局灶节段肾小球硬化症的基因复查及蛋白质组学研究

发布时间：2018-09-03 09:09

【摘要】：局灶节段肾小球硬化（focal segmental glomerulosclerosis，FSGS)是一组病理学上以仅累及部分肾小球及肾小球毛细血管袢部分小叶的硬化性病变为特征的临床病理综合征1，是成人肾病综合征（nephrotic syndrome，NS）和慢性肾衰竭最常见的原因之一，在经肾活检诊断的原发性肾小球肾炎中约占5.6~37.8%2-5。尽管存在一定地区差异，FSGS在各地区的发病率总体呈持续上升趋势6-8。FSGS常以大量蛋白尿为主要临床表现，可伴有镜下血尿、高血压和肾功能不全，治疗效果相对较差。患者确诊后5年和10年肾存活率分别约为83.5%和43.8%9。尤其是激素抵抗、激素依赖、反复复发的所谓“难治性”FSGS，如不能得到有效治疗，超过50%的患者将在3-8年内进展至终末期肾功能衰竭5。正因如此，FSGS已成为目前国际国内研究的热点之一。按不同病因，FSGS可分为原发性和继发性10，继发性FSGS的病因中包括家族性11。足细胞相关基因变异是家族性FSGS发病的重要原因，目前已知导致成人发病型家族性FSGS的基因主要有ACTN4、TRPC6和INF2基因12，但ACTN4、TRPC6基因在中国成人发病型家族性FSGS中的确切突变概率仍未可知。原发性FSGS约占80%13，而其发病机制尚有诸多未明。蛋白质组学(proteomics)技术的发展为寻找FSGS发病过程中的关键性物质，进一步阐明其发病机制和寻找新的治疗靶点提供了可能。近年来，采用相对和绝对定量同位素标记(isobaric tags for relative and absolute quantitation，iTRAQ)技术联合串联质谱的方法，可以对蛋白质的肽段进行比较精确的鉴别和定量14，与二维电泳相比，它具有重复性好，灵敏度高，标记效率高，可以进行定量检测等优点15-17。目前蛋白质组学已被广泛应用于肾脏疾病的诊断，发病机制的探索，疾病活动度以及预后的预判等方面18-23。其中尿液因取材简单，性质稳定，产生和排泄的途径与肾脏和泌尿道密切相关，是肾脏疾病蛋白质组学研究的理想标本。在本研究的第一部分，我们进行了国际单中心最大组的成年发病型家族性FSGS的TRPC6和ACTN6基因突变筛查（共80个家系），在80个先证者中共筛查出TRPC6基因Q889K突变2例，发现TRPC6基因SNP14种，未检出ACTN4突变，，筛出ACTN4基因SNP22种。报道了TRPC6基因在本组中国成年发病型家族性FSGS中的突变率为2.5%，ACTN4基因的突变率为0%。完善了成年发病型家族性FSGS中TRPC6和ACTN6基因突变率的数据。在本研究的第二部分，采用四联iTRAQ联合2D-nano-HPLC-MS/MS的方法在原发性FSGS、MCD患者、正常对照组尿液中检出并鉴定出蛋白质200种。其中包括细胞浆、细胞核、细胞膜、细胞外的多种酶类、生长因子、转录调节因子、受体蛋白、转运蛋白和离子通道蛋白等。应用Ingenuity Pathway Analysis（IPA）软件的Top canonical pathway对差异蛋白进行分析后发现FSGS组paxillin信号转导、肌动细胞细胞骨架信号转导通路受影响，提示这两条通路可能与FSGS发病机制有关。差异蛋白之一的vinculin经ELISA验证发现在原发性FSGS患者尿液中较对照组升高，结果有统计学差异，并与质谱结果一致。在本研究的第三部分，通过体外实验发现，阿霉素刺激24小时后足细胞中vinculin蛋白的表达水平升高；同时，免疫荧光结果显示FSGS患者肾组织vinculin荧光强度较轻微病变对照组高，提示vinculin蛋白可能参与FSGS的发病过程，或是FSGS肾损伤后表达量改变的蛋白之一。此外，我们对原发性FSGS患者尿vinculin水平与尿蛋白、血白蛋白、血肌酐值进行了相关分析，但未发现尿vinculin与上述指标存在相关性。总之，ACTN4和TRPC6基因突变的发病率较低，并不是成人发病型家族性FSGS的常见原因。蛋白质组学的发展为探索FSGS的发病机制带来新的启发，而vinculin蛋白可能参与FSGS的发病过程。
[Abstract]:Focal segmental glomerulosclerosis (FSGS) is a group of clinicopathological syndromes characterized by sclerotic lesions involving only part of the glomerular and part of the glomerular capillary loops. FSGS is one of the most common causes of adult nephrotic syndrome (NS) and chronic renal failure. Although there are some regional differences, the incidence of FSGS in various regions is generally on the rise 6-8. FSGS is often characterized by a large number of proteinuria as the main clinical manifestations, may be accompanied by microscopic hematuria, hypertension and renal insufficiency, the treatment effect is relatively poor. The annual and 10-year renal survival rates are about 83.5% and 43.8% respectively. Especially the so-called "refractory" FSGS with hormone resistance, hormone dependence and recurrence, if not effectively treated, more than 50% of the patients will progress to end-stage renal failure within 3-8 years. Therefore, FSGS has become one of the hot spots of international and domestic research.
According to different etiologies, FSGS can be divided into primary and secondary 10. Familial 11 is included in secondary FSGS. Podocyte-related gene mutation is an important cause of familial FSGS. It is known that ACTN4, TRPC6 and INF2 gene 12 are the main genes causing familial FSGS in adults, but ACTN4 and TRPC6 genes are found in Chinese adult pathogenic countries. The exact mutation probability in the family FSGS is still unknown.
