【作者】 黄朝晖；

【导师】 吴雄飞；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 研究背景粥样硬化性肾动脉狭窄(atherosclerotic renal artery stenosis, ARAS)是肾动脉狭窄( renal artery stenosis, RAS)最常见的病因。ARAS的进展导致肾动脉闭塞、肾小球滤过率(GFR)明显下降和肾脏缩小时,诊断为缺血性肾病。目前认为重度肾动脉狭窄和缺血性肾病的病理生理变化是狭窄后肾缺血,不断激活压力调节系统(包括激活肾素-血管紧张素系统、刺激肾上腺素分泌等)导致恶性高血压发生和肾小球滤过功能丧失,肾动脉进行性狭窄是导致缺血性肾病的发生和进展至终末期的主要原因。现行血管重建治疗的指征是肾动脉狭窄超过75%,但国内外大量文献提示:已经明显狭窄的ARAS患者血管支架术后肾功能无法逆转反而恶化,提示早期诊断和治疗ARAS非常重要,然而,由于对早期ARAS病理生理机制尚不清楚,目前还缺乏有效监测和治疗手段。现有研究成果表明:ARAS是全身动脉粥样硬化性疾病在肾动脉的表现,而心血管领域的研究早已证实动脉粥样硬化病变的实质是慢性进展性炎症反应,动脉粥样硬化斑块的形成、破裂及进行性增大与慢性进展性炎症反应密切相关;临床观察到轻中度ARAS其肾动脉狭窄程度与肾损害严重程度并非完全平行;国外学者业已在临床研究中发现ARAS患者肾动脉狭窄并不是导致肾实质损害的主要原因,推测ARAS患者的慢性肾损害可能是由血管狭窄以外的其它因素所致。这些研究结果提示我们:导致动脉粥样硬化的炎症反应是否同时作用于肾实质?除肾动脉狭窄引起肾脏慢性缺血性损害以外是否有炎症因素参与其下游肾脏实质的慢性损害?由此我们设想:①炎症反应可能是ARAS肾损害进展的重要机制之一;②通过下游肾脏病理的变化、肾功能损伤程度以及预测因子的动态变化,可预测上游ARAS的活动情况,为临床提供早期的治疗时机;③解除和延缓肾动脉狭窄同时控制可能存在的炎症反应也许会成为更有效的治疗策略。为验证这些设想,我们选择ApoE-/-小鼠――目前世界公认的研究动脉粥样硬化(AS)的成熟动物模型来建立ARAS模型,动态观察ApoE-/-小鼠在ARAS发病过程中肾脏损害的特点;对ApoE-/-小鼠ARAS肾脏炎症损害机制进行实验研究;探讨Rapamycin对ApoE-/-小鼠ARAS肾损害进展的干预作用;在动物实验研究的基础上,验证炎症机制在ARAS患者肾损害中的重要作用,并对肾动脉支架置入术时机进行初步的探讨。主要实验方法及结果一:ARAS小鼠模型的建立及ARAS肾损害特点观察选取同周龄C57BL/6J ApoE基因敲除小鼠及C57BL/6J野生型小鼠,取材25周龄至51周龄小鼠的肾动脉和肾脏,肾动脉连续切片、V.G染色,通过Image Pro-Plus图像分析系统(Media Chernetics Inc.USA)测量、计算肾动脉的狭窄程度。根据ARAS程度分3组(A组:<50%;B组:50%-75%;C组:>75%)。A组又根据斑块是否破裂分为A1(未破裂)和A2(破裂)组。肾脏常规HE、PAS和Masson染色,采用电镜及普通光镜动态观察各组ApoE-/-小鼠肾动脉病变及相应的肾脏病变。结果:1、利用ApoE-/-小鼠成功建立了粥样硬化性肾动脉狭窄动物模型;2、肾动脉和肾脏的变化是:①A1组未发现其下游肾脏病理改变;②A2组其下游肾内肾小管周围毛细血管减少,肾小管上皮细胞肿胀,电镜下可见细胞内线粒体肿胀;③B组和C组已发生斑块破裂,其肾内肾小管周围毛细血管减少明显,与A2组比较,差异显著(p<0.01,p<0.001);肾小管上皮细胞肿胀、脱落、坏死;肾小管间质病变严重;④微血栓形成;⑤斑块破裂后,肾动脉狭窄程度与肾小管周围毛细血管(PTC)密度、肾小管间质损伤(TI)程度明显负相关,PTC密度与TI程度明显负相关。结论:选择ApoE-/-小鼠成功建立ARAS模型;ARAS是进展性疾病;肾损害的出现不仅仅是狭窄造成的缺血性损害,缺血以外的损伤机制参与其中,极有可能是动脉粥样硬化病变的慢性进展性炎症反应。为此在实验第二部分对肾损害的炎症机制进行探讨。二:ApoE-/-小鼠肾动脉粥样硬化病变进展导致下游肾损害的炎症机制1、肾动脉粥样硬化病变活动造成肾损害的炎症因素的测定:选择各组ApoE-/-小鼠为实验组,同期喂养的野生型C57BL?6J小鼠作为对照组。取各组肾动脉及肾脏:采用Western blotting及免疫组化法检测NF-κBp65、ICAM-1及P-sel表达; ELISA检测IL-6和TF的表达;RT-PCR检测IL-6mRNA测定;免疫组化法检测巨噬细胞表达;采用激光共聚焦显微镜检测促炎症反应优势的单核巨噬细胞在各组小鼠肾组织中分布变化。2、ApoE-/-小鼠ARAS肾损伤与炎症因素的关系:分别取各实验组及对照组的肾组织,免疫组化方法检测α-SMA;偏振光显微镜观察ColⅠ、Col III在肾组织中表达情况;酶-底物直接显色法检测尿NAG;全自动生化分析仪检测血肌酐。研究炎症因素与肾脏纤维化及肾功能损害的相关性。结果:1、NF-кBp65在ARAS50%-75%的B组表达显著高于ARAS小于50%斑块已破裂的A2组,A2组明显高于ARAS小于50%斑块未破裂的A1组,而A1组与对照组相比,差异无统计学意义;其受控因子ICAM-1、P-sel、IL-6的表达变化与NF-кBp65的表达变化相似即B组表达显著高于A2组,A2组明显高于A1组,而A1组与对照组相比,差异无统计学意义;巨噬细胞及α-SMA的表达变化也与NF-кBp65的表达变化相似;尿NAG测定量变化也与NF-кBp65的表达变化相似。2、巨噬细胞在:A2亚组和B组的促炎优势单核巨噬细胞在肾小管间质及肾小球均有分布,但主要分布于肾小管、肾间质和肾血管,以肾间质最为明显;其分布数量于B组较A2组呈明显增多,且与肾功能减退呈正相关。B组肾小管间质区a-SMA及Ⅰ型胶元(Col I)、Col III蛋白质表达均明显增加,并与TIF程度以及促炎优势单核巨噬细胞分布数量呈正相关。对照组和A1亚组促炎优势单核巨噬细胞分布及数量明显少于A2组和B组, a-SMA、Ⅰ型胶元(Col I)、Col III蛋白质表达明显少于A2组和B组,无肾小管间质损害表现,肾功能正常。结论:炎症反应是ARAS进行性狭窄及其下游肾损害进展的重要机制之一。炎症反应在导致ARAS的同时,也同样损伤肾实质,因此炎症反应伴随在ARAS及其肾损害的始终,炎症机制是始动因素,缺血机制在后,共同控制着下游的肾损害。当ARAS到一定程度,缺血因素发生,同时又会促进下游炎性肾损害加剧,如此恶性循环导致ARAS肾损害加剧。为了进一步证实这一重要的炎症机制,我们在实验第三部分通过药物控制这种炎症反应,观察是否可以延缓ARAS肾损害的进程?三:免疫抑制剂雷帕霉素对ApoE-/-小鼠ARAS肾损害进展的干预作用。由于雷帕霉素可以抑制淋巴细胞的活化而控制炎症反应,同时雷帕霉素涂层支架在治疗动脉粥样硬化性狭窄后可以控制炎症反应,近期初步的临床实验证实雷帕霉素可以延缓慢性增殖性肾病,所以我们选择雷帕霉素作为本试验炎症反应控制药物。