【作者】 代鑫；

【导师】 王新良；

【作者基本信息】 河北医科大学 ， 儿科学， 2009， 硕士

【摘要】 目的:IgA肾病(IgA nephropathy,IgAN)是目前世界上最常见的慢性进行性的原发性肾小球疾病。其主要病理特征是IgA免疫复合物在系膜区沉积,导致系膜细胞的增殖和细胞外基质的沉积,引起一系列临床及病理生理改变,是导致终末期肾病(ESRD)的主要原因之一。随着肾脏活组织穿刺技术的逐渐推广,儿童IgA肾病的诊断率也在逐年提高,但目前尚无特效的治疗方法,临床仍以控制症状及经验性治疗为主。以往认为IgA肾病是一种良性疾病,预后好。但长期随访结果发现约20%～50%的成人患者最终发展至肾功能衰竭[1]。儿童患者预后较成人好,但起病5年、10年、15年后分别有5%、6%及11%的患儿产生慢性肾功能衰竭。因此如何积极防治IgA肾病在我国肾脏病研究中具有举足轻重的地位。大量的研究表明肾素-血管紧张素系统(Renin-Angiotensin System,RAS),尤其是血管紧张素Ⅱ(angiotensinⅡ,ATⅡ),是慢性肾脏疾病进行性发展的重要影响因素。RAS在IgA肾病的发病中也有重要作用。应用RAS阻滞剂可有效改善IgA肾病的临床症状,血管紧张素Ⅱ受体拮抗剂(AngiotensinⅡreceptor blockers,ARB)是肾素-血管紧张素系统(RAS)抑制剂,近年来被有效地用于IgA肾病的治疗。氯沙坦是特异性血管紧张素ⅡI型受体拮抗剂,对于IgA肾病患者,尤其是合并蛋白尿或高血压的患者,氯沙坦有利于保护肾功能延缓肾衰竭的发生。研究表明,p38MAPK信号转导通路可被血管紧张素Ⅱ等炎性因子激活,在炎症反应中发挥着重要的作用而且通过多种途径促进肾脏纤维化。而血管紧张素ⅡI型受体拮抗剂的肾脏保护作用是否通过阻断p38MAPK转导通路改善肾脏纤维化、延缓肾小球硬化的进程目前尚无明确定论。我们以IgA肾病大鼠为模型,观察氯沙坦对IgA肾病大鼠肾脏组织中p38MAPK表达情况的影响,初步探讨血管紧张素Ⅱ受体拮抗剂的肾脏保护作用机制及治疗IgA肾病的作用机制。方法:Wistar雄性大鼠,由河北医科大学实验动物中心提供(试验动物质量合格证编号:702022)。按汤颖等[2]建立的方法,选取体重100~120g雄性Wistar大鼠30只按随机数字表随机分成对照组(n=10);IgA肾病组(n=10);氯沙坦(losartan,杭州默沙东制药公司提供)组(n=10)。IgA肾病组和氯沙坦组大鼠以牛血清白蛋白400mg·kg-1·d-1隔天灌胃,持续6周;皮下注射蓖麻油0.5mL+四氯化碳0.1mL,每周1次,持续9周;分别于第6、8周以脂多糖0.05mg尾静脉注射,直至10周末。实验期间对照组动物自由进食饮水,不给予任何药物干预。建模后将IgA肾病组大鼠肾皮质组织切成3μm的冰冻切片,采用兔抗大鼠IgA和FITC标记羊抗兔IgG间接免疫荧光染色,荧光显微镜下观察到肾小球系膜区IgA沉积阳性,PAS和Masson染色显示系膜细胞增生和系膜基质的沉积表明造模成功。模型建成后,氯沙坦组大鼠给予氯沙坦20mg·kg-1·d-1灌胃四周,对照组给予等量蒸馏水灌胃四周,总实验周期为14周。各组大鼠处死前用代谢笼收集大鼠24小时尿液,测定24小时尿蛋白。股动脉放血分离血清测定肾功能。取部分肾组织置于4%多聚甲醛固定用于光镜观察及免疫组化检测;部分肾皮质组织用于提取肾组织总RNA和肾组织总蛋白。免疫组化检测肾皮质磷酸化p38蛋白激酶(p-p38MAPK)的表达特征。Western Blot检测肾脏磷酸化和非磷酸化p38MAPK的表达水平。RT-PCR检测大鼠肾组织中p38MAPK的mRNA含量。所有实验数据用均数±标准差(x±s)表示,用SPSS 11.5统计软件进行分析。组间差异用单因素方差分析(one way-ANOVA),有显著差异者用Newman-Kuels q检验进行两两比较,以P<0.05为有统计学意义。结果1生化指标显示,IgA肾病组大鼠24小时尿蛋白(24hUpro)、血尿素氮(BUN)和血肌酐(SCr)明显高于对照组(P<0.01),血白蛋白明显低于对照组(P<0.01);氯沙坦组与IgA肾病组相比各指标均有不同程度改善(P<0.05)。2免疫细胞化学结果显示,p-p38MAPK在对照组大鼠肾组织中有微弱表达,而在IgA肾病组的相对含量显著增加,与对照组相比有显著性差异(P<0.01);氯沙坦组蛋白表达较IgA肾病组降低,有显著性差异(P<0.01)。3 Western印迹结果显示,p38MAPK蛋白在IgA肾病组和对照组的表达无明显差异(P>0.05)。与对照组相比,IgA肾病组大鼠肾组织中p-p38MAPK的表达水平明显升高(P<0.01);氯沙坦组大鼠p-p38MAPK的蛋白表达较IgA肾病组明显降低(P<0.01)。4 RT-PCR结果显示,p38MAPK mRNA在对照组大鼠肾组织中有少量表达,IgA肾病组大鼠肾组织该蛋白表达明显上调(P<0.01);氯沙坦组大鼠p38MAPK mRNA表达显著下调(P<0.01)。结论1氯沙坦能有效的降低IgA肾病大鼠的24小时尿蛋白,并对IgA肾病大鼠肾功能改善有一定的作用。2在IgA肾病大鼠肾组织内p38MAPK被激活,p38MAPK信号转导途径的激活在IgA肾病的发生发展过程中可能发挥着重要的作用。3氯沙坦能够明显抑制IgA肾病大鼠肾组织p38MAPK信号蛋白的表达。4氯沙坦的肾脏保护作用可能是通过抑制p38MAPK的表达改善肾脏纤维化、延缓肾小球硬化来完成的。5 p38MAPK信号转导通路抑制剂可能成为临床治疗IgA肾病的新途径。更多还原

【Abstract】 Objectives:IgA nephropathy(IgAN), a chronic and prosessive Glomerular disease, is one of the major reasons of glomerulosclerosis and end-stage renal disease(ESRD), which induces a series of clinical and pathophysiological changes. IgAN is histologically characterized by overaccumulation of mensangial cell and extracellular matrix. With the development of renal tissue biopsy, diognosis ratio of children IgAN is increasing gradually. However, there is still no specific treatment. Empirical therapy is the main procedure for the IgAN patients in clinical practice. It was thought that IgAN is a benign disease and have a good prognosis. But long-term follow-up results found that about 20%~50% of adult patients will develop into the eventual development of renal failure. Prognosis of patients with children is better than adults. 