【作者】 杜君；

【导师】 孔垂泽；

【作者基本信息】 中国医科大学 ， 外科学， 2009， 博士

【摘要】 氨氯地平和依普利酮联合应用在缓解盐敏感高血压所致肾脏损害中作用机制的研究研究背景和目的世界范围内大约10亿人正在受到高血压的困扰。临床研究表明:大约50%的高血压患者为盐敏感高血压。而盐敏感高血压正是导致心血管疾病以及肾脏功能损害的最为重要的致病因素之一。有研究表明:肾功能不全的发生与肾间质纤维化具有极为密切的关系。一些研究发现:慢性肾间质缺氧在肾间质纤维化中发挥了重要作用并最终导致肾功能不全的发生。此外,一些导致慢性肾间质缺氧的诱因也已经查明,其中最为重要的是肾小管周毛细血管损伤以及血流减少。缺氧导致的ATP耗竭,会导致肾小管上皮细胞内钙离子浓度升高以及线粒体的损伤并进而导致细胞凋亡。L型钙通道阻断剂可以显著降低由缺氧所导致的肾小管上皮细胞凋亡。此外,有人发现L型钙通道阻断剂还可以显著降低由肾脏缺血再灌注所导致的肾脏各类细胞凋亡以及由血管紧张素Ⅱ所导致的肾小管周围缺血。以上研究暗示,一些临床研究中L型钙通道阻断剂的肾脏保护作用可能是通过对肾间质缺氧的抑制而实现的。然而氨氯地平在高血压中肾脏保护作用的机制尚需进一步研究。传统认为,醛固酮通过作用于远端肾单位来调节体内电解质平衡以及血压的稳定。但是最近人们对醛固酮的认识发生了变化。越来越多的证据表明,醛固酮在心血管以及肾脏损害过程中发挥了重要的作用。在肾脏损害方面,许多研究证明:醛固酮水平的升高可以导致蛋白尿、肾小球硬化以及肾间质纤维化。亦有研究发现,在醛固酮持续灌注SD大鼠中,肾小球表达的正常足细胞标记物podocin和nephrin显著降低,然而损伤足细胞标记物desmin表达却显著升高。盐皮质激素受体(MR)阻断剂依普利酮可以显著改善由醛固酮水平升高导致的足细胞损伤以及蛋白尿。因此,足细胞损伤的治疗将具有重要临床意义。以上研究提示,MR在足细胞损伤中可能发挥了重要作用,其可能机制尚需进一步研究。并且,联合应用氨氯地平和依普利酮是否能够提供更好的肾脏保护作用尚需研究。本研究首次研究了氨氯地平和依普利酮联合应用对于高盐诱导的Dahlsalt-sensitve(DS)大鼠盐敏感血压以及肾脏损害的治疗作用。并对其可能机制进行了深入探讨。方法高盐饲料诱导的盐敏感高血压合并肾脏损害动物模型的建立6周龄Dahl salt-sensitve(DS)大鼠饲以0.4%低盐饲料或4%高盐饲料,每两周行血压测定以及24小时尿蛋白测定。至第16周龄时,相对于低盐饲料喂养DS大鼠,高盐饲料喂养DS大鼠的收缩压(SBP)超过170 mmHg并且尿蛋白超过70 mg/24h。组织学检查可见严重肾小球硬化以及肾间质纤维化。以上结果证实:高盐饲料诱导的盐敏感高血压合并肾脏损害动物模型建立成功。实验动物分组:6周龄DS大鼠40只随机分为两组:对照组(8只,LS)喂以0.4%低盐饲料(Oriental Yeast,大阪,日本),实验组(32只,HS)喂以4%高盐饲料(Oriental Yeast,大阪,日本)。8周龄时将实验组大鼠进一步随机分为4组:阳性对照组(8只,HS),氨氯地平治疗组(8只,3 mg/kg/day,HS+A),依普利酮治疗组(8只,50 mg/kg/day,HS+E)和联合用药组(8只,HS+A+E),治疗时间均为8周。整个实验过程共计10周。实验期间,每两周采用套尾法测量血压以及代谢笼采集24小时尿液各一次。血浆和肾脏标本在苯巴比妥麻醉状态下采集,其中肾脏首先行生理盐水灌注冲洗,然后切片放入10%福尔马林(pH 7.4)固定石蜡包埋用于组织学检查,或者直接放入OCT冰冻切片包埋剂制作冰冻切片用于激光捕获微分离(LCM)。剩余肾脏组织放入液氮冷冻并且放入-80℃深低温冰箱保存。1.尿液及血液生化学检查通过24小时尿蛋白和尿肌酐测定以及血肌酐测定,了解肾脏功能损害情况。通过对血浆钠离子、钾离子和醛固酮水平测定了解体内电解质代谢状况。2.组织学检查通过PAS染色对肾小球硬化的程度进行评估。通过Azan染色对肾间质纤维化进行评估。3.免疫组织化学检查通过对缺氧标记物pimonidazole免疫组织化学染色对肾间质缺氧进行检测,aminopeptidase P免疫组织化学染色来评价肾间质毛细血管密度,desmin的免疫组化染色来检测足细胞损伤。4.肾小球的激光捕获微分离和总RNA提取通过激光捕获微分离技术分离肾小球组织并提取总RNA。5.Real-time PCR通过方法4中提取的总RNA合成cDNA文库。并通过Real-time PCR检测肾小球podocin、nephrin、MR和Sgk-1表达。6.统计学分析所有数据均以(?)±SE表示。各组间统计学比较通过方差分析(ANOVA)以及多重比较的Bonferroni事后检验进行。结果1.成功建立盐敏感高血压合并肾脏损害动物模型。2.收缩压(SBP)高盐饲料喂养的DS大鼠产生了严重的高血压。并且各个治疗组降低血压的水平相近。3.24h尿蛋白,24h尿蛋白/尿肌酐比值(Urinary protein/creatinine ratio,UPCR),血浆肌酐(Plasma creatinine)以及内生肌酐清除率(Creatinine clearance,Ccr)高盐饲料喂养DS大鼠发生了24h尿蛋白和UPCR的显著增加。单一用药可以显著降低24h尿蛋白和UPCR水平。但是,联合用药组可以更好的降低24h尿蛋白和UPCR水平。高盐饲料喂养DS大鼠同样也导致了血肌酐的增加以及内生肌酐清除率的降低。只有联合用药组可以显著抑制血肌酐的增加以及内生肌酐清除率的降低。4.组织学检查低盐饲料饲养DS大鼠的肾小球以及肾间质完全正常。高盐饲料喂养导致了DS大鼠严重的肾小球硬化以及肾间质纤维化。单一应用依普利酮可以显著降低肾小球硬化。然而,与依普利酮比较而言,单一应用氨氯地平可以很好的减轻肾间质纤维化。联合用药组对肾小球硬化以及肾间质纤维化均具有很好的治疗效果。5.MR激活导致的足细胞损伤高盐饲料喂养DS大鼠肾小球podocin和nephrin表达显著降低,然而desmin表达却显著升高。该结果提示着足细胞损伤的存在。单一应用依普利酮或者联合用药可以显著改善足细胞损伤。通过进一步检测MR表达以及MR活性标志物Sgk-1我们发现,高盐饲料喂养DS大鼠可以促进MR表达水平升高。尽管依普利酮不能够降低MR的表达水平。但是,单一应用依普利酮或者联合用药可以显著降低Sgk-1的表达。6.肾间质缺氧检测高盐饲料导致了肾间质pimonidazole阳性区域显著增加。这一结果提示肾间质缺氧的存在。氨氯地平可以部分改善肾间质缺氧的情况,但是依普利酮却没有任何效果。而联合用药可以显著改善肾间质缺氧的情况。已有研究表明,肾间质缺氧与肾间质毛细血管的缺失有重要关系。本实验发现,高盐饲料喂养DS大鼠同样导致了肾间质毛细血管的缺失。氨氯地平或联合用药可以显著改善肾间质毛细血管的缺失,而依普利酮却没有任何效果。结论1.肾小球硬化以及肾间质纤维化并不一定同时发生。二者均与肾功能密切相关,任何一种损伤均可以造成24小时尿蛋白水平升高。2.肾小球硬化以及肾间质纤维化的致病因素不完全相同。MR的激活所导致的足细胞损伤在肾小球硬化的发展中发挥了重要作用。而肾间质缺氧则在肾间质纤维化中则发挥了重要作用。3.通过不同的作用机制,氨氯地平和依普利酮联合应用可以显著改善由盐敏感高血压所致的肾脏足细胞损伤以及肾间质缺氧,进而抑制肾小球硬化、肾间质纤维化为代表的肾脏损害。本研究对于指导临床慢性肾脏损害治疗具有重要指导意义。更多还原

