糖酐酶(NAG)、水通道蛋白-2(AQP-2)的水平很少受各种因素的影响,且在肾功能减退早期阶段就开始发生改变,因此,作为早期肾功能减退的生化指标已用于临床和实验研究。血清CysC的浓度完全取决于肾小球滤过率;在各种炎症、免疫损伤等原因引起的肾小球滤过屏障损伤时,MA从尿液漏出会增多;当肾小管损伤,重吸收功能受损时,尿液中β2-MG、NAG的水平增加;肾脏AQP-2的水平直接影响肾脏对水分的重吸收,其中部分AQP-2可脱落于小管腔被尿液冲出。因此,尿液AQP-2的水平可能能间接反映肾脏功能。血清ET-1的水平严重影响着机体的血流动力学状态,而后者与肾功能呈正相关。因此,我们也检测了两组人员血清ET-1的水平。内皮素-1(ET-1)是Yanagisawa等1988年从猪的主动脉内皮细胞分离纯化出来的由21个氨基酸组成的血管活性肽。研究发现,内髓集合管上皮细胞产生的大量内皮素,可以降低肾脏对抗利尿激素的敏感性,抑制水的重吸收,减轻CHF患者心脏的前负荷。1993年Fushimi等发现了水通道蛋白-2(AQP-2),主要在肾脏集合管主细胞的管腔侧细胞膜和胞质内表达,是受精氨酸加压素(AVP)调控的最主要的肾脏水通道蛋白,其表达及穿梭调节与集合管重吸收密切相关。因此,集合管ET-1和AQP-2均在维持机体水平衡中起着重要作用。血管紧张素Ⅱ(AngⅡ)是肾素-血管紧张素系统的主要活性物质,在多种组织,如血管平滑肌、肾上腺、肾脏和心脏中与AT1受体结合,具有收缩血管和促进醛固酮释放等生物学效应,从而增加了心脏的前后负荷。AngⅡ受体拮抗剂(ARB)-氯沙坦钾,选择性作用于AT1受体,阻断了上述AngⅡ对机体产生的有害作用,从而有效地保护了CHF患者的心肾功能。本课题从临床病人、动物模型两个层面进行研究,并给予ARB-氯沙坦钾进行干预治疗,探讨CHF时早期肾功能减退的机制和氯沙坦钾的治疗作用。研究方法1.选取CHF患者和健康体检者分别作为CHF组和对照组。将CHF组患者随机分为常规治疗和氯沙坦钾两亚组,均给予8周治疗;体检健康人员作为对照组。治疗前检测两组血清ET-1的水平、心脏收缩功能,如左心室舒张末期内径(LVEDD)、左心室射血分数(LVEF)、左心室缩短分数(LVFS)及肾功能,如血清CREA、UREA、CysC,尿液AQP-2、MA、β2-MG、NAG的水平。治疗8周后再次检测CHF组两亚组心脏和肾脏功能各项指标的水平。2.将小鼠分为假手术组、CHF组和氯沙坦钾组。结扎左冠状动脉前降支建立心肌梗死(AMI)后CHF模型,假手术组小鼠只穿过不结扎左冠状动脉。分别于术前、术后2周检测3组小鼠血流动力学指标,如左心室舒张末期压(LVEDP)、左心室收缩末期压(LVESP)、左心室压力最大上升/下降速率(±dp/dtmax)。将血流动力学指标值的显著改变作为CHF模型建立成功与否的标志。将CHF小鼠随机分为CHF组和氯沙坦钾组。术后2周CHF组和假手术组小鼠予以饮用水1.5ml灌胃,氯沙坦钾组小鼠予以0.2%氯沙坦钾1.5ml灌胃,均1/日,连续8周。术后10周检测以上血流动力学指标,另外还检测以下指标:(1) RT-PCR法检测3组小鼠肾脏组织AQP-2、ET-1 mRNA的表达;(2)免疫组织化学法检测3组小鼠肾脏组织AQP-2、ET-1蛋白表达及分布;(3)Western blot法检测3组小鼠肾脏组织AQP-2、ET-1蛋白的表达。结果1.与对照组比较,治疗前CHF组LVEDD值显著增大,LVEF、LVFS值显著降低。与治疗前比较,治疗8周后CHF两亚组患者LVEDD值均显著减小,而LVEF、LVFS值均显著增高。治疗前CHF两亚组之间各项指标值差异无统计学意义;但在治疗8周后,与常规治疗组比较,氯沙坦钾组患者LVEDD值显著减小,LVEF、LVFS值显著增高。2.治疗前对照组、CHF组血清CREA、UREA、尿液NAG、β2-MG的水平均在正常值范围内。3.与对照组比较,治疗前CHF组患者血清CysC、ET-1、尿液MA、AQP-2的水平显著增高。与治疗前比较,治疗8周后CHF两亚组患者以上指标的水平均显著降低。治疗前CHF两亚组患者上述各项肾功能指标的水平差异无统计学意义,但在治疗8周后,与常规治疗组比较,氯沙坦钾组患者以上指标的水平均显著降低。4.与术前比较,术后2周假手术组小鼠血流动力学各项指标值差异无统计学意义,而术后2周CHF组、氯沙坦钾组小鼠LVEDP值均显著增高,LVESP、±dp/dt max值显著降低。5.与假手术组比较,术后10周CHF组小鼠LVEDP值显著增高,氯沙坦钾组LVEDP值较CHF组显著降低,但仍显著高于假手术组;与假手术组比较,术后10周CHF组小鼠LVESP、±dp/dt max值显著降低,氯沙坦钾组LVESP、±dp/dt max值较CHF组显著增高,但仍显著低于假手术组。6.HE染色光镜下可见三组小鼠肾组织肾小球结构均无明显病理改变。假手术组小鼠肾小管结构也正常;CHF组小鼠肾小管上皮细胞可见空泡形成;氯沙坦钾组小鼠肾组织病理改变较CHF组有所减轻,但肾小管上皮细胞仍可见水肿、变性改变。7.与假手术组比较,CHF组小鼠肾组织AQP-2mRNA、AQP-2蛋白的表达水平显著增强;氯沙坦钾组小鼠肾组织AQP-2mRNA、AQP-2蛋白的表达水平较CHF组显著减弱,但其表达水平仍显著高于假手术组。8.免疫组织化学染色后,光镜下可见肾组织AQP-2蛋白阳性表达呈棕黄色颗粒,主要位于集合管上皮细胞部位。假手术组小鼠肾组织AQP-2呈轻度棕黄色染色;CHF组小鼠肾组织AQP-2染色较假手术组显著增强;氯沙坦钾组小鼠肾组织AQP-2染色较CHF组染色稍减弱,但较假手术组显著增强。9.与假手术组比较,CHF组小鼠肾组织ET-1mRNA、ET-1蛋白的表达水平显著增强,且氯沙坦钾组小鼠肾组织ET-1mRNA、ET-1蛋白的表达水平较CHF组也显著增强。10.免疫组织化学染色后,光镜下可见肾组织ET-1蛋白阳性表达呈棕黄色颗粒,主要位于肾小球和肾小管上皮细胞。假手术组小鼠肾组织可见轻度棕黄色染色;CHF组较假手术组染色增强;氯沙坦钾组染色较CHF组染色也明显增强。结论1.血清CysC和尿液MA、AQP-2的水平显著增高,表明CHF患者存在以肾小球滤过功能受损为特征的早期肾功能减退。2.尿液NAG、β2-MG水平正常,表明CHF患者肾小管重吸收功能正常。3.临床上常用的肾功能生化指标血清,如血清CREA、UREA浓度的检测对CHF患者早期肾功能无诊断意义;早期肾功能指标,如血清CysC和尿液MA、AQP-2水平的联合检测对CHF患者早期肾功能减退具有诊断价值。4.血清ET-1水平显著增高,表明CHF患者存在明显血流动力学改变。5. CHF小鼠肾组织AQP-2基因、蛋白表达水平增加,促进了集合管对水的重吸收;CHF小鼠肾组织ET-1基因、蛋白表达水平降低,可能是容量负荷增加所致,抑制了集合管对水的重吸收。6.氯沙坦钾能有效改善CHF患者的心脏收缩功能及CHF患者、CHF小鼠血流动力学状态;7.氯沙坦钾能显著改善CHF患者的肾小球滤过功能。8.氯沙坦钾能下调CHF小鼠肾组织AQP-2基因、蛋白的表达;上调CHF小鼠肾组织ET-1基因、蛋白的表达,其意义在于抑制集合管对水的重吸收,促进水钠排泄,这可能是对此药延缓CHF病程进展作用机制的新解释。更多还原

【Abstract】 Background and ObjectivesIt was important to preserve renal function for delaying the progress of chronic heart failure (CHF). Epidemiology showed that mild renal hypofunction was a risk factor of poor prognosis of CHF. In recent years, the interaction between heart and renal in CHF patients has been widely paid close attention. It was indicated that there was renal dysfunction characteristic of damaged glomerular filtration and impaired baroreflex in CHF patients. Thus, it is significant to study the effect of renal hypofunction on the advancement of