二肽基肽酶4在常染色体显性多囊肾病发病中的机制与作用研究

发布时间：2018-01-31 21:11

本文关键词： 常染色体显性多囊肾病二肽基肽酶腺苷磷酸活化蛋白激酶哺乳动物雷帕霉素靶蛋白　出处：《第二军医大学》2016年博士论文　论文类型：学位论文

【摘要】：研究背景及目的常染色体显性多囊肾病(Autosomal dominant polycystic kidney disease, ADPKD)是人类发病率最高的单基因遗传性肾脏病。接受替代治疗的终末期肾病(ESRD)中7-10%是ADPKD患者。尽管在发现此病500年来进行了大量的研究,直至今日仍然无法治愈且对其机制尚不明了。所有人种皆不能幸免于ADPKD,7-10%的ESRD因此而起。全球范围,接受肾脏替代治疗(RRT)的第四大原因就是ADPKD。在不同的国家,ADPKD导致的ESRD发病率有所不同,在日本是每年百万分之4.8,在美国是每年百万分之7.9,欧洲各国每年百万分之3.9到15.3。在丹麦哥本哈根[4]和美国奥姆斯特德进行的流行病学调查显示,80岁以下人口中,ADPKD的患病率是400到1000分之一。此后在欧洲和日本进行的其他研究显示不同地区ADPKD患病率波动于543到4000分之一。鉴于上述研究地理位置、观测时间、采样方法、诊断标准、家系调查、人口多寡都有不同,故而差异虽大尚可理解。然则,来自尸检报告的数据要高于1/500,为1/339-1/492。这说明大量患者终其一生未能确诊。ADPKD由多囊肾病基因1(Polycystic kidney disease 1 gene, PKD1)和多囊肾病基因2(Polycystic kidney disease 2 gene, PKD2)两个基因突变造成,此二基因分别编码多囊蛋白-1(Polycystin-1, PC1)及多囊蛋白.2(Polycystin-2, PC2)。其中PKD1突变的患者占80～85%,PKD2突变的患者占15-20%。所谓PKD3,经过对相关病例的重新分析,已经基本排除了其存在的可能性。PC1与PC2在肾小管上皮细胞初级纤毛上相互作用构成复合体,后者属于瞬时受体电位(transient receptor potential, T P)家族中的一种非选择性钙离子渗透通道。PC1的C末端结构域可以易位至细胞核的结构,并充当基因转录的共激活剂。PC1和PC2在纤毛经受机械刺激时引发Ca2+瞬间内流。ADPKD的发病机制尚未清楚,有前途的治疗方法主要集中于通过阻断过度活化mTOR通路和cAMP通路来延缓疾病进展。动物实验发现mTOR抑制剂(依维莫司和西罗莫司)改善啮齿类动物模型囊性肾病的效果非常显著,但依维莫司和西罗莫司都是强效的免疫抑制剂,可能带来致命的副作用,且临床试验并未取得良好的疗效。所以我们需要探索更为安全有效的手段。大量报道证实AMP活化蛋白激酶(AMP-activated protein kinase, AMPK)下调是：mTOR通路过度活化的原因之一,通过上调MPK水平可以抑制过度活化的mTOR通路,并在动物模型中取得了良好的效果。通过抑制二肽基肽酶4(dipeptidyl peptidase 4, DPP4)的活性可以上调AMPK水平是毫无疑问的,但能否抑制过度激活的mTOR通路改善ADPKD, ADPKD患者和动物模型中是否存在DPP4过表达从而过度激活了mTOR通路,这两个问题尚无解答。本课题的目的是观察ADPKD患者和实验动物肾组织中DPP4是否存在表达上调,以及使用DPP4抑制剂,是否能同过上调AMPK,抑制mTO R活性,达到治疗ADPKD的目的。研究方法1、DPP4在ADPKD患者及动物模型中的表达水平检测：搜集ADPKD患者肾组织与移植供肾组织标本,用western blot技术测定两者的DPP4蛋白表达量,并进行比较；繁育多囊肾模型Han:SPRD大鼠,鉴定出野生型(WT)和发病模型(cy-／+),正常饲养至12周,取肾组织,采用western blot测定DPP4蛋白表达量,进行比较。2、动物实验验证抑制DPP4活性：干预药物包括AMPK激活剂二甲双胍(Metformin), DPP4抑制剂维格列汀(Vildagliptin)和利格列汀(Linagliptin)。繁育多囊肾模型Han:SPRD大鼠,出生后鉴定出野生型(WT)和发病模型(cy-/+)。将发病模型大鼠分为对照组(Ctrl)以及二甲双胍(MET)组、维格列汀(VIG)组、利格列汀(LIG)组。后3组大鼠自出生后4周给药,于第4/8/12周取血查肾功能,第12周代谢笼测24小时尿量,并处死动物,取双肾测肾重体重比,提取蛋白,送病理行全片扫面。3、细胞实验观察抑制DPP4活性对细胞增殖的影响：采用人肾囊肿衬里上皮细胞株WT9.12进行培养。以MTT方法观察在不同浓度的二甲双胍、维格列汀、利格列汀作用下,24小时、48小时、72小时细胞增殖情况。4、抑制DPP4活性治疗ADPKD的机制研究：取不同浓度二甲双胍、维格列汀、利格列汀处理的WT9.12细胞,Western blot技术分析干预后AMPK的蛋白含量变化。取前述实验提取WT、Ctrl、MET、VIG、LIG组大鼠肾组织蛋白,用western blot技术测定AMPK、Akt、p70S6K蛋白含量。研究结果1、ADPKD患者肾脏组织中DPP4的表达量显著高于移植供肾(1.97 vs.0.60,P=0.002),升高了大约3倍。多囊肾模型大鼠(cy-/+)肾脏中情况类似,发病的模型大鼠肾脏中DPP4表达量升高了接近3倍(7.49 vs 1.14,P=0.001)。2、与对照组相比,二甲双胍、维格列汀和利格列汀均可显著降低Han:SPRD大鼠肾重体重比(KW/BW)、囊肿指数,改善肾功能。3组给药组之间上述指标没有统计学差异。3、二甲双胍、维格列汀、利格列汀都可以抑制WT9.12的增殖。三种药物都存在浓度依赖性,在24小时、48小时、72小时三个时间节点50nM浓度的二甲双胍对WT9.12细胞的增长抑制率分别为34%,60%,74%；20nM维格列汀对WT9.12细胞的增长抑制率分别为14%,20%,28%；1nM利格列汀对WT9.12细胞的增长抑制率分别为79%,86%,93%。4、经给药后,给予二甲双胍、维格列汀、利格列汀的模型大鼠肾组织中pAMPK的表达均显著高于Ctrl组(P0.001),分别升高了2.7倍、3.5倍和5倍;而且维格列汀组显著高于二甲双胍组(3.5 vs.2.7,P0.001),而利格列汀组又显著高于维格列汀组(5.1 vs 2.7,P0.001)。和对照组相比,二甲双胍(4.5 vs 5.1,P=0.029)、维格列汀(3.9 vs 5.1,P0.001)、利格列汀(3.0 vs 5.1,P0.001)都可以使模型大鼠肾组织中过度活化的1nTOR被抑制,下游产物p70S6K表达量显著下降；维格列汀组p70S6K蛋白表达量低于二甲双胍组(3.9 vs4.5,P=0.045),利格列汀组低于维格列汀组(3.0vs.3.9,P=0.001)。结论本课题发现/DPKD患者和多囊肾模型大鼠肾组织中DPP4过表达。使用DPP4抑制剂,可以上调AMPK抑制过度激活的mTOR通路,抑制囊肿细胞增殖,延缓病情进展。DPKD患者的DPP4过表达引起的mTOR过度激活可能是其发病机制之一,抑制DPP4活性,是治疗ADPKD潜在的靶点。