1、体内实验:选择①33周龄组ApoE-/-小鼠;②药物组:33周龄ApoE-/-小鼠,予雷帕霉素100ug/kg(BW)/day灌胃8周至41周龄;③41周龄组ApoE-/-小鼠;④对照组:41周龄野生型C57BL?6J小鼠。每组6只,均为雄性。检测各组肾动脉的狭窄程度、肾动脉和肾脏M1型巨噬细胞浸润情况、NF-κBp65及IL-6mRNA的表达情况及肾脏纤维化程度。2、体外培养:采用雷帕霉素及LPS共同刺激小鼠腹腔巨噬细胞,检测各组小鼠腹腔巨噬细胞的NF-κBp65和MHCⅡ的表达情况及IL-6mRNA的转录水平变化。研究雷帕霉素对单核巨噬细胞促炎症优势的影响。结果:1、雷帕霉素能显著下调ApoE-/-小鼠肾脏α-SMA和Ⅰ型、Ⅲ型胶原纤维的表达;显著减少肾动脉斑块处及肾脏间质M1型巨噬细胞浸润数量;下调肾动脉及肾脏组织NF-κBp65的表达;减少NF-κBp65下游IL-6的表达。2、小鼠巨噬细胞经LPS刺激1h后,其核内的荧光强度(代表的NF-κBp65表达量)显著增强.与LPS刺激组相比,不同浓度的雷帕霉素+LPS组均能使NF-κBp65的荧光强度值降低,有显著性差异,雷帕霉素使NF-κBp65的荧光强度值降低呈剂量依赖性。结论:雷帕霉素可以控制ARAS所致的肾脏炎症反应,延缓肾脏纤维化,再次证实ApoE-/-小鼠ARAS肾损害的重要发生机制之一是炎症反应。四:临床实验:在临床ARAS患者中初步验证动物实验结论。观察尿NAG/Ucr、尿液巨噬细胞阳性率、肾活检应用于ARAS筛查及监测治疗效果的可能性。检测17例ARAS患者、20例合并≥2项动脉粥样硬化危险因素的动脉粥样硬化疾病患者及20例同年龄健康者的尿NAG/Ucr、尿液巨噬细胞阳性率、ARAS组行3例肾活检等检测肾小管间质炎症反应指标,观察支架置入术前后及未行支架术患者的尿NAG/Ucr、尿液巨噬细胞阳性率的变化。结果:1、尿NAG/UCr、尿液巨噬细胞阳性率能反映ARAS肾小管间质的受损情况,对监测ARAS进展及观察支架术治疗的效果有一定帮助,但与ARAS狭窄程度不一定完全平行;2、3例肾活检提示ARAS患者肾损害的重要发生机制仍然是炎症反应,其首先累及肾小管间质。由于收集样本时间有限造成样本量过小,需加进一步大样本量研究。结论:尿NAG/UCr、尿液巨噬细胞阳性率及肾脏病理可以作为预测ARAS活动的指标,提示控制炎症治疗和放置血管支架的时机。全文结论炎症反应是ARAS病变及其下游非缺血肾损害的重要机制之一。早期控制炎症反应不但可以延缓或阻断ARAS的进展,同时也是保护ARAS患者肾功能的重要手段。更多还原

【Abstract】 BackgroundARAS is the most common cause for renal artery stenosis. As ARAS results in renal artery occlusion, significant reduction in the glomerular filtration rate (GFR) and kidney atrophy, ischemic nephropathy can be diagnosed. It is currently thought that the pathophysiology of severe renal artery stenosis and ischemic nephropathy is stenosis-induced renal ischemia, malignant hypertension secondary to continual activation of the blood pressure regulating systems (the renin-angiotension system and adrenal glands) and glomerular filtration dysfunction. Progressive renal artery stenosis is a major cause for end-stage ischemic nephropathy. Current indication for renal angioplasty is renal artery stenosis > 75%. Nevertheless, it is suggested that vascular stenting worsens renal function of patients with significant ARAS. Therefore, early diagnosis and treatment of ARAS is crucial. Nevertheless, since the mechanism of early ARAS remains unclear, no effective method to monitor and treat ARAS is available. It is demonstrated that ARAS is a manifestation of systematic atherosclerosis in the renal artery, and that atherosclerotic changes are essentially chronic progressive inflammatory reactions. The formation, rupture and progressive enlargement of atherosclerotic plaques are closely associated with chronic progressive inflammatory reactions. Clinical observations showed that the degree of mild to moderate ARAS does not parallel to the degree of renal damage. Clinical research has demonstrated that ARAS is not the main cause for renal parenchymal damage. Hence, it is postulated that chronic renal damage may result from other factors than renal artery stenosis in ARAS patients. These results suggest that atherosclerotic inflammatory reaction may also act on the renal parenchyma.Hence, we postulate that:①inflammatory reaction may be one of the important mechanisms for renal