【Abstract】 Renoprotective Effects of Amlodipine in Combination with eplerenone in Hypertensive Dahl RatsBackground and ObjectiveHypertension affects around 1 billion people worldwide,and suboptimal control of blood pressure is the number one attributable risk for death throughout the world. Clinical studies have demonstrated that around 50%of hypertensive patients have salt-sensitve hypertension,a well-known independent risk factor for increasing cardiovascular morbidity and mortality,and progressive renal functional impairment. Renal dysfunction and injury have been reported to show better correlations with structural damage to the renal tubuloinsterstitium than with damage to the glomeruli.It has been demonstrated that chronic hypoxia in the tubulointerstitium is a final common pathway to end-stage renal failure.In addition,a number of mechanisms that induce tubulointerstitial hypoxia have been suggested;occurring as a consequence of a reduction in peritubular capillary（PTC） blood flow,i.e.,peritubular ischemia and injury. In hypoxia,ATP depletion causes cellular Ca²⁺ increase,mitochondrial injury and apoptosis in renal tubular cells.Tanaka et al.showed that an L-type Ca²⁺ channel blocker（CCB） attenuates mitochondrial injury and apoptosis in hypoxic renal tubular cells.Furthermore,treatment with CCBs restored ischemic/reperfusion-induced apoptosis and angiotensinⅡ-induced peritubular ischemia.These data suggest that the reno-protective effects of CCBs reported in clinical studies may be mediated through inhibition of tubulointerstitial hypoxia.However,the mechanisms by which amlodipine elicits reno-protective effects in hypertensive subjects are not clear.Aldosterone is traditionally viewed as a hormone regulating electrolyte and blood pressure homeostasis by acting on the distal nephron.Recently,there has been a paradigm shift regarding the role of aldosterone.Growing evidence suggests that aldosterone plays an important pathogenetic roles in cardiovascular and renal injury.In the kidney, aldosterone promotes proteinuria,glomerulosclerosis,arteriopathy,and renal fibrosis. In aldosterone-infused rats,glomerular expressions of slit diaphragm-associated molecules nephrin and podocin were markedly decreased,whereas expression of a damaged podocyte marker desmin was upregulated.Proteinuria and podocyte damage were completely reversed by selective aldosterone blocker eplerenone.Thus,podocytes can be an important therapeutic target.In addition,the combined effects of amlodipine and eplerenone have not been investigated.In the present study,we examined the effects of amlodipine and eplerenone on the blood pressure and renal function in DS rats.In addition,we evaluated the therapeutic efficacy of the combination of amlodipine and eplerenone.Furthermore,we try to investigate the possible mechanisms of the beneficial effects of amlodipine in combination with eplerenone.MethodsSetting up salt-induced hypertension and renal injury model in Dahl salt-sensive rats:6-week-old Dahl salt-sensitive（DS） rats,weighing 165-180g,received 4% high-salt diet for 10 weeks.SBP was measured in conscious rats by tail-cuff plethysmography,and 24-hour urine samples were collected at every two weeks.At the end of the treatment,DS rats showed severe hypertension（SBP > 170 mmHg） and proteinuria（>70 