【作者】 黎承杨；

【导师】 邓耀良；

【作者基本信息】 广西医科大学 ， 泌尿外科， 2009， 博士

【摘要】 草酸钙肾结石的病因复杂。近年来发现,草酸、草酸钙晶体可导致肾脏上皮和间质损伤,其机制与活性氧簇(ROS)大量生成引起的氧化应激(OS)有关。研究认为肾脏上皮损伤是促进结石形成的关键因素,而间质损伤则是慢性肾病发展和最终肾功能衰竭的最重要的危险因素之一。因此,探讨肾脏OS损伤机制的研究应该是结石防治工作的重要内容,抗氧化疗法对于肾结石的防治及肾脏的保护可能会有好处,这是结石研究的新思路之一。本课题分五个部分,通过建立高草酸尿症和草酸钙晶体沉积的肾结石大鼠模型,并分别采用夹竹桃麻素(apocynin)、氯沙坦(losartan)和牛磺酸(taurine)三个特异性的抗氧化剂进行干预治疗,重点观察NADPH氧化酶在肾脏中的表达和作用;同时建立与晶体沉积相关的巨噬细胞体外培养体系,观察草酸钙晶体刺激下巨噬细胞NADPH氧化酶的表达和活性变化。研究发现,在高草酸尿症大鼠模型中,肾脏出现明显的OS损伤,而NADPH氧化酶是肾脏ROS形成的重要来源之一,血管紧张素Ⅱ通过刺激NADPH氧化酶的表达而促进肾脏OS的发展;研究结果还提示在低剂量诱石剂诱导下肾脏线粒体的改变早于NADPH氧化酶表达水平的变化;在体外细胞培养体系中,具有吞噬功能的巨噬细胞在草酸钙晶体刺激下其NADPH氧化酶的表达和活性也增加,并引起细胞OS损伤。本研究结果提示抗氧化疗法对高草酸尿症大鼠肾脏具有保护效果。第一部分摘要:高草酸尿症大鼠模型的建立和肾脏氧化应激损伤的观察目的:建立高草酸尿症大鼠模型,观察高草酸尿和草酸钙晶体沉积导致肾脏氧化应激(OS)损伤的情况。方法:高草酸尿症模型通过给予雄性SD大鼠(190-210g)自由饮用含0.8%乙二醇的饮水4周而诱导建立。动物随机分2个组(n=8):A,空白组,饮用正常饮水;B,高草酸尿症组,饮用含0.8%乙二醇的饮水。四周后收集大鼠24h尿液,分别检测尿草酸、尿钙、尿肌酐、尿H2O2、尿8-IP,采血测血清肌酐,取肾脏组织进行病理分析(HE染色、钙盐染色、免疫组化、电镜观察)。结果:与空白组比较,高草酸尿症组大鼠尿草酸排泄量明显增多,尿钙减少,尿8-IP和H2O2增多,而尿肌酐排泄量减少,肌酐清除率下降;肾脏病理分析示肾组织中大量含钙结晶体沉积,肾小管腔扩大和上皮损伤,肾间质大量炎性细胞浸润,肾组织CD68、TGF-β、α-SMA的表达均明显增强。结论:采用0.8%乙二醇的饮水法成功诱导建立高草酸尿症大鼠模型,同时观察到高草酸尿症大鼠肾脏OS发展和肾脏损伤出现。第二部分摘要:NADPH氧化酶在高草酸尿症大鼠肾脏中的表达和作用研究目的:观察NADPH氧化酶在高草酸尿症大鼠肾脏中的表达和作用。方法:采用0.8%乙二醇饮水法诱导建立高草酸尿症大鼠模型。动物随机分4个组(n=8):A,空白组;B,高草酸尿诱导组;C,NADPH氧化酶特异抑制剂夹竹桃麻素干预组,高草酸尿诱导+夹竹桃麻素(0.2g·kg-1·d-1,灌胃给予);D,夹竹桃麻素对照组,夹竹桃麻素(0.2g·kg-1·d-1,灌胃给予)。4周后检测各组大鼠尿液、肾组织中的氧化应激(OS)指标水平,免疫组化观察NADPH氧化酶亚基p47phox蛋白在肾脏中的表达位置,RT-PCR检测肾组织NADPH氧化酶亚基p47phox、gp91phox、Nox-1, p22phox, Nox-4 mRNA的表达水平,免疫印迹检测肾组织p47phox蛋白的表达水平。结果:免疫组化证实p47phox在各组大鼠肾脏中均有表达,表达部位在肾小球、集合管、远曲和近曲小管、髓袢等。与空白组比较,诱导高草酸尿症后大鼠肾脏p47phox、gp91phox和Nox-1 mRNA表达显著增多,p22phox, Nox-4 mRNA的表达未发现明显变化,p47phox蛋白表达也明显增多,伴肾脏OS损伤;诱导高草酸尿症的同时使用夹竹桃麻素则肾脏表达p47phox、Nox-1 mRNA及p47phox蛋白减少,但gp91phox mRNA的表达未明显减少,而肾脏的OS损伤程度减轻,但仍高于对照组水平。结论:NADPH氧化酶是高草酸尿症大鼠肾脏中ROS形成的来源之一。第三部分摘要:血管紧张素Ⅱ刺激高草酸尿症大鼠肾脏NADPH氧化酶的表达和作用目的:观察血管紧张素Ⅱ(AngⅡ)和NADPH氧化酶在高草酸尿症大鼠肾脏氧化应激(OS)形成中的相互作用。方法:采用0.8%乙二醇饮水法诱导建立高草酸尿症大鼠模型。动物随机分6个组(n=8):A,空白组;B,高草酸尿症组;C,高草酸尿症+夹竹桃麻素治疗组;D,单纯夹竹桃麻素治疗组;E,高草酸尿症+氯沙坦治疗组;F,单纯氯沙坦治疗组。后4组分别灌胃给予夹竹桃麻素(0.2g·kg-1·d-1)或氯沙坦(30mg·kg-1·d-1)。4W后检测大鼠尿液、肾组织中的氧化应激(OS)指标,放免法检测肾组织AngⅡ的含量,免疫组化法观察p47phox蛋白在肾脏中的表达位置,RT-PCR法检测肾组织p47phox mRNA的表达水平。结果:p47phox在各组大鼠肾脏中都有广泛表达,表达部位包括肾皮质、内髓、外髓。与A组比较,B组尿液8-IP明显增多,肾组织SOD活性降低,肾组织AngⅡ含量增多,p47phox mRNA在肾组织中的表达水平也明显增多。使用夹竹桃麻素(C组)和氯沙坦(E组)均可抑制肾组织p47phox mRNA的表达,同时肾脏的OS程度减轻。结论:在高草酸尿症大鼠模型中,肾脏p47phox mRNA表达增多,导致肾脏OS;同时肾脏的肾素血管紧张素系统(RAS)也被激活,后者可通过刺激p47phox mRNA的表达而导致肾脏OS程度增加。第四部分摘要:低剂量诱石剂诱导条件下大鼠肾脏线粒体的改变先于NADPH氧化酶表达的变化目的:高草酸尿和肾脏内晶体沉积可导致肾脏氧化应激(OS)的发展,线粒体和NADPH氧化酶均被认为是肾脏内活性氧簇(ROS)形成的来源。牛磺酸是一种重要的抗氧化剂。本研究观察在低剂量诱石剂诱导下肾脏线粒体和NADPH氧化酶的改变情况,探讨肾脏ROS的主要来源,同时观察牛磺酸对肾脏的保护作用。方法:采用定量灌胃2.5%乙二醇+2.5%氯化铵(4ml/d,分2次给予)并控制饮水4周诱导建立草酸钙肾结石大鼠模型。动物随机分4个组(n=8):空白组(A组)、结石诱导组(B组)、牛磺酸干预组(C组)和牛磺酸对照组(D组),其中B、C组定量灌胃诱石剂,A、D组灌胃相同量的饮水;A、B组喂食标准颗粒饲料;C、D组喂食含2.0%牛磺酸的颗粒饲料。各组的日饮水量均控制在20ml/只。