RENAL DISEASES IN CHILDHOOD

儿童期肾病

• 批准号: 2003143
• 负责人: ALFRED F MICHAEL
• 金额: $ 34.46万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-12-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2003143
• 关键词: Alport syndrome; basement membrane; child (0-11); collagen; congenital deafness; congenital kidney disorder; enzyme linked immunosorbent assay; gender difference; gene mutation; genetic mapping; glomerulonephritis; human genetic material tag; human tissue; immunocytochemistry; immunoelectron microscopy; immunopathology; kidney transplantation; laboratory mouse; laboratory rabbit; monoclonal antibody; nephritis; nucleic acid probes; nucleic acid sequence; polyanion; protein structure; radiotracer; surface antigens; tissue /cell culture; western blottings

The long-term objectives of this research proposal are to understand the biologic role of a group of related novel collagen chains with particular attention to the structure of specialized renal basement membranes and the changes related to certain diseases especially, Alport familial nephritis and other renal diseases. (1) A crucial role is the characterization of alpha5(IV) protein and its relationship to the Alport antigen. The latter, a 26 kD peptide, is defined by antibody probes that discriminate X-linked Alport syndrome and in common with alpha3(IV) NC1 and alpha4(IV) NC1 is not detected in Alport glomerular basement membrane (GBM). The gene for alpha5(IV) (COL4A5) has been assigned to the Xq22 locus of the X chromosome. We have shown that an alpha5(IV) NC1 peptide codistributes with the Alport antigen in normal GBM and is missing from Alport GBM. We propose to characterize this molecule, define its distribution, and determine its relationship to the Alport antigen, and its interaction with other novel chains in dimers and helices. The various methods used will involve biochemical and immunochemical techniques to isolate monomers and dimers, the development of monoclonal and polyclonal antibody probes to peptides and NC1 and the use of immunohistochemical techniques. (2) The sequencing of cDNA and genomic DNA of the Alport antigen, alpha4(IV) and alpha3(IV) is an important aspect of this proposal since it will define the genetically-discriminating Alport peptide and allow us to understand the interrelationships among these molecules. Since this component is missing from most males with Alport syndrome and present in a mosaic pattern in affected females, the definition of whether it is the same as alpha5(IV) NC1 or is an homologous related chain-is crucial. Screening of a library constructed from mRNA of transformed bovine endothelial cells (a line that makes alpha3(IV) NC1 and the Alport antigen) will be carried out with our antibody probes, and subsequent nucleotide sequencing, Northern analysis,and genomic analysis. Similarly, the cDNA of alpha4(IV) NCI will be sought-initially using nucleotides developed from N-terminal sequences of the isolated chains as well as antibody screening. (3) A major component of this research is to define further the genetic defect in Alport syndrome for mutations in novel collagen genes; to characterize further the relationship of these mutations to Alport phenotypes, and the occurrence of post-transplant anti-GBM nephritis. This will include investigations of the mutations involving the gene encoding alpha5(IV) NC1, the Alport antigen, and alpha4(IV) NC1. Further, we will address the hypothesis that patients with Alport syndrome that develop anti-GBM nephritis following transplantation may have unique mutations that prevent expression of certain epitopes and the development of immunologic tolerance during fetal life. These studies will involve a number of a molecular biologic techniques. (4) The recognition that these novel chains of type IV collagen in contrast to alpha1(IV) and alpha2(IV) have common distributions in renal basement membranes, late ontogenic expression, exhibit temporal and spatial segregation in disease-strongly suggest the presence of molecular interactions in a network and functional specialization. Using biochemical and immunochemical techniques, we will study the interactions of these chains in cultured cells and isolated basement membranes.

这项研究建议的长期目标是了解 一组相关的新型胶原蛋白链的生物学作用 注意专门的肾脏基底膜的结构和 尤其是与某些疾病有关的变化，特别是家族性肾炎 和其他肾脏疾病。 （1）至关重要的作用是α5（IV）蛋白及其的表征 与Alport抗原的关系。 后者是26 kD肽，是 由抗体探针定义的，该抗体探针区分X连锁ALPORT综合征和 在Alport中未检测到与alpha3（iv）NC1和alpha4（iv）NC1共同的共同点 肾小球基底膜（GBM）。 α5（IV）（COL4A5）的基因具有 已分配给X染色体的XQ22基因座。 我们已经证明了 alpha5（iv）NC1肽与正常GBM中的Alport抗原共同分布 并缺少Alport GBM。 我们建议表征该分子， 定义其分布，并确定其与Alport的关系 抗原及其与二聚体和螺旋中其他新型链的相互作用。 所使用的各种方法将涉及生化和免疫化学 分离单体和二聚体的技术，单克隆的发展 和肽和NC1的多克隆抗体探针以及使用 免疫组织化学技术。 （2）Alport抗原的cDNA和基因组DNA的测序， alpha4（iv）和alpha3（iv）是该提案的重要方面 将定义遗传上歧视的Alport肽，并允许我们 了解这些分子之间的相互关系。 自此 大多数患有Alport综合征的男性都缺少成分，并且存在于 受影响女性的马赛克模式，是否是 与alpha5（iv）NC1相同或是同源相关的链，至关重要。 筛选由转化牛的mRNA构建的库 内皮细胞（使Alpha3（IV）NC1和Alport抗原的线） 将使用我们的抗体探针和随后的核苷酸进行 测序，北方分析和基因组分析。 同样， 使用从 分离链的N末端序列以及抗体筛选。 （3）这项研究的主要组成部分是进一步定义遗传 新型胶原基因突变的Alport综合征缺陷；到 进一步表征这些突变与Alport的关系 表型和移植后抗GBM肾炎的发生。 这 将包括研究涉及基因编码的突变 Alpha5（IV）NC1，Alport抗原和alpha4（IV）NC1。 此外，我们会的 解决了发展的假设 移植后抗GBM肾炎可能具有独特的突变 防止某些表位表达和免疫学的发展 胎儿生活中的宽容。 这些研究将涉及许多 分子生物学技术。 （4）认识到这些新型IV胶原蛋白的新链相比 到alpha1（iv）和alpha2（iv）在肾脏基底具有共同的分布 膜，晚期的表达，表现出时间和空间 疾病的分离表明存在分子 网络和功能专业化中的交互。 使用生化 和免疫化学技术，我们将研究这些技术的相互作用 培养细胞和孤立的基底膜中的链。