Narrowing Genetic Regions on Rat Chromosome Twelve Involved in the Development of Hypertension and Kidney Disease

Hypertension is a disease which afflicts a third of U.S. adults over the age of 20, and 90-95% of the adult hypertensive cases are characterized as primary or essential hypertension, meaning that the underlying cause of the elevations in blood pressure are unknown. In addition, hypertension is one of the leading risk factors for chronic kidney disease, which affects 16.8% of adults over the age of 20 in the U.S. Unfortunately, substantial progress in the treatment and prevention of both hypertension and chronic kidney disease have not been made, partially due to their genetic complexity. Studies with inbred rat models, such as the SS (Dahl salt-sensitive) hypertensive and BN (Brown Norway) normotensive rat strains can help dissect the genetic complexity of hypertension and kidney disease. The SS strain develops hypertension and renal damage on elevated salt diets. Previous work has shown that a 6.1 Mb SS region on rat chromosome 12 significantly increases blood pressure and renal damage compared to the SSBN12 control rat strain, which was derived by transferring BN rat chromosome 12 onto the SS background.;This thesis examines the following hypotheses: (1) A gene(s) located between 13.41-19.55 Mb on rat chromosome 12 contribute to blood pressure regulation and/or renal damage and (2) the causative gene(s) and pathway(s) can be identified through positional cloning approaches and sequence/expression analysis.;As there were 133 genes and 12,673 single nucleotide variants between SS and BN within the 6.1 Mb candidate region for hypertension and renal disease on rat chromosome 12, smaller sub-congenic strains were developed and characterized in order to physically narrow the list of potential candidate genes and variants. By generating three overlapping sub-congenic strains and excluding regions that did not appear to elevate blood pressure and renal disease, we reduced the candidate region for hypertension and renal disease by 86% to an 830 kb region with 14 genes. Furthermore, we observed indicators of renal damage before the development of hypertension in our rat models.;Whole genome sequencing, whole transcriptome analyses, and qRT-PCR were used to further prioritize candidate genes. Analysis of whole genome sequencing data from several different rat strains showed a 100 kb region with 96 single nucleotide positions where SS had a different allele at every position compared to the normotensive BN, SR (Dahl salt-resistant), and WKY (Wistar Kyoto) rat strains (which all shared the same allele at all 96 positions). Only two genes, Grifin and Chst12, are present within the 100 kb region. Moreover, whole transcriptome and qRT-PCR analyses nominated Grifin and Chst12 as novel candidate genes for hypertension and/or renal disease: Grifin was found to be significantly up-regulated in mesenteric vessels between our smallest congenic and the control SSBN12 strain while Chst12 was significantly down-regulated in congenic mesenteric vessels, renal cortex, and renal medulla compared to the control SSBN12 strain. In addition, sequence, structural, and evolutionary analysis of the Grifin gene showed that the current annotation of the human GRIFIN gene as a pseudogene is incorrect.;Finally, we examined the resistance vasculature of our congenic and consomic control strains prior to the development of hypertension to determine whether there were any functional differences between mesenteric arteries from these strains since both Grifin and Chst12 were found to be differentially expressed in mesenteric vessels. Third order mesenteric arteries from the 6.1 Mb congenic and a smaller congenic strain were significantly more reactive to the vasoconstrictors, phenylephrine and serotonin, compared to the SSBN12 control. No differences in response to the endotheliumdependent (acetylcholine) or endothelium-independent (sodium nitroprusside) vasodilators were observed between congenic and consomic mesenteric arteries. In addition, congenic mesenteric arteries were stiffer and had significantly higher collagen to elastin ratios compared to consomics. In summary, these results showed that congenic mesenteric arteries had enhanced sensitivity to vasoconstrictors and structural differences prior to the development of hypertension.;Collectively, the studies in this dissertation nominated two novel genes, Grifin and Chst12, for hypertension and renal disease, in addition to implicating the resistance vasculature and the renal system as contributors to elevations in blood pressure in our congenic strains in response to high salt diets. Future studies need to validate the role of Grifin and/or Chst12 in hypertension and kidney disease.