4周后收集各组大鼠24h尿液检测草酸、肌酐和8-异前列腺素(8-IP)水平;采血测血清肌酐;取肾脏分离线粒体,比色法测定超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性,荧光比色法检测线粒体氧化损伤水平;肾组织HE和钙盐染色观察晶体沉积和上皮损伤情况,透射电镜下观察肾小管上皮的超微病理改变,免疫组化和RT-PCR观察NADPH氧化酶亚基p47phox在肾脏中的定位和基因表达水平;同时检测肾组织中血管紧张素Ⅱ的水平。结果:与空白组比较,使用诱石剂诱导结石后大鼠尿8-IP显著增加,肌酐清除率降低,肾脏/体重比值增加,肾组织线粒体SOD、GSH-Px活性下降伴线粒体损伤,肾脏内广泛结晶体沉积并上皮损伤表现;同时使用牛磺酸进行干预则尿8-IP生成减少,肌酐清除率增加,肾脏/体重比值减少,肾组织线粒体SOD、GSH-Px活性增加而线粒体损伤程度减轻,肾小管上皮病理损伤情况改善。但是,肾组织p47phox mRNA的表达水平,以及肾组织匀浆液血管紧张素Ⅱ的水平,在各组中均无明显的差异。结论:在此动物模型中,线粒体而非NADPH氧化酶与肾脏OS损伤有关;牛磺酸通过线粒体途径发挥其抗氧化剂的作用。第五部分摘要:草酸钙晶体刺激巨噬细胞NADPH氧化酶表达和活性的体外细胞培养研究目的:在实验动物模型和肾结石患者的肾脏内均发现晶体沉积的周围有大量的单核/巨噬细胞浸润,提示单核/巨噬细胞也参与肾脏内晶体的沉积过程。本研究观察草酸钙晶体刺激下巨噬细胞NADPH酶的表达和活性变化及其作用。方法:建立鼠巨噬细胞株体外培养体系,在体系中加入一水草酸钙(COM)晶体共同培养,并使用NADPH氧化酶特异抑制剂夹竹桃麻素进行干预处理。实验分4个组:A组,空白组,单纯巨噬细胞;B组,巨噬细胞+COM;C组,巨噬细胞+COM +夹竹桃麻素组;D组,巨噬细胞+夹竹桃麻素。共同培养6h后,采用光镜、扫描电镜和透射电镜观察巨噬细胞粘附、吞噬COM情况;同时采用比色法检测各组细胞培养基中MDA、LDH的水平,荧光比色法检测各组细胞内ROS水平,比色法检测各组细胞NADPH氧化酶的活性,免疫印迹检测各组巨噬细胞表达NADPH氧化酶亚基p47phox蛋白的水平。结果:①光镜和电镜下观察到巨噬细胞粘附和吞噬COM。②与A组比较,B组细胞培养液中MDA、LDH水平均明显升高,细胞内ROS生成增多,细胞NADPH氧化酶活性显著增加;使用夹竹桃麻素(C组)则可减少COM刺激下巨噬细胞培养液中MDA、LDH的水平,细胞内ROS生成减少,细胞NADPH氧化酶活性降低。③免疫印记检测结果发现,与A组比较,B组细胞表达p47phox蛋白明显增多,使用夹竹桃麻素(C组)则可抑制COM刺激下巨噬细胞p47phox蛋白的表达水平。结论:在体外巨噬细胞培养体系中,COM晶体可刺激巨噬细胞NADPH氧化酶的表达和活性,引起细胞氧化应激损伤。这可能是肾间质晶体沉积导致组织损伤的机理之一。更多还原

【Abstract】 Hyperoxaluria and crystal deposition induce renal oxidative stress (OS) and renal tubular injury which in turn contribute to the development of nephrolithiasis, both mitochondria and NADPH oxidase are considered to be the source of reactive oxygen species (ROS).This study focused on the role of NADPH oxidase in the development of renal oxidative stress induced by oxalate and calcium oxalate crystals in vitro and vivo. Apocynin, losartan and taurine were chosen here to act as the special intervenors (antioxidants). Five related parts of the study were discussed respectively. Results of this study suggest that renal angiotensinⅡand its stimulation of NADPH oxidase may partially account for the development of OS in kidney in a rat model of hyperoxaluria. We also detected evidences that the changes of mitochondria preceded that of the expression of the NADPH oxidase in kidney. In vitro, COM stimulate the expression and activity of NADPH oxidase in macrophage which may also account for the renal OS, since macrophages were seen commonly accumulating in the interstitium of kidney with crystal deposition. Part I: Establishment of a rat model of calcium oxalate nephrolithiasis, and the development of renal oxidative stress in the modelObjective To establish a rat model of calcium oxalate nephrolithiasis, and observe the development of renal oxidative stress (OS) in this model.Methods Animal model of hyperoxaluria and crystal deposition was established in adult male Sprague-Dawley