Beta-2 Adrenergic Receptor Polymorphic Variants: An Endotype of Salt Sensitive Hypertension (Beta2AR).

1. Background and Significance
2. Importance of the Problem. Over 1/3 of adults in the USA have hypertension (HTN), of which 51.3% (over 34 million Americans) have uncontrolled blood pressure (BP)1, leading to a high prevalence of cardiovascular (CV) complications mediated by elevated BP1. Among the major hurdles to be addressed in HTN are: 1) the lack of specificity in treatment programs, and 2) the unclear mechanisms by which genetic and environmental factors (e.g. dietary Na+ intake) affect BP and CV function. Clearly, the first hurdle is - for the most part - a consequence of the second. A recent document from NIH provides the following guide to address these issues. "To address interindividual variability [responder/ non-responder phenomena] in […] complex diseases [e.g., obesity, asthma, type 2 diabetes, hypertension, chronic kidney disease, and cardiovascular diseases], it has been instructive to take a lesson from the asthma field, noted for its early recognition of prioritizing elucidation of disease endotypes over phenotypes"3. Like asthma, HTN "is now considered an umbrella diagnosis for several diseases with distinct mechanistic pathways [endotypes] \& variable clinical presentations [phenotypes]. A phenotype covers the clinically relevant or obvious properties of the disease [for example, HTN could be salt resistant, salt sensitive, orthostatic, pregnancy induced, etc.]. However, phenotypes do not necessarily show the direct relationship to underlying disease etiology and pathophysiology that may be a key for treatments to work; endotypes [or endophenotypes which require a genetic component] are meant to do that. Another aspect is that several endotypes can lead to the same phenotype or umbrella diagnosis. Classifying complex diseases [e.g., HTN] using pathogenetic mechanism-based subtypes (endotypes/endophenotypes) may have other advantages in epidemiological, genetic, and treatment-related studies"3. Recent reviews have reported that for nearly 50% of the essential (primary) HTN population, all of whom have salt sensitive blood pressure (SSBP), a dozen or more endotypes potentially underpin their pathophysiologic mechanisms. To date, the pathophysiologic mechanisms of these endotypes ultimately involve dysfunction in sodium/volume homeostasis and the components of the two major pathways that regulate this process--- the renin-angiotensin-aldosterone (Aldo) system (RAAS) and the renal vasculature, i.e., renal plasma flow (RPF)10,11. In most cases, one or the other system is adversely affected, and the alternate system tends to compensate for the altered sodium handling. This project focuses on one of the less understood endotypes: beta-2 adrenergic receptor (β2AR) gene-mediated dysfunction in SSBP. This endotype is unique because preliminary and published data suggest that the mechanism(s) causing the SSBP association with a common β2AR risk diplotype (Arg16Arg/Gln27Gln) may or may not be the direct consequence of the primary effect of the diplotype---a gain of function for the β2AR gene. On one hand, increased β2AR levels could directly activate the two major Na+/volume homeostatic pathways (Aldo and RPF) resulting in Na+ retention and SSBP. However, it is possible that the cause of the SSBP may be indirect: maladaptation of the Na+/volume homeostatic systems that "believe" that the β2AR-induced vasodilation is the result of volume depletion and therefore Na+ retention is required - a maladaptation like that documented in other CV conditions - most notably, in congestive heart failure (CHF). Either of the two mechanisms could lead to SSBP, but with quite different therapeutic implications. Thus, the project herein will assess the two contrasting hypotheses regarding the mechanism(s) underlying the β2AR endotype-associated SSBP. These two different possibilities are reminiscent of the apparent contradictory results for the same diplotype - in asthma. The "risk" diplotype (Arg16Arg/Gln27Gln) is associated with bronchodilation, and as such should act as a protective mechanism (akin to an endogenous beta agonist); however, its prevalence is surprisingly higher in asthma12. The opposing diplotype that we call "non-risk" (Gly16Gly/Glu27Glu) associates with lower asthma risk but it also describes improved responsiveness to exogenous beta agonists13. The numerous β2AR-endotype investigations in the asthma field successfully converged on genotyping patients as a measure to ensure better responsiveness to beta agonist therapy14. However, in the CV system the apparent contradictory results have yet to be leveraged. The prevalence of the "non-risk" diplotype is higher in heart failure patients, yet it confers better responsiveness to beta-blockers15. In contrast, the "risk" diplotype (Arg16Arg/Gln27Gln) displays decreased vasodilation response to isoproterenol infusion9,16,17, potentially related to an already overstimulated vasodilation pathway at baseline (see our Preliminary Results for RPF). These apparently conflicting results for the β2AR diplotype remain unresolved in the CV field. While many PubMed publications link β2AR gene variance to asthma and CV function, none connect it to SSBP, except those from our group - a knowledge gap we aim to begin addressing in this proposal.