impairment progression in ARAS;②the severity of renal impairment ought to be consistent with that of renal artery atherosclerosis. Current methods to examine the renal artery cannot accurately reflect renal artery atherosclerosis and renal impairment. Nevertheless, the degree of ARAS can be inferred according to renal pathological changes and renal functional impairment, particularly tubular functional impairment. Regarding the timing for ARAS treatment, renal pathology and function should be stressed. In particular, dynamic detection of tubular function may help identify ARAS progression early;③ARAS-caused renal impairment is not always consistent with the severity of RAS; hence, the timing of vascular stenting cannot be determined according to the severity of renal artery stenosis.In the present study, ARAS models were established in ApoE-/- mice, and then the characteristics of renal impairment were dynamically observed during ARAS. The mechanisms of renal impairment in ARAS and the effect of rapamycin, an anti-inflammatory drug, on the progression of renal impairment were explored. Furthermore, in order to investigate the role of inflammation in ARAS-caused renal impairment and the timing for renal artery stent placement, renal arteriography, biopsy and tubular function testing were carried out in ARAS patients.Methods and results1. Establishment of ARAS models and observations of ARAS-caused renal impairment.The renal arteries and kidneys were harvested from C57BL/6J ApoE-knockout mice and wild-type mice of the same age. The renal arteries were embedded in paraffin, sectioned successively, and subjected to V.G staining. The sections were analyzed for renal artery stenosis using Image Pro-Plus image analysis system (Media Chernetics Inc. USA). Based on the severity of ARAS, the animals were divided into 3 groups (A: renal arterial luminal stenosis <50%; B: 50% -70%; C: >70%). In addition, group A was subdivided to A1 (without atherosclerotic plaque rupture) and A2 (with atherosclerotic plaque rupture) according to the status of atherosclerotic plaques. Kidney sections were subjected to conventional HE, PAS and Masson staining, and observed dynamically under electronmicroscopy and light microscopy for renal artery lesions and renal lesions.Results: 1. ARAS models were established successfully in ApoE-/- mice; 2. with the progression of renal artery atherosclerotic lesions, i.e., rupture and progressive enlargement of atherosclerotic plaque, tubular interstitial impairment occurred and aggravated, which resulted in tubular interstitial fibrosis and nephron destruction. 2. Mechanisms of renal impairment due to progression of renal artery atherosclerotic lesions in ApoE-/- mice.(1) ApoE-/- mice from groups A1 and A2 and wild-type mice of the same age were raised under the same conditions. In order to investigate the mechanism of renal impairment due to rupture of renal artery atherosclerotic plaques, NF-κB p65, ICAM-1 and P-sel expression was detected by Western blotting; IL-6 expression was detected by ELISA; IL-6 mRNA was detected by RT-PCR; macrophages were observed immunohistochemically; urine NAG was detected by direct enzyme-substrate coloration.