rats by administration of 0.8% ethylene glycol (EG) in drinking water for 4 weeks. At the end of the study, markers for the state of OS, urinary 8-IP and H2O2, and the creatine clearnance and kidney/body weight were examined. Histological examinations of kidneys were performed to check the tubular injury and interstitium injury, as well as the crystal deposition in kidney. Renal tubular ultrastruture changes were analyzed under TEM. The expression of CD68、TGF-βandα-SMA in kidney were evaluated by immunohistochemistry.Results Compared with the control, renal OS developed significantly in rats received EG. Histological examinations of kidneys show renal tubular injury, with increased expression of CD68、TGF-βandα-SMA in kidney.Calcium crystal deposits in kidney were detected evidently.Conclusions Rat model of hyperoxaluria and crystal deposition was established successfully. Renal OS and renal injury, and calcium crystal deposits were detected evidently. Part II: Role of NADPH oxidase in the development of renal oxidative stress in a rat model of calcium oxalate nephrolithiasisObjective To investigate the role of NADPH oxidase in the development of renal oxidative stress (OS) in a rat model of calcium oxalate nephrolithiasis. Methods Rat model of hyperoxaluria and crystal deposition was established by administration of 0.8% ethylene glycol (EG) to the male SD rats in drinking water for 4 weeks. Apocynin, one of the selective NADPH oxidase inhibitor, was also administrated in the experimental group(0.2g·kg-1·d-1). At the end of the study, markers for the state of OS, urinary 8-IP and H2O2, and the kidney/body weight were examined. Immunohistochemistry, RT-PCR and Western blotting analysis were made respectively to evaluate the expression of NADPH oxidase subunits (p47phox, gp91phox, Nox-1, p22phox, Nox-4) in kidneys.Results Compared with the control, OS and renal injury occurred in the rats recived EG, in accordance with the increased expression of NADPH oxidase subunits p47phox, gp91phox, Nox-1 mRNAs and p47phox protein in kidneys. Simutanious treatment with apocynin can partially reduce OS and renal injury, as well as the expression of p47phox and Nox-1 mRNAs and p47phox protein in kidneys.Conclusions The increased expression of NADPH oxidase was suggested to be partially accounted for the development of OS in kidney in this rat model. Part III: AngiotensinⅡstimulate the expression and activity of NADPH oxidase in a rat model of calcium oxalate nephrolithiasisObjective To investigate the roles of angiotensinⅡand NADPH oxidase in the development of renal oxidative stress (OS) in a rat model of calcium oxalate nephrolithiasis.Methods Animal model of hyperoxaluria and crystal deposition was established in adult male Sprague-Dawley