5%, 6% and 11% children patients in 5 years, 10 years and 15 years after onset have chronic renal failure. Therefore, the active prevention of IgAN has a pivotal position in study of kidney disease. A lot of research shown Renin-Angiotensin System has important influence in the chronic renal disease, especially for angiotensinⅡ. RAS also play an important role in the pathogenesis of IgAN. Application of RAS blockers is effective in improving clinical symptoms of IgAN patients. AngiotensinⅡreceptor blockers is RAS blockers and has been effectively used for the treatment of IgAN in recent years. Losartan is a kind of angiotensinⅡI type receptor antagonist. The study shows that for patients with IgAN, especially in the merger or urinary protein in patients with hypertension, losartan can protect renal function and delay the occurrence of renal failure. The results show that p38 mitogen-activated protein kinase(p38MAPK) signal transduction pathway can be activated by many kinds of inflammatory factors, such as angiotensinⅡ. p38MAPK play a important role in inflammatory response and promote renal fibrosis by multiple channels. The block of p38MAPK signal transduction pathway can improve renal fibrosis and delay glomerulosclerosis. There is no definite conclusion about the renal protective effect of angiotensinⅡI type receptor antagonist whether by block of p38MAPK signal transduction pathway or not. We observe the effect of losartan on expression of p38MAPK in IgAN rats renal tissue by IgA nephropathy rats model and preliminary study the mechanism of the renal protective effect of AngiotensinⅡreceptor blockers.Methods:30 male Wistar rats (provided by animal experiment center of Hebei Medical University) with 100~120g of weight were randomly divided into three groups: control group(n=10), IgAN group(n=10) and losartan group(n=10). The rats of IgAN group and losartan group had been administered with bovine serum albumin by gavage at a dose of 400mg·kg-1·d-1 every other day for 6 weeks and injected 0.4ml castor oil plus 0.1ml CCl4 once a week for 9 weeks. Lipoplysaccharides were injected at 6th and 8th week. These rats were observrd until 10th week. The rats of control group were free of eating and drinking. Afte 10 weeks, renal tissue was cut into 3μm frozen secion. IgA accumulation in glomerular mensangial areas was examined under the microscope after stained with flurorescein-conjugated affinipure goat anti-rabbit IgG and rabbit anti-rat IgA by immunofluorescence. Proliferation of mensangial cell and overaccumulation of extracellular matrix were observed by PASM and Masson stains. These results indicated that the successful ratio of the