(2) ApoE-/- mice were used in the experimental group, and wild-type mice were included in the control group. In order to investigate the effect of progressive lesion enlargement after renal artery atherosclerotic plaque rupture on renal inflammatory impairment and tubular interstitial fibrosis in ApoE-/-mice,α-SMA, ICAM-1, P-sel, NF-κBp65 expression and macrophages (F4/80) in the kidney tissue were detected immunohistochemically; IL-6 and TF expression was detection by ELISA; urine NAG was detected by direct enzyme-substrate coloration; blood creatinine was determined using an automatic biochemistry analyzer.(3) In order to investigate the role of mononuclear macrophages in ARAS-caused renal impairment of ApoE-/- mice, kidney tissue of groups A1, A2, B and of the control group were observed under laser confocal scanning microscopy for changes in the distribution of proinflammatory mononuclear macrophages;α-SMA was detected immunohistochemically; the expression of Col I and Col III was observed under polarizing microscopy. Results:①NF-κB p65 dominated ARAS progression, which played important roles in tubular-interstitial cell phenotypic transformation, tubular interstitialα-SMA expression upregulation, extensive necrosis of tubular epithelial cells and interstitial fibrosis, and inflammatory cell infiltration, as well as interstitial inflammatory impairment.②with rupture and enlargement of renal artery atherosclerotic plaques, pro-inflammatory, activated macrophages accumulated at the site of plaque ruptures and in tubular interstitium.③Aggravation of inflammatory reactions was the major cause for rupture and progressive enlargement of renal artery atherosclerotic plaques and subsequent renal artery stenosis, and was also one of the key factors causing progressive tubular interstitial impairment and nephron destruction. 3. Effect of rapamycin on the progression of ARAS-caused renal impairment in ApoE-/- mice.(1) In vivo study: 24 male mice were used in the study:①33-week-old ApoE-/- mice;②medication group: 33-week-old ApoE-/-mice, which were intragastrically administered 100ug/kg rapamycin daily for 8 to 41 weeks;③41-week-old ApoE-/-mice;④control group: 41-week-old wild-type mice (n=6 for each group). The severity of renal artery stenosis, M1-type macrophage infiltration in the renal artery and kidney, NF-κBp65 and IL-6mRNA expression, and the severity of renal fibrosis were investigated in each group.