In the rat, two distinct angiotensin type la receptor (AT1aR) mRNAs are synthesized from a single ATtaR Lene by alternative splicing. These transcripts are encoded by exons 1 and 3 (E1,3) and exons 1, 2, and 3 (E1,2,3). We found that the E1,3 variant is more efficiently translated and results in higher AT1R densities in cell membranes and greater Ang II-induced signal transduction than the E1,2,3 transcript. We also found that salt-Loaded hypertensive DahL salt-sensitive (DS) rats had higher ratios of E1,3/E1,2,3, even though no significant differences in the total mRNA Levels were observed. This increased ratio corresponds with higher AT1R densities in the renal cortex compared to normotensive DS and Dahl-salt resistant (DR) animals. In this grant, we propose to investigate the mechanisms and functional consequences of these alternatively spliced variants.
In Aim 1, we will determine the molecular mechanisms responsible for exon 2-mediated repression of AT1aR translation. We will test the hypotheses that the rat E1,2-,3 transcript is translated less efficiently in vivo than the E1,3 transcript due to an inhibitory RNA cis element within exon 2, which results in significantly less AT1R protein expression and AT1R-mediated function compared to the E1,3 variant; and, that exon 2 contains a regulatory element that serves to modulate rates of AT1R translation by interacting with exon 2 specific RNA binding proteins.
In Aim 2, we will determine the cell and tissue specificity of the altered expression of AT1aR transcripts observed in the salt-Loaded hypertensive DS rat and the time course of the alterations in splicing induced by sodium Loading. We will test the hypothesis that increased AT1aR E1,3/E,1,2,3 ratios observed in DS rats maintained on a HS diet, occurs in a cell specific manner within the kidney. In addition, we hypothesize that those cells and kidney structures exhibiting elevated E1,3/E1,2-,3 ratios will also exhibit abnormal expression of nitric oxide synthase (NOS) isoforms and nicotinamide adenine dinucteotide phosphate (NADPH) oxidase subunits since Ang II modulates these pathways through the AT1R and because DS rats exhibit abnormalities in NOS and NADPH oxidase in the kidney. We also hypothesize that the time course of saltinduced hypertension and changes in NOS and NADPH oxidase expression and activity will correlate with induction of altered At1R splicing.
In Aim 3 we will determine which factors associated with salt loading induce the alteration in ATlaR splicing that is observed in the hypertensive DS rat. We will test the hypothesis that intra-rena factors associated with sodium loading, rather than elevated MAP, induce the alteration in AT1R splicing. Even though the Literature suggests alternative splicing occurs in all mammalian angiotensin receptors, few investigators have focused on the role of alternative splicing in physiological and pathophysiological control of angiotensin receptor gene expression. Thus, these proposed studies will yield new insights into gene regulation of the AT1R. The fact that inhibitors of AT1Rs are very effective in treating hypertension and associated cardiovascular and renal disease, suggests that these studies could ultimately lead to new pharmaceutical targets to treat these age-related pathologies.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Cardiovascular and Renal Study Section (CVB)
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Lin, Michael
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Georgetown University
Internal Medicine/Medicine
Schools of Medicine
United States
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Sandberg, Kathryn; Ji, Hong; Einstein, Gillian et al. (2016) Is immune system-related hypertension associated with ovarian hormone deficiency? Exp Physiol 101:368-74
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