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.
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