[Abstract]:Background and objective: autosomal dominant polycystic kidney disease (Autosomal dominant polycystic kidney disease, ADPKD) is a human disease incidence of single gene hereditary kidney. The highest hormone replacement therapy of end-stage renal disease (ESRD) in 7-10% ADPKD patients. Although the disease has done a lot of research for 500 years, until today still can not the cure and the mechanism remains unknown. All races are not immune to ADPKD, 7-10% and ESRD. So the world, received renal replacement therapy (RRT) is the fourth leading cause of ADPKD. in different countries, the incidence of ESRD caused by ADPKD is different in Japan every year is 4.8 ppm, in the United States is the year 7.9 ppm, according to European countries every year 3.9 ppm to 15.3. in Denmark and the United States of Copenhagen [4] Olmsted epidemiological survey, the population under the age of 80, ADPKD The prevalence rate is 400 to 1000 points. One of the other studies since then in Europe and Japan show that different regions ADPKD prevalence rates from 543 to one in 4000. In view of the above research location, observation time, sampling methods, diagnostic criteria, family survey, population size are different, so the difference is big is understandable. However, from the autopsy report data for 1/339-1/492. is higher than 1/500, indicating a large number of patients in his life was not diagnosed.ADPKD from polycystic kidney disease 1 (Polycystic kidney disease 1 gene, PKD1) and polycystic kidney disease 2 (Polycystic kidney disease 2 gene, PKD2) two gene mutation caused the two gene encoding polycystins respectively -1 (Polycystin-1, PC1) and.2 (Polycystin-2, PC2, polycystins). The patients with PKD1 mutations accounted for 80 ~ 85%, PKD2 mutation patients accounted for 15-20%. called PKD3, after re analysis of related cases, Have basically ruled out the possibility of.PC1 and PC2 are interaction in renal tubular epithelial cells of primary cilia form complexes, which belong to the transient receptor potential (transient receptor potential, T P) is a non selective calcium channel family penetration in the.PC1 C terminal domain structure can be translocated to the nucleus, and act as the gene transcription coactivator.PC1 and PC2 subjected to mechanical stimulation in the cilia caused pathogenesis of.ADPKD Ca2+ within the moment is not yet clear, promising therapies focus on blocking the excessive activation of the mTOR pathway and cAMP pathway to delay the progression of the disease. Animal experiments showed that mTOR inhibitors (Bea Vimal S and sirolimus) improve the rodent