【作者】 王瑞；

【导师】 丁国华；

【作者基本信息】 武汉大学 ， 内科学， 2010， 博士

【摘要】 随着人民生活水平的不断提高,高脂血症的发生率不断升高,脂质异常对肾脏的损害作用日益为人们所重视,脂质异常是肾脏损害进展的独立危险因素,已经证实食源性高胆固醇血症大鼠有蛋白尿的发生及肌酐清除率的下降,肾脏病理显示除肾小球外,小管及小管间质损害亦较突出,表现为肾小管上皮细胞肥大,炎性细胞浸润,细胞因子产生,小管间质纤维化,我科既往的实验已证实OX-LDL可通过P13K通路诱导肾小管上皮细胞肥大,他汀类药物可抑制这种作用,高胆固醇血症大鼠肾小管损伤明显,有OPN和MCP-1表达,但脂质引起小管上皮细胞损伤的具体机制仍尚不十分清楚。诸多研究表明肾小管上皮细胞发生转分化是肾小管间质纤维化进展的主要机制,但脂质是否可通过诱导肾小管上皮细胞转分化导致肾小管间质纤维化还罕有报道。Sawamura首先在牛内皮细胞上发现LOX-1是一种ox-LDL受体,后来相继在巨噬细胞、血管平滑肌细胞、单核细胞、培养的大鼠和人软骨细胞、人INT-407肠细胞等多种细胞中都发现有LOX-1表达,在这些细胞中LOX-1可介导ox-LDL的摄取与降解,还可作为一种粘附分子,参与细胞-细胞间的相互作用和黏附,参与炎症发生。已经发现LOX-1与动脉粥样硬化、缺血再灌注、心肌梗塞、类风湿性关节炎和骨关节炎等多种疾病有关,而抗LOX-1治疗可逆转病理损害,认为LOX-1可能是多种疾病治疗的一个新的靶点。极少数的有关LOX-1和肾脏的研究也提示LOX-1可能在慢性肾脏疾病进展中起作用,但肾小管上皮细胞是否有LOX-1表达尚有争议,是否被OX-LDL上调并介导其进入细胞内并进一步导致肾小管损伤以及其可能的机制尚需进一步研究。他汀类药物是羟甲基戊二酰辅酶A还原酶抑制剂,一方面通过降低总胆固醇和低密度胆固醇保护肾脏,另一方面体内体外实验证实它还有降脂以外的肾保护作用,机制亦不是十分清楚。为探讨以上问题,本课题分为四个部分：第一部分主要探讨肾小管上皮细胞表达LOX-1的水平及氧化低密度脂蛋白的诱导作用；第二部分探讨LOX-1和ROS在ox-LDL诱导的肾小管上皮细胞转分化的作用；第三部分探讨辛伐他汀对ox-LDL诱导的肾小管上皮细胞转分化的作用；第四部分探讨辛伐他汀对高胆固醇血症大鼠肾保护作用的机制。第一部分ox-LDL诱导肾小管细胞表达LOX-1目的：探讨肾小管上皮细胞表达LOX-1的水平及氧化低密度脂蛋白的诱导作用。方法：体外培养NRK-52E细胞,加入不同浓度(Oug/ml,25ug/ml,50ug/ml, 100ug/ml)的氧化低密度脂蛋白(ox-LDL)刺激24h,以及用50ug/ml ox-LDL分别孵育NRK52E细胞0h,6h,12h,24h,或先用LOX-1阻滞剂多聚肌苷酸(polyinosonic acid, polyⅠ)和爱兰苔胶(carrageenan)及N-acetyl-L-cysteine (NAC)预处理后与50ug/ml ox-LDL共同孵育24h,以实时定量PCR (realtime PCR)测定LOX-1 mRNA的表达水平,激光共聚焦和Western blotting测定LOX-1蛋白表达,油红O法检测细胞内脂质,激光共聚焦检测细胞内ROS水平。结果：1.激光共聚焦显示LOX-1低水平表达于正常NRK-52E细胞膜上。2.Realtime-PCR和Western blotting显示25,50,100 ug/ml ox-LDL都可以增加LOX-1mRNA转录和蛋白的表达(P<0.05),并且随刺激浓度的增加而增高,(P<0.05)；50ug/ml ox-LDL 6h即可刺激NRK-52E细胞膜LOX-1 mRNA转录和蛋白的表达,并且随刺激时间延长而转录和表达增加。3.随着细胞LOX-1表达的增加,细胞内脂质增多,ROS生成增多。4. polyⅠ、carrageenan和NAC预处理后再与50ug/ml ox-LDL一起孵育,50ug/ml ox-LDL刺激的LOX-1表达、ROS生成及细胞内脂质均减少。结论：正常NRK-52E细胞膜低水平表达LOX-1, ox-LDL能刺激LOX-1表达增加,而LOX-1可促进细胞脂质摄入和活性氧生成。第二部分LOX-1和ROS在ox-LDL诱导的肾小管上皮细胞转分化中的作用目的：研究LOX-1和ROS在ox-LDL诱导肾小管上皮细胞转分化中的作用。方法：体外培养NRK-52E细胞,加入不同浓度(0ug/ml,25ug/ml,50ug/ml, 100ug/ml)的氧化低密度脂蛋白(ox-LDL)刺激24h,以及先用LOX-1阻滞剂多聚肌苷酸(polyinosonic acid, polyⅠ)和爱兰苔胶(carrageenan)及N-acetyl-L-cysteine(NAC)预处理后与50ug/ml ox-LDL共同孵育24h,以Western blotting测定a-SMA和E-cadherin表达,荧光显微镜检测a-SMA表达。结果：随着ox-LDL刺激浓度的增加,细胞开始表达a-SMA并逐渐增多,而E-cadherin的表达逐渐减少,细胞发生转分化。用poly I和carrageenan阻滞LOX-1表达或用抗氧化剂NAC减少ROS生成后,a-SMA和E-cadherin的变化得到部分逆转。结合第一部分,E-cadherin蛋白与LOX-1和ROS呈密切负相关,相关系数分别为-0.94和-0.82,a-SMA蛋白与LOX-1和ROS呈密切正相关,相关系数分别为0.97和0.87。结论：LOX-1和ROS在ox-LDL诱导肾小管上皮细胞转分化中起重要作用。第三部分辛伐他汀对ox-LDL诱导肾小管上皮细胞转分化作用的影响目的：观察辛伐他汀对ox-LDL诱导肾小管上皮细胞转分化作用的影响。方法：体外培养NRK-52E细胞,分为正常组(Oug/ml ox-LDL)、ox-LDL组(50ug/ml ox-LDL)、辛伐他汀组(50ug/ml ox-LDL+10μmol/L辛伐他汀),孵育24小时后以Western blotting测定LOX-1蛋白、a-SMA和E-cadherin表达,激光共聚焦检测细胞内ROS水平,荧光显微镜检测a-SMA表达。结果：与正常组相比,ox-LDL组NRK-52E细胞LOX-1表达增高,ROS生成增多,a-SMA并逐渐增多,而E-cadherin的表达逐渐减少,细胞发生转分化。辛伐他汀可明显抑制LOX-1表达,减少ROS生成,a-SMA和E-cadherin的变化也得到部分逆转。