3. Scientific Premise: Recent scientific advances can now be leveraged to identify, within the complex HTN population, homogeneous subsets that share a common, often novel, root basis. As noted above, understanding the mechanism(s) responsible for the altered physiology in these subsets will provide an opportunity to hone treatment programs to an individual level, with the anticipation of better attainment of BP goals. We recently identified a novel hypertensive endotype involving β2AR gene variation, and its likely effects on receptor function. In two hypertensive cohorts, individuals homozygous for the β2AR risk diplotype Arg16Arg/Gln27Gln display SSBP4-6. Importantly, the prevalence of this diplotype is 13-32%, i.e. 8-21 million hypertensive Americans18. This functional diplotype inhibits β2AR downregulation7-9 and drives increased β2AR expression (gain of function). Further, to assess mechanisms at a cellular level, we generated a mouse model that carries the risk (Arg16Arg/Gln27Gln) β2AR diplotype (β2AR Mut), which displays SSBP and therefore is translationally relevant. The following preliminary and published data provide support for our hypotheses:
4. Both mice and human β2AR risk homozygotes have SSBP.
5. In human β2AR risk homozygotes, the SSBP is caused by a fall in BP on the restricted salt diet (RES), not the traditional rise in BP on the liberal salt diet (LIB).
6. In humans, the β2AR risk vs. non-risk diplotype displays higher RPF on a LIB diet, yet an inappropriate change in RPF response to Angiotensin II (AngII) in the face of SSBP (no difference from non-risk homozygotes).
7. In human β2AR risk vs. non-risk homozygotes, Aldo levels on a LIB diet are inappropriately high.
8. In the mice carrying the β2AR risk diplotype, males have an appropriate decrease in the renal artery resistive index (RI) on the LIB diet - i.e. an increase in RPF - while the females have an inappropriate increase in RI - i.e. a decrease in RPF. We had insufficient data to address this question in the human observational cohort. These data suggest at least two likely mechanisms for the β2AR endotype-associated SSBP (Fig. 1). The primary defect in the endotype (increased β2AR levels): either 1) directly activates the two major Na+/volume homeostatic pathways (Aldo and RPF) resulting in Na+ retention or 2) indirectly activates volume mediating sensors inappropriately signaling volume depletion. Thus, in response to the gain of function - vasodilation induced in the in β2AR risk homozygotes - the two key salt retaining mechanisms Aldo production and renovascular function appear to inappropriately increase Na+ reabsorption, volume expansion and SSBP. While either process could lead to SSBP, the therapeutic implications for the two mechanisms are quite different.
9. Effect on Scientific Knowledge/Clinical Practice This translational research project will elucidate the mechanisms by which a functional β2AR gene diplotype interacts with Na+ intake to modify Aldo levels and/or renovascular function and thus affect SSBP. Successful completion of this project may modify scientific knowledge/clinical practice in at least three ways. First, a genotype approach would allow early identification of individuals at risk for inappropriate circulating Aldo levels and/or renovascular dysfunction during Na+ replete conditions, with subsequent salt sensitive HTN and HTN-related CV complications. Second, this project may lead to changes in the treatment of HTN from the present non-specific, "trial and error", algorithm-based approach to a more specific, genotype-directed drug paradigm. If our hypotheses prove correct, this mechanistic project will provide the scientific basis for a future clinical trial to determine the efficacy of chronic β2AR-specific blockade in improving Aldo levels, renovascular function and SSBP in individuals homozygous for the β2AR diplotype. Third, the in-depth mechanistic knowledge to be gained from this project may lead to the identification of novel signaling pathways in the adrenal that are dysfunctional in the β2AR diplotype carriers, thus generating novel pharmacological targets for specific, individualized therapeutic approaches. Specifically, this may include the development of new drugs that target the adrenal β2AR signaling mechanisms or β2AR-AT1R interactions. This is important since current beta-blockers are mostly β1AR-specific, which may limit their clinical usefulness in this patient population.
10. Changes in the Field if Proposed Aims are Achieved. Despite substantial observational data that the mechanisms responsible for the prevalence and severity of various diseases differ in women than men, the current algorithmic-based approaches do not reflect these differences clinically or in research studies. Success in documenting the validity of the hypotheses being evaluated herein would encourage physicians and scientists to perform similar three-step approaches. 1) Acknowledge that chronic illnesses are NOT necessarily diseases but syndromes of a collection of individual diseases. 2) The underlying specific mechanisms can be determined using a translational approach combining deep phenotyping of a controlled human cohort with focused preclinical studies. 3) Creating a four-step paradigm to achieve specific, individualized therapy:

