Genomics and Proteomics for Pathogensis and Pathophysiology of IgA Nephropathy

本课题旨在通过基因组学和蛋白质组学的研究，阐明人IgA肾病的发病机制和病理生理机制。首先，制备了人肾小球cDNA文库，并对大约6,000个克隆进行了测序。肾小球基因已经被储存和注释，以建立一个肾小球基因数据库。利用基因芯片技术寻找IgA肾病患者肾组织中表达上调或下调的基因。我们关注了一个基因，因为它还没有被表征，并且在人IgA肾病肾组织中显著和独特地上调。据推测，该基因编码一个膜蛋白，具有单一的跨膜结构域和粘蛋白样结构域。通过使用一种假定的多肽的抗体的免疫组织化学方法，该蛋白定位于正常人肾组织中近端小管的基底外侧膜。为了了解该分子的生理功能，用酵母双杂交系统搜索了其他可能与该蛋白结合的分子。其中一个杂交分子是生长激素的受体，在人类肾脏组织中的同一位置表达。在使用高度表达这些基因的细胞的培养中，研究了这些分子之间的相互作用。我们发现，当生长因子与受体结合后，这种新分子在胞质区域相互作用时，干扰或减弱了信号转导。用2D凝胶电泳法和MALDI-TOF质谱仪(MS)或LC MS/MS进行蛋白质组学分析，在正常人肾小球样本的1500个斑点中鉴定出大约500个斑点。每个分子的注释斑点数据库已准备好通过互联网开放。采用双向凝胶电泳法和质谱法对IgA肾病标本进行蛋白质组学分析时，由于样本量的模拟存在一定的困难。

This project was aimed to clarify pathogenesis and pathophysiology of human IgA nephropathy by genomics and proteomics. First, human glomerulus cDNA library was prepared and approximately 6,000 clones were sequenced. The glomerular genes have been banked and annotated to make a glomerular gene database. By cDNA microarray technique unique genes up-or down-regulated in human kidney tissues from patients with IgA nephropathy were searched. We paid an attention on one gene since it had not been characterized yet and was significantly and uniquely up-regulated in human IgA nephropathy kidney tissues. The gene was presumed to encord a membrane protein with a single membrane spanning domain and mucin-like domain. By immunohistochemistry using an antibody against a presumed peptide, the protein was localized at basolateral membrane of proximal tubules in normal human kidney tissues. To understand a physiologic function of this molecule, other molecules which might bind with this protein was searched by a yeast two-hybrid system. One of hybridized molecules was a receptor for a growth hormone expressed at the same site in human kidney tissues. An interaction between these molecules was examined in culture using cells highly expressed with these genes. We found that this new molecule interfered or attenuated signal transduction after binding of he growth factor to the receptor when interacted at the cytoplasmic domain. By proteomics using 2D gel electrophoresis and MALDI-TOF mass spectrometer (MS) or LC MS/MS approximately 500 spots out of 1500 spots were identified in normal human glomerulus samples. Database of the spots with annotation of each molecule was prepared to open through the internet. Proteomic analysis of IgA nephropathy samples by 2D gel electrophoresis and MS was difficult because of imitation of sample amounts.