结论：辛伐他汀可能通过下调LOX-1和ROS抑制ox-LDL诱导的肾小管上皮细胞转分化。第四部分辛伐他汀他汀对高胆固醇血症大鼠的肾保护作用目的：评价辛伐他汀对高胆固醇血症大鼠的肾保护作用方法：将SD大鼠分为正常组、高脂组、治疗组,12周后处死。高脂组饲以高脂饮食,治疗组除高脂饮食外给予10mg·kg-·d-1的辛伐他汀灌胃,12周后观察肾小球病理形态变化；检测肾皮质丙二醛(malondialdehyde,MDA)含量、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase GSH-Px)的活性并用免疫组化方法观察LOX-1、α-SMA和E-cadherin表达的变化。结果：与正常组相比,模型组的大鼠肾皮质MDA水平升高,SOD、GSH-Px、活性下降,肾小管上皮细胞LOX-1和a-SMA表达增高,E-cadherin表达下降,辛伐他汀治疗后可以降低肾皮质MDA水平的升高,增强SOD、GSH-Px活性,减少LOX-1和a-SMA,增加了E-cadherin在小管的表达,改善了肾小管上皮细胞转分化。结论：辛伐他汀可能通过下调LOX-1表达,降低肾皮质氧化应激水平抑制肾小管上皮细胞转分化起到保护脂质肾损害。更多还原

【Abstract】 Dyslipidemia is the independent risk factor for the progression of renal damage. It has been confirmed that proteinuria increased and creatinine clearance rate decreased in diet-induced hypercholesteremic rats. Renal pathology disclose obvious tubule-interstitial injuries, which display with tubular epithelial cell hypertrophy, inflammatory cell infiltration, cytokine generation and tubular interstitial fibrosis, but the mechanism is unclear.Many research indicate that tubular epithelial-myofibroblast transdifferentiation (TEMT) is the major mechanism in the development of tubular interstitial fibrosis, However, there are few reports regarding the TEMT induced by ox-LDL.LOX-1 is a recently discovered specific ox-LDL receptor that is found to be expressed in macrophage, vascular smooth muscle cell, monocyte endothelial cell, cartilage cell and many other cells. It has been demonstrated that LOX-1 not only mediated the digestion and degradation of ox-LDL, but also mediate multiple pathological injuries,such as ischemia-reperfusion,injured myocardium, atherosclerosis, rheumatoid arthritis.A few studies on LOX-1 expression in the kidney have suggested that LOX-1 might have an effect on the development of chronic renal disease. However,there is some controversy whether the LOX-1 is expressed in the tubular epithelial cells and mediate ox-LDL into tubular epithelial cells to lead to tubule injury. Statins is a HMG-CoA reductase inhibitors.It protects kidney through decreasing TG and LDL.On the other hand,it has many lipid-lowering-independent effects which mechanism is not clear.To approach the above question, present study was designed as following four parts:Part I was to evaluate the expression of LOX-1 on tubular epithelial cells and the effect of ox-LDL on LOX-1 in vitro; partⅡwas to investigate the effect of oxidized low density lipoprotein receptor-1 (LOX-1) and ROS on TEMT induced by oxidized low-density lipoprotein (ox-LDL);part III was to investigate the renal protective effect of simvastatin on TEMT induced by oxidized low-density lipoprotein; Part IV was to investigate the renal protective effect of simvastatin on tubular epithelial-myofibroblast