mg/24h）.Histological analysis also found severe glomerularsclerosis and tubulointerstitial fibrosis.These evidences confirmed the success of this animal model.Experimental groups:Male DS rats at 6 weeks of age were randomly selected to receive rat diet containing high salt（HS;4%NaCl;n=32;Oriental Yeast,Osaka,Japan） or low salt（LS;0.4%NaCl;n=8;Oriental Yeast） for 10 wk.The DS rats fed with high-salt diet were then randomly divided into the following 4 groups（8 rats in each group）:control（HS）;amlodipine 3mg/kg/day by gavage（HS+A）;eplerenone 50mg/kg/day by gavage（HS+E） and amlodipine 3mg/kg/day combined with eplerenone 50mg/kg/day（HS+A+E）.The treatments lasted from 3 to 10 weeks after the start of high-salt diet feeding.SBP was measured in conscious rats by tail-cuff plethysmography,and 24-hour urine samples were collected at every two weeks.Blood and kidney samples were collected at the end of week 10 under sodium pentobarbital anesthesia.Kidneys were perfused with chilled saline solution,then kidney sections were either fixed in 10% paraformaldehyde（pH 7.4） and embedded in paraffin for histological examination or frozen in Tissue-Tek O.C.T.compound for laser capture microdissection（LCM）. Remaining renal tissues were snap-frozen in liquid nitrogen and stored at -80 8888888.1.Urine and blood biochemistryDetermine the renal functional impairment by examining the urinary protein and creatinine excretion,plasma creatinine level and creatinine clearance.We also determine the electrolyte homeostasis by checking the plasma sodium,potassium and aldosterone levels.2.Histological examinationEvaluate the glomerular size and glomerularsclerosis by PAS staining.We also evaluate the tubulointerstitial fibrosis by Azan staining.3.ImmunohistochemistryRenal hypoxia was examined by pimonidazole,which binds to tissues with pO₂ levels below 10 mmHg.PTCs loss was examined by immunohistochemistry of aminopeptidase P.Podocyte injury was examined by immunohistochemistry of desmin.4.LCM and RNA isolationWe microdissected the glomeruli tissues by LCM.Glomerular RNA was extracted using RNAqueous-Micro kits.5.Real-time PCRTotal RNA was reverse transcripted into cDNA.We examined the glomerular expression of podocin,nephrin,MR and Sgk-1 by Real-time PCR.6.Statistical analysisValues are presented as mean±SE.Statistical comparisons of the differences between treatments were performed using one-way analysis of variance（ANOVA） combined with the Bonferoni post hoc test for multiple comparisons.A value of P< 0.05 was considered statistically significant.Results1.Salt-sensitive hypertension and renal injury animal model was established successfully. 2.SBPHypertension developed progressively in the rats fed with high-salt diet,but didn’t develop in the rats fed with low-salt diet.The hypotensive effects were comparable among all the treatment groups.3.Urinary Protein/Creatinine,Plasma Creatinine and Creatinine Clearance After 10 wk of treatment with a high-salt diet,rats showed a higher urinary protein/creatinine ratio as compared with rats received low-salt diet.Monotherapy with amlodipine or eplerenone could reduce urinary protein/creatinine ratio.Combination of amlodipine and eplerenone exhibited the best improvement of urinary