animal model cystic kidney disease, the effect is very significant, but Bea Vimal S and are potent immunosuppressant sirolimus, may lead to fatal side-effects, and Clinical trials have not achieved good results. So we need to explore a more safe and effective means. A large number of reports confirmed that AMP activated protein kinase (AMP-activated protein kinase, AMPK) is one of the reasons: the down-regulation of mTOR pathway activation, by increasing the level of MPK can inhibit the excessive activation of the mTOR pathway system, and have achieved good results in in the animal model. By two inhibition of dipeptidyl peptidase 4 (dipeptidyl peptidase 4, DPP4) activity can increase the level of AMPK is no doubt, but can inhibit the excessive activation of the mTOR pathway to improve the ADPKD, whether the DPP4 expression of mTOR pathway leading to excessive activation of ADPKD patients and animal models, these two issues is no answer. The purpose of this study is to observe the renal tissue of ADPKD patients and animal in the presence of DPP4 expression, and the use of DPP4 inhibitors, whether can the same on AMPK inhibition The mTO activity of R, achieve the purpose of treatment of ADPKD. Methods: 1. To detect the expression of DPP4 in ADPKD patients and animal models: collect donor kidney tissue samples in patients with ADPKD and renal tissue transplantation, determination of expression of both DPP4 protein by Western blot technology, and compared the breeding of polycystic kidney; Han:SPRD rat model, identification the wild type (WT) and disease model (cy- / +), normal feeding to 12 weeks, the renal tissues, DPP4 protein expression was detected by using Western blot, compared to.2, the animal experiments to inhibit the activity of DPP4: drug intervention including AMPK activator (Metformin), metformin DPP4 inhibitor (Vildagliptin) and Leigh Glenn Dean Vee Glenn Dean (Linagliptin) breeding. Han:SPRD rat model of polycystic kidney, after the birth of identified wild type (WT) and model (cy-/+). The incidence of the disease model rats were randomly divided into control group (Ctrl) and metformin (MET) group, vildagliptin (VIG) group, linagliptin (LIG) group. After 3 groups of rats from birth 4 weeks after administration, in the 4/8/12 week blood check renal function, Twelfth Zhou Dai Xie cage was measured 24 h urine volume, and kill the animal, the two kidneys measured kidney weight / body weight ratio, protein extraction, pathology the line scanning.3 cells, experimental observation of effect of DPP4 on cell proliferation by human renal cyst lining epithelial cells WT9.12 were cultured. Vee Glenn Dean observed in different concentrations of metformin, using MTT method, under the action of linagliptin, 24 hours, 48 hours, 72 hours, the proliferation of.4 cells, inhibiting mechanism DPP4 activity in the treatment