rats by administration of 0.8% ethylene glycol (EG) in drinking water for 4 weeks. Simultaneous treatment with apocynin (0.2g·kg-1·d-1) or losartan (30mg·kg-1·d-1) by intragastric administration were performed in rats respectively. At the end of the study, markers for the state of OS, urinary 8-IP and the enzymatic activity of superoxide dismutase (SOD) in kidney homogenates were assessed. The concentration of angiotensinⅡin kidney homogenates was determined using radioimmunoassay method.Expression of NADPH oxidase subunit p47phox protein and mRNA in kidney were localized and evaluated by immunohistochemistry and real time-PCR respectively.Results P47phox express widely in the kidneys of this rat model, including renal cortex, inner medulla and outer medulla.Compared with the control, OS developed significantly in rats received EG, with increased expression of p47phox mRNA in kidneys. Renal angiotensinⅡalso increased significantly. Treatment with apocynin or losartan significantly reduced the excretion of urinary 8-IP, restored the SOD activity, with decrease in the expression of p47phox mRNA in kidney, but the levels of those OS markers in apocynin or losartan treated rats were still higher than that of the normal controls.Conclusions Results suggest that renal AngⅡand its stimulation of NADPH oxidase may partially account for the development of renal OS in this rat model.Part IV: Mitochondria but not NADPH oxidase account for the renal oxidative stress in a rat model of calcium oxalate nephrolithisisObjective Hyperoxaluria and crystal deposition induce oxidative stress (OS) in kidney; both mitochondria and NADPH oxidase are considered to be the source of reactive oxygen species (ROS). Taurine is known as an antioxidant. We investigate the putative source of ROS, as well as the effects of taurine treatment on renal protection in a rat model of calcium oxalate nephrolithisis.Methods Animal model of nephrolithiasis was established in adult male Sprague-Dawley rats by intragastric administration of 2.5% ethylene glycol+2.5% Ammonium Chloride 2ml twice daily, with restriction on intake of drinking water(20ml /per rat daily) for 4 weeks. Simultaneous treatment with taurine (2.0% mixed with the chow) was performed. At the end of the study, indexes of OS and renal injury were assessed. Renal tubular ultrastruture changes were analyzed under TEM. Crystal deposition in kidney was analysed under light microscopy. Expression of NADPH oxidase subunit p47phox protein and mRNA in kidney were localized and evaluated by immunohistochemistry and real time-PCR respectively. AngiotensinⅡin kidney homogenates was determined by radioimmunoassay method. Results Compared with the control, oxidative injury of kidney occurred in rats