CHF.Now, levels of creatinine and urea nitrogen in serum as conventional biochemical indicators have been used to evaluate renal function clinically, but they were affected by such factors as muscle, diet and some morbid states and were above normal values only when renal function was seriously damaged, so they could not be used to diagnose early renal hypofunction in CHF patients. Levels of cystatin(CysC)in serum and microamount albumn(MA),β2-microglobulin(β2-MG), N-acetyl-β-glucosaminidase(NAG), aquaporin -2(AQP-2) in urine were seldomly influenced by various kinds of factors and began to change at an early stage of renal hypofunction, so they have already been used as early renal hypofunction biochemical indicators in clinical and experimental research. Concentration of cysC in serum was completely determined by glomerular filtration rate. MA leaking out to urine increased when glomerular filtration barrier damaged resulting from some factors, such as inflammation, immunologic injury. The levels ofβ2-MG、NAG in urine increased when nephric tubule damaged and its reabsorption was lesioned. Level of AQP-2 of kidney directely affected water reabsorption and a part of AQP-2 ablating to nephric tubule lumens was flushed by urine, so the level of AQP-2 in urine might indirectly reflected renal function. Level of endothelinin-1(ET-1) in serum seriously influenced hemodynamics states which positively correlated with renal function, so, we measured level of ET-1 in serum of people in two groups too.ET-1, vasoactive peptide constituted of 21 amino acids, was detached and purified from pig arteriae aorta endotheliocyte in 1988. It was found that epithelial cell of medulla interna collecting duct secreted a large amount of endothelin-1 (ET-1) which weakened renal sensitivity to antidiuretic hormone (ADH) and so inhibited water reabsorption and eased heart preload. AQP-2, expressed on cytolemma and intracytoplasm at the side of lumens of principal cell of renal collecting duct, was a main renal aquaporin regulated by arginine vasopressin (AVP). Its expression and shuttling back and forth regulation closely correlated with the collecting duct reabsorption. Therefore, ET-1 and AQP-2 of collecting duct played an important role in maintaining body fluid balance.AngiotensinⅡ(AngⅡ) was a main active compound of renin-angiotonin system and combined with AT1 receptor of such kinds of tissue as vascular smooth muscle, adrenal gland, kidney and heart. It possessed biological effect of vasoconstriction and promoting aldosterone release, so it added heart preload and afterload. Angiotensin receptor blocker (ARB)-losartan potassium combined selectively with AT1 receptor and blocked above adverse effects caused by AngⅡ, thus, it could protect heart and renal function.This experiment was based on research conducted on two levels of clinical patients and animal model, both with ARB-losartan potassium intervention. Mechanism of early renal hypofunction in CHF and the therapy effect of losartan potassium on renal function were explored in this study.Methods1. CHF patients and healthy people in health examination were selected as CHF group and control group respectively. Patients in CHF group were randomly divided into two subgroups of conventional therapy and losartan potassium therapy, both with 8 weeks of drug treatment. Before drug treatment, level of ET-1 in serum and heart contractile function, such as values of left ventricular end-diastolic dimension (LVEDD), left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS) and renal function, such as levels of CREA, UREA, CysC in serum and AQP-2, MA,β2-MG and NAG in urine were measured in both groups. After 8 weeks of drug treatment, above indicators were measured again in two subgroups of CHF group. 