• Identify the specific mechanism of the individual diseases;
• Identify the precise treatment/prevention plan that is mechanistically based;
• Assess if there is a biological sex component;
• Identify the specific person with the disease (using genotype).

1. Specific Aims and Objectives

Hypertension (HTN) treatment is moving towards a more refined individualized approach because the present algorithmic, population-based approach has met with limited success. More than half of hypertensives do not achieve blood pressure (BP) goals1,2. In recent decades, scientific advances have been used to identify specific, homogeneous subsets of HTN that share a common root basis, now referred to as endotypes 3. Linking genetic factors to phenotypes, endotypes help identify more precise therapeutic mechanisms. One novel hypertensive endotype involves the beta-2 adrenergic receptor (β2AR), where individuals homozygous for the Arg16Arg/Gln27Gln β2AR risk diplotype display salt sensitivity of BP (SSBP)4-6. Importantly, the prevalence of this diplotype of 13-32%, i.e. 8-21 Million hypertensive Americans1. This functional diplotype inhibits β2AR downregulation7-9 and drives increased β2AR expression. We generated a mouse model that carries the risk (Arg16Arg/Gln27Gln) β2AR diplotype (β2AR Mut), which displays SSBP and therefore is translationally relevant.

As we have reported in other SSBP endotypes, one or both of the two key sodium (Na+)/volume homeostatic systems may be dysregulated, increasing Na+ reabsorption, volume expansion and SSBP. 1) Aldosterone (Aldo) production. The β2AR risk diplotype associates with increased β2AR expression in human adrenals and with increased Aldo levels on a liberal Na+ (LIB) diet 4-6 in young, but not old, hypertensives. 2) Renovascular function. The β2AR risk diplotype associates with higher renal plasma flow (RPF) on a LIB diet in young, but not old, hypertensives. This effect (likely related to increased vascular β2AR expression) is a potential normal RPF response to SSBP. However, on a restricted Na+ (RES) diet, the β2AR risk and non-risk homozygotes had a nearly identical percent change in RPF response to angiotensin II (AngII)--an inappropriate response to SSBP.

These data suggest two alternate hypotheses for the β2AR endotype-associated SSBP. The primary defect in the endotype (increased β2AR levels): either 1) directly activates the two major Na+/volume homeostatic pathways (Aldo and RPF) resulting in Na+ retention or 2) indirectly activates volume mediating sensors inappropriately signaling volume depletion. Either process could lead to SSBP, but with quite different therapeutic implications. We will address these contrasting hypotheses in three translationally structured Aims:

Aim 1: Hypothesis - The β2AR risk diplotype identifies, in humans, a subset of young hypertensives with SSBP, higher β2AR tissue expression and altered urine Na+ excretion. Approach: In β2AR risk vs. non-risk hypertensives (<51 years), we will assess prospectively: 1) SSBP, 2) levels of β2AR expression (ex vivo in PBMCs and superficial inferior epigastric arteries (SIEA)) and 3) the renal response to an acute salt load. Results: Risk homozygotes will show higher β2AR levels and increased SSBP, with lower BP on a RES (but not on LIB diet). Delayed Na+ excretion in the β2AR risk vs non-risk group would suggest a RPF restriction, while an accelerated excretion would suggest increased Aldo. Differences in responses by sex are not anticipated.

