Molecular & Functional Regulatory Network in Hypertension

摘要 原发性高血压影响超过5000万美国人，并且血压盐敏感性增加是某些高血压患者人群，特别是非洲裔美国人的突出特征。虽然高血压的常见形式是多因素的（多基因和环境），但这种疾病常见形式的遗传基础仍然难以捉摸。Dahl盐敏感（SS）大鼠是一种广泛使用的动物模型，其概括了人类盐敏感性高血压和相关肾损伤的许多方面。项目1的目标是使用整合系统方法研究基因和蛋白质表达的相互作用，以确定肾脏单细胞类型的功能（肾髓质厚Henle升支= mTAL）。已知mTAL在SS大鼠和人类高血压的肾髓质功能、钠排泄和高血压中发挥重要作用。我们假设13号染色体同源区域内的基因序列变异（SS. 13 [BN 26]; BN等位基因取代SS）改变了影响mTAL功能的分子调控网络，从而导致盐敏感性高血压和肾损伤。在目标1中，将对同源区（SS. 13 [BN 26]; 13.2 Mb）进行测序，以获得高质量的成品 组装该区域并注释以搜索基因序列变体。将开发亚同源菌株以鉴定将盐敏感性减弱至~ 5 Mb的狭窄区域，然后将使用序列捕获芯片对另外14种与SS和BN具有不同进化历史的菌株中的该区域进行后续测序。将使用以下方法过滤序列差异（SNP和其他基因组变体）： SS中的偶然变异应仅与其他盐敏感菌株共同的标准。目的2：构建mTAL上皮细胞的分子生理调控网络（BayeN），并利用该网络模型研究SS大鼠盐敏感性高血压和肾损伤的相关通路和基因。目标3将根据目标1和2的结果选择候选基因， 使用锌指核酸酶（ZFN）和/或使用转基因方法过表达该基因，以研究该基因对转录组/蛋白质组/代谢组和相关功能网络的影响。 几个技术飞跃和概念方法是这个项目所独有的。这些包括1）下一代测序，以提供SS和同源SS. 13 [BN 26]菌株的13.2 Mb区域的极高分辨率测序，与NimbleGen序列捕获芯片偶联，用于对其他大鼠菌株进行测序; 2）转录组分析（Affyxin）和使用分离和纯化的mTAL细胞的质谱亚蛋白质组和代谢组分析，比较SS和耐盐同类大鼠; 3）使用“知识受限”的大规模贝叶斯分析来寻找使用转录组和功能数据隐藏的未知途径; 4）有效敲除被发现对mTAL调控网络有重要贡献的基因的新技术（锌指核酸酶），提供了验证和表征功能相关性的明确方法。 项目1解决了高血压领域面临的一个关键挑战：确定导致盐敏感性高血压的肾功能改变的复杂成分（基因，蛋白质和途径）。它与项目2和项目3高度协同，项目2测试了非蛋白质编码基因（microRNA）可能在高血压和肾损伤中起重要作用的创新概念，项目3旨在确定远离肾素基因chr 13的突变调节SS大鼠中肾素活性和血管生成的机制。所有项目都极大地受益于Cores 8和C提供的基因组、遗传、蛋白质组和计算技术进步。

ABSTRACT Essential hypertension affects more than 50 million Americans and increased blood pressure salt-sensitivity is a prominent feature in certain populations of hypertensive patients, especially African Americans. Although it is evident that the common forms of hypertension are multiifactorial (polygenic and environmental), the genetic basis of the frequent forms of this disease remain elusive. The Dahl salt-sensitive (SS) rat is a widely used animal model that recapitulates many aspects of human salt-sensitive hypertension and associated renal injury. The goal of Proiect 1 is to study the interplay of gene and protein expression using integrative systems approaches to determine the functionality of a single cell type of the kidney (renal medullary thick ascending limb of Henle = mTAL). The mTAL is known to play an important role in renal medullary function, sodium excretion, and hypertension in the SS rat and in human hypertension. We hypothesize that gene(s) sequence variants within a congenic region of chromosome 13 (SS.13[BN26]; BN alleles substituted for SS) alter molecular regulatory networks affecting function of the mTAL thereby contributing to salt-sensitive hypertension and renal injury. In Aim 1, the congenic region (SS.13[BN26]; 13.2 Mb) will be sequenced to obtain a finished high-quality assembly of this region and annotated to search for gene sequence variants. Subcongenic strains will be developed to identify a narrow region that attenuates salt-sensitivity to ~ 5 Mb and a sequence capture chip will then be utilized for follow-up sequencing of this region in 14 additional strains that have a different evolutionary history from the SS and BN. The sequence differences (SNPs and other genomic variants) will be filtered using the criteria that a casual variant in the SS should only be in common with other salt-sensitive strains. Aim 2 will construct a molecular and physiological regulatory network (BayeN) of the mTAL epithelial cell and use that model to identify pathways and genes that may contribute to salt-sensitive hypertension and renal injury in SS rats. Aim 3 will select a candidate gene based on results from Aims 1 and 2 and either knock the gene out using zinc finger nucleases (ZFN) and/or over-express the gene using transgenic approaches to study the impact of this gene upon the transcriptome/proteome/metabolome and associated functional network. Several technological leaps and conceptual approaches are unique to this Project. These include 1) Next generation sequencing to provide very high resolution sequencing of a 13.2 Mb region of SS and congenic SS.13[BN26] strains coupled with a NimbleGen sequence capture chip for sequencing of additional rat strains; 2) Transcriptome analysis (Affymetrix) and a mass spectrometry sub-proteome and metabolome analysis using isolated and purified mTAL cells comparing the SS and salt-resistant congenic rats; 3) The use of a large-scale Bayesian analysis which is "knowledge-constrained" to seek yet unknown pathways hidden using the transcriptome and functional data; 4) A novel technology (zinc finger nucleases) to efficiently knock out a gene found to contribute importantly to the mTAL regulatory network, providing a definitive way to validate and characterize functional relevance. Project 1 addresses a critical challenge facing the field of hypertension: to identify the complex components (genes, proteins and pathways) that are responsible for alterations in kidney function leading to salt-sensitive hypertension. It is highly synergistic with Project 2 which tests the innovative concept that non-protein-coding genes (microRNA) may play an important role in hypertension and renal injury and Project 3 which aims to Identify the mechanisms by which mutations distant from the renin gene on chr 13 regulate the activity of renin and angiogenesis in the SS rat. All projects benefit greatly from genomic, genetic, proteomic and computational technological advances provided by Cores 8 and C.