2. All mouse were randomly divided into three groups: sham operation group, CHF group and losartan potassium group. The CHF mouse model after acute myocardial infarction (AMI) was set up by ligating left anterior descending coronary artery, while left anterior descending coronary artery was not ligated, but was through in sham operation group. Before operation and 2 weeks after operation, hemodynamics indicators, such as values of left ventricular end-diastolic pressure (LVEDP), left ventricular end-systolic pressure (LVESP), left ventricular pressure max upstroke/downstroke velocity(±dp/dtmax) were measured in three groups. Significances of above indexes were regarded as a signal of successful CHF model. CHF mouse were randomly divided into CHF group and losartan potassium group. After 2 weeks of operation, 1.5ml water was given by intragastric administration in sham operation group and CHF group. 1.5ml 0.2% losartan potassium was given by intragastric administration in losartan potassium group. All administration was once a day and with 8 weeks. 10 weeks after operation, above hemodynamics indicators were measured again and additional following indicators were detected: (1) Expressions of AQP-2mRNA, ET-1mRNA of mouse renal tissue were detected with reverse transcription polymerase chain reaction (RT-PCR); (2) Expressions and distribution of protein of AQP-2 and ET-1 of mouse renal tissue were detected with immunohistochemical method;(3) Expressions of protein of AQP-2 and ET-1 of mouse renal tissue were detected with western blot.Result1. Before treatment, the value of LVEDD increased significantly and the values of LVEF and LVFS decreased notably in CHF group compared with control group. The value of LVEDD was much lower and the values of LVEF and LVFS were much higher after 8 weeks of drug treatment than before treatment in two subgroups of CHF group respectively. There were no significances of the values of all above indictors between two subsets of CHF group before treatment, but after 8 weeks of drug treatment, the value of LVEDD was much lower and the values of LVEF and LVFS were much higher in losartan potassium group than in conventional therapy group.2. The levels of CREA and UREA in serum and NAG、β2-MG in urine were in ranges of normal values in control group and CHF group before treatment. 3. Before treatment, the levels of CysC,ET-1 in serum and MA, AQP-2 in urine increased significantly in CHF group compared with control group. The levels of above biochemical indicators were much lower after 8 weeks of drug treatment than before treatment in two subgroups of CHF group respectively. There were no significances of the levels of these detection indexes between two subgroups of CHF group before treatment, but after 8 weeks of drug treatment, the levels of these indictors were much lower in losartan potassium group than in conventional therapy group.4. There were no significances of all hemodynamics indexes of mouse between before operation and 2 weeks of operation in sham operation group, but the value of LVEDP increased significantly and the values of LVESP and±dp/dt max decreased notably after 2 weeks of operation than before operation in CHF group and losartan potassium group.5. After 10 weeks of operation, compared with in sham operation group, the value of LVEDP increased significantly in CHF and the value of it was much lower in losartan potassium group than in CHF group, but was still much higher than in sham operation group; the values of LVESP、±dp/dt max