protein/creatinine ratio.Plasma creatinine level was enhanced in the rats fed with high-salt diet compared with rats received low-salt diet.Monotherapy slightly decreased plasma creatinine.Combination treatment could significantly decrease the plasma creatinine level.In accordance with plasma creatinine,creatinine clearance was decreased in rats fed with high salt.Only the combination of amlodipine and eplerenone could significantly increase creatinine clearance compared with other monotherapy.4.Histological findingsRats that received low-salt diet showed almost normal glomeruli and tubulointerstitium;however,rats that received high-salt diet exhibited injured glomeruli characterized by sclerosis and enlarged glomerular size.In rats that received high-salt diet,severe tubulointerstitial fibrosis were also observed.Monotherapy with eplerenone but not amlodipine significantly inhibited glomerularsclerosis.However,single treatment with amlodipine could provide better improvement of tubulointerstitial fibrosis compared with eplerenone.Surprisingly,the combination of amlodipine and eplerenone dramatically reduced both glomerularsclerosis and tubulointerstitial fibrosis.5.Podocyte Injury caused by MR Upregulation and ActivationIn rats receiving a high-salt diet,glomerular expressions of podocin and nephrin were markedly decreased,whereas expression of a damaged podocyte marker desmin was upregulated.Treatment with eplerenone or combining amlodipine with eplerenone improved the podocyte injury.Furthermore,we found that glomerular MR expression was upregualted by high-salt loading.Although the MR expression can not be suppressed by eplerenone,treatment with eplerenone or eplerenone in combination with amlodipine dramatically decreased the upregulation of Sgk-1.6.Assessment of Renal HypoxiaThe pimonidazole-positive area in the renal cortex was markedly increased in control DS rats compared to low salt-fed rats.Treatment with amlodipine partially attenuated the increase in pimonidazole staining,while eplerenone treatment had no effect.The combination treatment markedly improved the increased pimonidazole positive area in renal cortex.It is proposed that the loss of peri-tubular capillary is one of the causes for renal hypoxia.The area positive for anti-endothelial aminopeptidase P antibody was smaller in the renal cortex of control rats.The decreased aminopeptidase P immunoreactivity was partially restored by amlodipine or the combination treatment,whereas eplerenone treatment had no effect on the loss of immunoreactivity by high salt feeding.Conclusion1.Glomerularsclerosis and tubulointerstitial fibrosis don’t always co-exist in renal injury.Any of them could cause renal injury alone.2.Glomerularsclerosis and tubulointerstitial fibrosis can be caused by different pathological factors.For example,activation of MR and podocyte injury may play a important role in glomerularsclerosis.However,tubulointerstitial hypoxia may be crucial for tubulointerstitial fibrosis.3.Amlodipine in combination with eplerenone provide better renoprotective effects characterized by reducing both glomerularsclerosis and tubulointerstitial fibrosis through the improvement of both podocyte injury and tubulointerstital hypoxia.更多还原