transdifferentiation (TEMT) in diet-induced hypercholesteremic rats. PartⅠox-LDL induce tubular epithelial cells to express LOX-1Objective:To evaluate in vitro the expression of LOX-1 in tubular epithelial cells and the effect of ox-LDL on LOX-1 expression.Methods:NRK-52E cells were incubated with ox-LDL (0,25,50,and 100ug/ml) for 24 hours or incubated with ox-LDL of 50ug/ml for 0h,6h,12h,24h or pre-treated with the chemical inhibitor of the LOX-1 receptor polyinosinic acid (polyⅠ) and carrageenan or the antioxidant N-acetyl-L-cysteine (NAC), the cells were then exposed to 50ug/ml of ox-LDL.The expression of LOX-Ⅰ, and reactive oxygen species (ROS) were analyzed by real-time PCR, western blotting, immunofluorescence and confocal laser scanning microscopy. Lipid was examined by oil red O.Results:1. LOX-1 was expressed on the cytomembrane of NRK-52E at low level. 2. Ox-LDL stimulated the expression of LOX-1 mRNA and protein in dose-and time-dependend manners.3. Following the increase in the LOX-1 protein level, the lipid intake and ROS generation were increased.4. Pre-treatment with polyⅠ, carrageenan or NAC could inhibit ox-LDL induced LOX-1 expression, lipid intake and ROS generation.Conclusion:Ox-LDL increased the expression of LOX-1 mRNA and protein in a dose-and time-dependent manners. LOX-1 can promote lipid intake and ROS generation.PartⅡ:Effect of LOX-1 and ROS on Oxidized low-density lipoprotein induced tubular epithelial-myofibroblast transdifferentiationObjective:To explore the effect of LOX-1 and ROS on oxidized low-density lipoprotein induced tubular epithelial-myofibroblast transdifferentiation.Methods:NRK-52E cells were incubated with ox-LDL (0,25,50, and 100ug/ml) for 24 hours or pre-treated with the chemical inhibitor of the LOX-1 receptor polyinosinic acid (polyⅠ) and carrageenan or the antioxidant N-acetyl-L-cysteine (NAC), the cells were then exposed to 50ug/ml of ox-LDL.The expression of a-SMA, and E-cadherin were analyzed by Western blotting and immunofluorescence. Results:1. Ox-LDL increased the a-SMA protein expression and decreased the E-cadherin protein expression in a dose-dependent manner from Oμg/L to 100μg/L (P<0.05).2. When LOX-1 was blocked by chemical inhibitor of the LOX-1 receptor poly I and carrageenan or ROS was inhibited by NAC,the increased a-SMA and decreased E-cadherin protein were reversed. Combined with part I, a-SMA protein has a positive relationship with LOX-1 and ROS(r=0.97,0.87), E-cadherin protein has a negative relationship with LOX-1 and ROS(r=-0.94,-0.82).Conclusion:LOX-1 and ROS play a vital role in oxidized low-density lipoprotein induced tubular epithelial-myofibroblast transdifferentiation.PartⅢ:Effect of simvastatin on oxidized low-density