A myriad of physiological data confirms that renin is a critical component of the normal angiogenesis response seen in skeletal muscle with electrical stimulation. Using SS-13BN/Mcwi (SS-13BN) consomic rats and a series of congenic rats, we have demonstrated that restoration of a small region surrounding the renin gene confers normal renin levels and restores the normal angiogenic phenotype in the Dahl S (SS) rat. Despite this data, no difference has been observed in the coding region or the classically defined promoter region of the renin gene that would explain the differences in expression. Therefore it is the goal of this application to determine the sequence variants in the region, demonstrate which of these variants impact renin gene regulation in vitro, and using a transgenic approach, demonstrate that this candidate sequence variant eliminates normal renin regulation and the angiogenic phenotype in a subcongenic rat. This will be accomplished through a series of experiments comprising three specific aims.
In specific Aim 1, we will begin with a congenic rat line SS.BN- (D13rat123-D13rat101)/Mcwi (referred to as line 9) in which a region of less than 4.5 Mbp surrounding the renin gene region has been introgressed from the BN (Brown Norway) genome onto the SS/JrHsdMcwi (SS) background. Using well-established techniques of marker-assisted selection to identify recombinants, we will reduce this candidate region to 1-2 Mbp.
In specific Aim 2, we will identify candidate sequence variants within this reduced region by sequencing the SS and other closely related strains that do not share the antiangiogenic phenotype, including the Dahl salt resistant (SR), the Lyon Normotensive (LN), the Fawn Hooded Hypertensive (FHH) and the BN rats. Using the five strains, we have shown that we can reduce the number of potential causative strain-specific sequence variants within the candidate region to a manageable number, theoretically as low as 10, with a probability of having a false positive candidate mutation remaining below 1%. The candidate variants identified by sequencing and validated by a bioinformatics approach will be tested in vitro. This will be achieved by the use of a cell based system in which endothelial cells derived from the subcongenic line carrying the reduced region (Aim 1) will be transfected with SS Bacterial Artificial Chromosomes (BACs) and renin regulation will be assessed. We will use the surrogate phenotype of serum starvation-induced renin expression to assess the efficacy of the targeted mutation in modulating the renin phenotype.
Specific Aim 3 will focus on the final identification and validation of the causative strain-specific sequence variant in this region. Our final experiment will be to use a transgenic rescue approach with a bacterial artificial chromosome transgene harboring the SS allele. Substitution of the allele identified in previous experiments will be performed on the background of the reduced subcongenic to test for confirmation of the loss of the angiogenic phenotype in vivo.
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