(2) In vitro cell culture: In order to investigate the effect of rapamycin on the proinflammatory properties of mononuclear macrophages, mouse peritoneal macrophages were co-stimulated with rapamycin and LPS, and the changes in NF-κBp65 and MHCII expression and IL-6mRNA transcription were detected in each group. Results:①rapamycin significantly reduced M1-type macrophage infiltration in renal artery atherosclerotic plaques and renal interstitium, downregulated NF-κBp65 expression by the renal artery and renal tissue, the transcription of IL-6 (a central regulator of inflammatory reaction), and the expression of renalα-SMA and types I, III collagen;②rapamycin inhibited the activation by LPS of MHCII and NF-κBp65 expression by mouse peritoneal macrophages in a dose dependent manner. These findings support inflammatory reaction as an important pathological mechanism of ARAS caused renal impairment.4. In patients on high risk of ARAS, tubular interstitial inflammatory reaction was assessed by determining urine NAG/Ucr and the urine macrophage detection rate and performing renal biopsy, if necessary. ARAS patients were screened through color Doppler of the renal artery or/and nephrogram, and were treated by renal artery stenting after definite diagnosis by arteriography. Changes in urine NAG/Ucr and the urine macrophage detection rate were observed before and after stent placement and in patients who did not undergo stenting.Results: 1. Inflammatory reaction was found to be an important mechanism for ARAS caused renal impairment, which affected tubular interstitium first; 2. Urine NAG/UCr and the urine macrophage detection rate reflected tubular interstitial impairment, thus helping monitor ARAS progression and assess the therapeutic effect of stenting; 3. The severity of ARAS stenosis was not an indicator for treatment, but active ARAS lesions should be treated.Conclusions1. ARAR models were established successfully in C57BL/6J ApoE-/- mice. Active ARAS lesions in ApoE-/- mice, i.e., rupture and progressive enlargement of atherosclerotic plaques, are an important factor promoting and aggravating renal lesions, and tubular interstitium is the first to be affected.2. Aggravation of inflammatory reaction acts as an important mechanism of active ARAS lesions, i.e., rupture and progressive enlargement of renal artery atherosclerotic plaques, and ARAS-caused renal impairment is mediated by this mechanism. Early control of renal artery inflammatory reaction is beneficial for delaying or suppressing the occurrence of tubular interstitial lesions and protecting renal function.3. Both in vivo and in vitro experiments indicated that rapamycin①reduces the accumulation of macrophages in the renal artery and renal tissue of ApoE-/- mice and suppresses the proinflammatory action of macrophages;②downregulates the expression of inflammatory factors;③reduces renal mesenchymal cells. Therefore, rapamycin suppresses renal artery atherosclerosis and tubular interstitial fibrosis, and protects renal function. These results support inflammatory reaction as an important pathogenetic mechanism of ARAS.4. Clinical studies preliminarily indicate that:①active ARAS lesions should be treated;②urine testing and renal biopsy may indicate active renal artery lesions, thus helping guide the treatment;③a preliminary strategy to treat ARAS is to combine anti-inflammatory therapy with early renal artery stenting.更多还原