The development of proteomics technology has made it possible to find the key substances in the pathogenesis of FSGS, further elucidate its pathogenesis and find new therapeutic targets. In recent years, relative and absolute quantitative isobaric tags for rela have been used. Compared with two-dimensional electrophoresis, it has the advantages of good reproducibility, high sensitivity, high labeling efficiency, and can be used for quantitative detection. At present, proteomics has been widely used in kidney. Diagnosis, pathogenesis, activity and prognosis of the disease are 18-23. Urine is an ideal specimen for proteomics of kidney diseases because of its simple material, stable nature, and the way of production and excretion is closely related to the kidney and urinary tract.
In the first part of this study, we screened for TRPC6 and ACTN6 gene mutations in adult onset familial FSGS in the largest international single center group (80 families). Two cases of Q889K mutations in TRPC6 gene were screened out among 80 probands. Fourteen kinds of SNP14 mutations were found in TRPC6 gene, no ACTN4 mutations were detected, and 22 kinds of SNP22 of ACTN4 gene were screened out. The mutation rates of TRPC6 and ACTN4 genes in this group of Chinese adult onset familial FSGS were 2.5% and 0%, respectively.
In the second part of this study, we detected and identified 200 proteins in urine of patients with primary FSGS, MCD and normal controls by quadruple iTRAQ and two-dimensional-nano-HPLC-MS/MS. These proteins include cytoplasm, nucleus, cell membrane, extracellular enzymes, growth factors, transcription regulators, receptor proteins, transporters and ion channels. Top canonical pathway of Ingenuity Pathway Analysis (IPA) software was used to analyze the differential proteins. It was found that paxillin signal transduction and actinocyte cytoskeleton signal transduction pathway were affected in FSGS group, suggesting that the two pathways may be related to the pathogenesis of FSGS. In patients with primary FSGS, the urine levels were higher than those in the control group, and the results were statistically significant.
In the third part of this study, we found that the expression of vinculin protein in podocytes increased after 24 hours of adriamycin stimulation. At the same time, the immunofluorescence results showed that vinculin fluorescence intensity in renal tissue of FSGS patients was higher than that of the control group with mild lesions, suggesting that vinculin protein may participate in the pathogenesis of FSGS, or FSGS renal injury. In addition, we analyzed the correlation between urinary vinculin and urinary protein, serum albumin and serum creatinine in patients with primary FSGS, but found no correlation between urinary vinculin and the above indexes.
In conclusion, the low incidence of ACTN4 and TRPC6 mutations is not a common cause of familial FSGS in adults. The development of proteomics provides new insights into the pathogenesis of FSGS, and vinculin may be involved in the pathogenesis of FSGS.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692.6

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