lipoprotein induced tubular epithelial-myofibroblast transdifferentiationObjective:To explore the effect of LOX-1 and ROS on oxidized low-density lipoprotein induced tubular epithelial-myofibroblast transdifferentiation.Methods:NRK-52E cells were divided into three groups:control group(0ug/ml ox-LDL), ox-LDL group(50ug/ml ox-LDL), simvastatin group(50ug/ml ox-LDL+10 umol/L simvastatin). Following incubation for 24h, expression of LOX-I, a-SMA and E-cadherin and production of reactive oxygen species (ROS) were analyzed by Western blotting, immunofluorescence and confocal laser scanning microscopy.Results:1. Compared with control group, ox-LDL stimulated expression of LOX-1 and a-SMA and ROS generation, but inhibited expression of E-cadherin.2. Simvastatin inhibited LOX-1 expression and ROS generation. Simvastatin also reversed the increased a-SMA and the decreased E-cadherin expression (P<0.05)Conclusion:Simvastatin inhibited oxidized low-density lipoprotein induced tubular epithelial-myofibroblast transdifferentiation through down regulating LOX-1 and ROS. PartⅣ:Simvastatin inhibit tubular epithelial-myofibroblast transdifferentiation in hypercholesteremic rats through down regulation of LOX-1 and oxidative stressObjective:To investigate effect of Simvastatin on tubular epithelial-myofibroblast transdifferentiation in hypercholesteremic rats and the possible mechanism.Methods:The SD rats were divided into three groups as follows:normal group,high fat diet group and simvastatin group(rats were fed with high fat diet plus 10 mg·kg-1·d-1 simvastatin). Six rats in each group were sacrificed at 12th week. Levels of total cholesterol (TC) and triglyceride(TG) in serum were measured by enzymatic colormetric methods.The injury of renal tubulointerstitium was observed under microscope with PAS and masson staining and the expression of renal LOX-1,α-SMA and E-cadherin were determined by immunohistochemistry.In addition, the activity of antioxidant including SOD, GSH-PX and level of MDA in the cortex of kidney were measured.Results:1. Compared with normal group, MDA levels in the cortex of kidney was higher and SOD、GSH-Px activities in the cortex of kidney were lower in high fat diet group.2. Compared with normal group, LOX-1 and a-SMA expression were higher and E-cadherin expression were lower in renal tubular epithelial cell.3. Simvastatin could desease MDA generation and expression of LOX-1 and a-SMA, but increased SOD and GSH-Px activities and E-cadherin expression with improvement of tubular epithelial-myofibroblast transdifferentiation.Conclusion:Simvastatin can ameliorate tubular epithelial-myofibroblast transdifferentiation via the inhibition of LOX-1 and oxidative stress in hypercholesteremic rats.更多还原