decreased significantly in CHF and the values of them were much higher in losartan potassium group than in CHF group, but were still much lower than in sham operation group.6. After HE stain, there was no obviously pathological structure change of renal glomeruli of mouse in three groups. Normal structure of renal tubule was observed in sham operation. There was vacuolization of renal tubule in CHF group and the pathological change of renal tubule in losartan potassium group eased, but there were still changes of oedema and degeneration under light microscope.7. Compared with in sham operation group, the expressions of AQP-2 mRNA and AQP-2 protein of mouse renal tissue increased significantly in CHF group. The expressions were much lower in losartan potassium group than in CHF group, but were still much higher than in sham operation group.8. After immunohistochemistry stain, positive expression of AQP-2 protein of mouse renal tissue, presented with buffy grains, mainly distributed in epithelial cells of collecting tube under light microscope. There was light amber-coloured stain in mouse renal tissue in sham operation group and there was obviously augmented stain in CHF group. There was lighter colored stain in losartan potassium group than in CHF group, but was still much deeper than in sham operation group.9. Compared with in sham operation group, the expressions of ET-1 mRNA and ET-1 protein of mouse renal tissue increased significantly in CHF group. The expressions were much higher in losartan potassium group than with in CHF group too.10. After immunohistochemistry stain, positive expression of ET-1 protein of mouse renal tissue, presented with buffy grains, mainly distributed in renal glomeruli and epithelial cells of collecting tube under light microscope. There was light amber-coloured stain in sham operation group and there was obviously augmented stain in CHF group compared with in sham operation group. There was also much deeper in losartan potassium than in CHF group.Conclusions1. The levels of CysC in serum and MA, AQP-2 in urine increased significantly, showing that there was early renal hypofunction characteristic of glomerular filtration functional lesion in CHF patients.2. The levels of NAG、β2-MG in urine were in ranges of normal values, showing that there was normal tubular reabsorption function in CHF patients.3. Such common biochemical indicators as the levels of CREA、UREA in serum had no diagnostic function, while combined measurement of the levels of such early renal function biochemical indicators as the levels of CysC in serum and MA, AQP-2 in urine had diagnostic value for early renal hypofuncion in CHF patients.4. The level of ET-1 in serum increased significantly, showing that there was obviously hemodynaics change in CHF patients.5. The increasing gene and protein expressions of renal AQP-2 of CHF mouse promoted collecting duct to reabsorb water; while the increasing gene and protein expressions of ET-1 of CHF mouse, resulted from increasing volume load, inhibited collecting duct from reabsorbing water.6. Losartan potassium could effectively improve heart systolic function of CHF patients and hemodynamics states of patients and mouse with CHF.7. Losartan potassium could improve glomerular filtrateion function of CHF patients. 8. Losartan potassium could downregulate gene and protein expressions of AQP-2 and upregulate gene and protein expressions of ET-1 of renal tissue of CHF mouse, so it inhibited collecting duct from reabsorbing water and promoted kidney to excrete water and sodium, which maybe a new explanation of its mechanism of action of delaying the course of CHF.更多还原