Molecular & Functional Regulatory Network in Hypertension

摘要 原发性高血压影响着5000多万美国人，血压升高对盐敏感是某些高血压患者，特别是非裔美国人的显著特征。尽管高血压的常见形式显然是多因素的(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，它概括了人类盐敏感高血压及其相关肾损伤的许多方面。项目1的目标是使用综合系统方法研究基因和蛋白质表达的相互作用，以确定肾脏单一细胞类型的功能(肾髓质厚的Henle=mTAL)。已知mTAL在SS大鼠的肾脏髓质功能、钠排泄和高血压以及人类高血压中发挥重要作用。我们假设基因(S)序列变异在13号染色体的同源区域(SS。13[BN26]；取代SS的BN等位基因)改变影响mTAL功能的分子调控网络，从而导致盐敏感型高血压和肾脏损伤。在目标1中，将对同源区域(SS.13[BN26]，13.2Mb)进行测序，以获得该区域的高质量组装，并对其进行注释以寻找基因序列变异。将开发亚同源菌株来识别一个能减弱对~5Mb盐敏感性的狭窄区域，然后将利用序列捕获芯片对另外14个与SS和BN具有不同进化历史的菌株进行该区域的后续测序。序列差异(SNPs和其他基因组变异)将使用SS中的偶然变异只能与其他盐敏感菌株相同的标准进行筛选。目的2将构建一个mTAL上皮细胞的分子和生理调控网络(BAYEN)，并使用该模型来确定可能导致SS大鼠盐敏感型高血压和肾脏损伤的途径和基因。AIM 3将根据AIMS 1和AIMS 2的结果选择一个候选基因，并使用锌指核酸酶(ZFN)敲除该基因和/或使用转基因方法过度表达该基因，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该项目独一无二的。这包括1)下一代测序，以提供对SS和同源SS.13[BN26]菌株的13.2Mb区域的非常高分辨率的测序，并结合NimbleGen序列捕获芯片对更多的大鼠品系进行测序；2)转录组分析(Affymetrix)，以及使用分离和纯化的mTAL细胞对SS和耐盐同源大鼠进行比较的质谱亚蛋白质组和代谢组学分析；3)使用大规模的贝叶斯分析，这种分析是“知识受限的”，以寻找利用转录组和功能数据隐藏的未知途径；4)一种新的技术(锌指核酸酶)，可以有效地敲除被发现对mTAL调控网络有重要贡献的基因，为验证和表征功能相关性提供了一种明确的方法。 项目1解决了高血压领域面临的一个关键挑战：确定导致盐敏感型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)可能在高血压和肾脏损伤中发挥重要作用的创新概念，项目3旨在确定远离ChR 13上肾素基因的突变调节SS大鼠肾素活性和血管生成的机制。所有项目都从核心8和C提供的基因组、遗传学、蛋白质组和计算技术进步中受益匪浅。

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 multifactorial (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 Project 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.