IgA nephropathy model was 90%. The rats of losartan group had been administered with losartan by gavage at a dose of 20mg·kg-1·d-1 everyday for 6 weeks. The total experimental period was 14 weeks. Rats in each group were collected 24-hour urine for 24 hour urine protein. Blood was drawn from femoral to separate serum for determination renal function. Partial renal tissue was fixed in 4% formaldehydum and embeded with paraffin. Partial renal cortices were used to abstract total RNA and protein. The protein localization of p-p38MAPK was evaluated by immunohistochemistry. The protein levels of p38MAPK and p-p38MAPK was evaluated by Western blot; The mRNA levels of p38MAPK was measured by reverse transcription and polymerase chain reaction(RT-PCR). All data were expressed as x±s. F and T test was separately used to analyse the difference between two groups and within one group, which was finished with SPSS11.5 software.Results:1 Biochemical indexes: 24 hour urine protein, BUN and SCr in the rat of IgAN group were obviously higher than the rat of control group(P<0.01). ALB in the rat of IgAN group was obviously lower than the rat of control group(P<0.01). The biochemical indexes in the rat of losartan group was significantly improve.2 Immunohistochemically: p-p38MAPK has weak expression in the control group of rats kidney. Compared with those of control rats, the expressions of p-p38MAPK was obviously higher in IgAN group(P<0.01). Compared with those of IgAN group, the expressions of p-p38MAPK was obviously lower in losartan group(P<0.01).3 Western blot: Compared with those of control rats, the expressions of p38MAPK have no significant difference in IgAN group(P>0.05); Compared with those of control rats, the expressions of p-p38MAPK was obviously higher in IgAN group(P<0.01); Compared with those of IgAN group, the expressions of p-p38MAPK was obviously lower in losartan group(P<0.01).4 Real-time quantitative polymerase chain reaction(RT-PCR): p38MAPK mRNA has weak expression in the control group of rats kidney. Compared with those of control rats, the expressions of p38MAPK mRNA was obviously higher in IgAN group(P<0.01). Compared with those of IgAN group, the expressions of p38MAPK mRNA was obviously down-regulated in losartan group(P<0.01).Conclusion:1 Losartan can obviously decrease 24-hour urine protein of IgA nephropathy rats and improve the renal function of IgA nephropathy rats.2 P38MAPK signal transduction pathway was activated in renal tissue of IgAN rats. The activation of p38MAPK signal transduction pathway may play an important role in the pathogenesis of IgA nephropathy.3 Losartan can obviously inhibit expression of p38MAPK in renal tissue of IgA nephropathy rats.4 The renal protective effect of Losartan may be by regulating the expression of p38MAPK to improve renal fibrosis and delay glomerular sclerosis.5 Inhibitor of p38MAPK signaling pathway may be a new routin for treating IgA nephropathy.更多还原