of ADPKD: different concentrations of metformin, Vee Glenn Dean, linagliptin treated WT9.12 cells, changes of protein content in Western intervention AMPK blot technology. The experiment from extraction of WT, Ctrl, MET, VIG, renal tissue in rats of LIG protein, AMPK was determined by Western blot technology, Akt, The content of p70S6K protein. Results: 1. Expression of DPP4 in renal tissue of ADPKD patients was significantly higher than that of donor kidney transplantation (1.97 vs.0.60, P=0.002), increased by about 3 times. The rat model of polycystic kidney (cy-/+) similar to the kidney, the expression of DPP4 increased by nearly 3 times the onset of kidney in model rats (7.49 vs 1.14, P=0.001.2), compared with the control group, metformin and vildagliptin and Leigh Glenn Dean could significantly decrease the Han:SPRD of rat kidney weight to body weight ratio (KW/BW), cyst index, improve renal function in.3 group administered group no significant difference between the index of.3, metformin, Vee Glenn Dean, linagliptin can inhibit the proliferation of WT9.12. Three kinds of drugs are concentration dependent, in 24 hours, 48 hours, three time node growth metformin concentration of 50nM for 72 hours on the WT9.12 cell inhibition rates were 34%, 60%, 74%; 20nM vildagliptin on WT9.12 cells increased The inhibition rates were 14%, 20%, 28%; Leigh Glenn Dean 1nM on WT9.12 cell growth inhibition rate were 79%, 86%, 93%.4 after treatment, metformin, Vee Glenn Dean, pAMPK expression of linagliptin in renal tissues in model rats were significantly higher than Ctrl group (P0.001), were increased by 2.7 times, 3.5 times and 5 times; and vildagliptin group was significantly higher than that of metformin group (3.5 vs.2.7, P0.001), while the Leigh Glenn Dean group was significantly higher than that of vildagliptin group (5.1 vs 2.7, P0.001). Compared with the control group, metformin (4.5 vs 5.1, P=0.029), Vee Glenn Dean (3.9 vs 5.1, P0.001, Leigh Glenn Dean) (3 vs 5.1, P0.001) can make the 1nTOR excessive activation of renal tissue in the rat model was inhibited, the expression of p70S6K downstream products decreased significantly; vildagliptin group p70S6K protein expression was lower than that of metformin group (3.9 vs4.5, P=0.045), linagliptin group was lower than that of group 3.0vs (vildagliptin .3.9, P=0.001). Conclusion: This study found that overexpression of DPP4 in renal tissue of rats with /DPKD and polycystic kidney model. Using the DPP4 inhibitor, can inhibit the mTOR pathway activated by upregulation of AMPK, inhibition of cyst cell proliferation, progression of.DPKD in patients with DPP4 overexpression caused by excessive activation of mTOR may be involved in its pathogenesis. Inhibition of DPP4 activity is potential therapeutic target for ADPKD.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R692.1

【相似文献】