Aim 2: Hypothesis - In humans and mice, the β2AR risk diplotype identifies a SSBP subset mechanistically characterized by dysfunctional RPF responses to changes in Na+ intake. Approach: In β2AR risk vs. non-risk hypertensives (<51 years) and β2AR Mut vs. wild type (WT) mice on LIB and RES diets, we will assess: 1) in vivo RPF and BP basally (humans and mice) and in response to AngII and angiotensin-converting enzyme inhibition (ACEi) (humans); 2) ex vivo β2AR and AngII levels in human SIEA and mouse carotid vessels, as well as vascular reactivity studies in the same vessels (β2AR-specific agonism/blockade, AngII/ACEi). 3) in β2AR Mut vs. WT mice, we will contrast BP and RPF responses to in vivo treatment with β2AR-specific blocker ICI 118,551 vs. ACEi Enalapril. Results: Vascular β2AR and AngII levels will be higher in risk vs non-risk homozygotes, with reduced RPF responses to AngII, and decreased ex vivo vasoconstriction/vasorelaxation responses to AngII and β2AR-specific stimulation, respectively. Finally, it is uncertain whether the β2AR-specific blocker or the ACEi will result in a lower BP in β2AR Mut vs. WT mice.

Aim 3: Hypothesis - In humans and mice, the β2AR risk diplotype identifies a SSBP subset mechanistically characterized by dysfunctional Aldo secretion. Approach: In β2AR risk vs. non-risk hypertensives (<51 years) and β2AR Mut vs. WT mice on LIB and RES diets (see Aim 2), we will assess: 1) in vivo Aldo and PRA levels basally (humans and mice) and in response to AngII and upright posture (humans); 2) In mouse Zona Glomerulosa cells, we will assess Aldo responses to secretagogues (including a β2AR-specific agonist) and measure the β2AR and Aldo biosynthetic enzymes. 3) in β2AR Mut vs. WT mice, we will contrast Aldo responses to the β2AR-specific blockade vs. ACEi (see Aim 2). Results: In risk vs. non-risk homozygotes, Aldo will be increased at baseline and in response to AngII. In β2AR Mut vs. WT mice, adrenal β2AR will be increased, with higher serum Aldo levels and Aldo responses to agonists, with no changes in enzyme levels. it is uncertain whether one of the treatments will result in greater Aldo change in β2AR Mut vs. WT mice.

Both hypotheses explored in our Aims suggest that the β2AR risk diplotype activates the Na+/volume homeostatic systems. However, whether this activation is direct or indirect has profound therapeutic implications.

1. General Description of Study Design

A.1. Rationale: Our approach is based on two observational studies in hypertensives, in which individuals homozygous for the Arg16Arg/Gln27Gln β2AR risk diplotype, display SSBP and increased Aldo levels4-6. Further, our preliminary data suggest that these associations are driven by the effect of the diplotype in young, but not old, hypertensives on a LIB diet. In silico assessments in two eQTL databases also suggest that the β2AR diplotype associates with increased β2AR expression in human tissues. Additional preliminary studies in mice that carry the risk diplotype (β2AR Mut) show that - as compared to WT, β2AR Mut mice have SSBP. These data suggest two alternate hypotheses for the β2AR endotype-associated SSBP. The primary defect in the endotype (increased β2AR levels) can: either 1) directly activate the two major Na+/volume homeostatic pathways (Aldo and RPF) resulting in Na+ retention or 2) indirectly activate volume mediating sensors inappropriately signaling volume depletion with stimulation of these homeostatic pathways. The purpose of this Basic Experimental Study Involving Humans (BESH) is to determine, prospectively, the endotype driven by the β2AR risk diplotype and specifically document three critical steps by associating the risk β2AR diplotype with: 1) increased β2AR expression and protein levels in vasculature and mononuclear cells; 2) a specific response to an acute salt load: either accelerated or delayed Na+ excretion, suggesting either a RPF effect or an Aldo effect, respectively; and 3) increased SSBP.