induced nephrolithiasis. Hyperplasia of mitochondria developed in renal tubular epithelium .The activities of SOD and GSH-Px in mitochondria decreased significantly and the mitochondrial membrane showed oxidative injury. Taurine treatment significantly alleviated oxidative injury of kidney and its mitochondria, restored SOD and GSH-Px activities, with relative slight morphological changes in kidney. We could not detect statistically changes in the renal p47phox mRNA expression, as well as the renal angiotensinⅡin those rats. Conclusions Results suggest that mitochondria but not NADPH oxidase account for the OS in kidney, taurine protected the kidney from oxidative injury by the mitochondrial-linked pathway in this rat model.Part V: Calcium oxalate crystals stimulate the expression and activity of NADPH oxidase in macrophage in vitroObjective The NADPH oxidase was originally discovered in neutrophils, where it is a potent source of millimolar quantities of superoxide during phagocytosis and plays a vital role in nonspecific host defense. Macrophages were seen commonly accumulating around the crystals in the interstitium of kidney, which suggest that macrophage involve in the crystal deposition in kidney.We test our hypothesis that COM crystals stimulate the expression and activity of NADPH oxidase in macrophage which may also account for the renal oxidative stress (OS) and injury.Methods Confluent cultures of macrophage cells (Ana-1) were exposed to COM crystals for 6 h .Markers for the state of oxidative stress, MDA and LDH in the culture medium were measured to investigate the involvement of ROS. Cellular ROS and the activity of NADPH oxidase in macrophage cells were assessed by ?uorescence spectrophotometrical analysis and spectrophotometrical analysis, respectively. We also investigated the effect of the NADPH oxidase inhibitor apocynin on the COM crystal-induced expression of p47phox protein using western blot analysis. Cell-crystal reaction was detected by light microscopy and TEM.Results Exposure of macrophage cells to COM crystals resulted in increased expression of p47phox protein, with increased MDA and LDH in the culture medium. Cellular ROS and the activity of NADPH oxidase in macrophage cells also increased significantly. Treatment with apocynin reduced the crystal-induced expression of p47phox protein, inhibited the activity of NADPH oxidase in macrophage cells, in accordance with decrease of those OS markers. We also detected the phagocytosis of crystal by macrophage under light microscopy and TEM.Conclusions Results indicated that COM crystals stimulate the expression and activity of NADPH oxidase in macrophage .This may be one of the the mechanism of renal interstitium injury induced by crystal deposition.更多还原