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In salt-resistant phenotypes, chronic elevated dietary sodium intake evokes suppression of renal sodium retaining mechanisms to maintain sodium homeostasis and normotension. We have recently shown that brain Gαi2 protein pathways are required to suppress renal sympathetic nerve activity and facilitate maximal sodium excretion during acute intravenous volume expansion in Sprague-Dawley rats. Here we studied the role of brain Gαi2 proteins in the endogenous central neural mechanisms acting to maintain fluid and electrolyte homeostasis and normotension during a chronic elevation in dietary salt-intake. Naïve or bilaterally renal denervated adult male Sprague-Dawley rats were randomly assigned to receive an intracerebroventricular scrambled or Gαi2 oligodeoxynucleotide infusion and then subjected to either a normal salt (0.4%) or high-salt (8.0%) diet for 21-days. In scrambled oligodeoxynucleotide-infused rats, salt-loading, which did not alter blood pressure, evoked a site-specific increase in hypothalamic paraventricular nucleus Gαi2 protein levels and suppression of circulating norepinephrine content and plasma renin activity. In salt-loaded rats continuously infused intracerebroventricularly with a Gαi2 oligodeoxynucleotide, animals exhibited sodium and water retention, elevated plasma norepinephrine levels, and hypertension, despite suppression of plasma renin activity. Furthermore, in salt-loaded bilaterally renal denervated rats, Gαi2 oligodeoxynucleotide-infusion failed to evoke salt-sensitive hypertension. Therefore, in salt-resistant rats subjected to a chronic high salt diet, brain Gαi2 proteins are required to inhibit central sympathetic outflow to the kidneys and maintain sodium balance and normotension. In conclusion, these data demonstrate a central role of endogenous brain, likely paraventricular nucleus specific, Gαi2–subunit protein gated signal-transduction pathways in maintaining a salt-resistant phenotype.
Salt-sensitive hypertension, which occurs in approximately 50% of hypertensive patients, results in a 3-fold increase in the risk of adverse cardiovascular events1–3. In normotensive salt-resistant subjects, neural (renal sympathetic) and circulating humoral (angiotensin-aldosterone) sodium-retaining mechanisms are suppressed to counter the influence of dietary salt-intake on central sympathetic outflow and systemic cardiovascular hemodynamics4–6. In contrast, increased sympathetic nervous system activity and avid sodium and water retention at the level of the kidneys is pivotal in the development of salt-sensitive hypertension3, 4, 6, 7. Alterations in body fluid osmolality/Na+ concentration are detected by osmoreceptor/sodium-sensitive receptors located in the hypothalamic paraventricular nucleus (PVN) and the circumventricular organs4, 8, 9. The PVN is a key component of the neural network that regulates central sympathetic outflow, fluid/electrolyte homeostasis, and the long-term regulation of systemic arterial blood pressure10, 11.
CNS G-protein coupled receptor (GPCR) systems (e.g. the α2-adrenoceptor, the GABAB receptor) participate in the maintenance of fluid and electrolyte balance in salt-resistant subjects by inhibiting renal sympathetic nerve activity, which facilitates natriuresis and normotension12, 13. However, the role(s) that downstream Gα-subunit proteins play in mediating the central GPCR-evoked renal sympathoinhibitory and natriuretic responses to high salt-intake remains unknown. Our laboratory has demonstrated that a moderate increase in sodium intake (0.9% saline drinking water) for 7 days evokes an endogenous, hypothalamic PVN-specific increase in Gαi2 protein expression14. Further, central Gαi2 protein pathways are required to mediate the natriuresis produced by both central α2-adrenoceptor stimulation and an acute i.v. isotonic saline load14, 15. Additionally, we have demonstrated the renal sympathoinhibitory, and consequently natriuretic, responses to isotonic saline volume expansion are dependent on central Gαi2 protein pathways14.
Based on our prior observations, we hypothesize that in a salt-resistant phenotype (e.g. Sprague-Dawley rat) subjected to a chronic elevation in dietary sodium intake, brain Gαi2-subunit protein-gated signaling pathways are augmented as a mechanism to maximize inhibition of central sympathetic outflow to the kidneys and thereby facilitate sodium excretion. Further, we predict that blockade/inhibition of this central Gαi2 protein sympathoinhibitory pathway will trigger sodium and water retention and the development of salt-sensitive hypertension.
Male Sprague-Dawley rats (Harlan Laboratories Inc., IN), 275–300 g, were housed individually under a 12-h light/dark cycle. Following completion of chronic surgical procedures (see below) rats were randomly assigned to a standard rodent diet (total Na+ content 0.4% [174 mEq Na+/kg]) or high sodium diet (total Na+ content 8% [1.378 mEq Na+/kg]) and provided ad libitum access to tap drinking water for a 21-day experimental period. All procedures were conducted in accordance with the National Institutes of Health, the Louisiana State University Health Sciences Center and the Boston University School of Medicine Institutional Animal Care and Use Committee guidelines for the Care and Use of Laboratory Animals.
Down-regulation of brain Gαi2 protein expression levels in rats was achieved by continuous i.c.v. infusion of a phosphodiesterase oligodeoxynucleotide (ODN) probe that selectively targets Gαi2 proteins (5’-CTT GTC GAT CAT CTT AGA-3’)14, 15 (The Midland Certified Reagent Company Inc., TX). Control studies involved i.c.v. infusion of a scrambled (SCR) ODN (5’-GGG CGA AGT AGG TCT TGG-3’). For chronic ODN infusion, animals were anesthetized (ketamine, 30 mg/kg intraperitoneally [i.p.] in combination with xylazine, 3 mg/kg i.p.) and stereotaxically implanted with a stainless steel cannula into the right lateral cerebral ventricle, which was connected via silastic tubing to a miniosmotic pump (model 2004; Durect Corporation, CA). ODNs were dissolved in isotonic saline and infused i.c.v. at 25µg/6µl/day.
Metabolic balance studies were conducted in all treatment groups on day-21 of the dietary sodium intake period14, 16. Following completion of the metabolic balance study, the same animals were then randomly assigned to a subgroup in which they were 1) surgically instrumented for blood pressure measurement and acute cardiovascular studies (see below; N=6/treatment group/diet/acute study), or 2) sacrificed for collection of blood (N=6/treatment group/diet). In all cases whole brains were collected and frozen for measurement of Gα-subunit proteins.
After a 21-day high dietary sodium challenge, animals were anaesthetized (sodium methohexital, 20 mg/kg, i.p., supplemented with 10 mg/kg intravenously, [i.v.] as required) and instrumented with catheters in the left femoral artery and left femoral vein for the measurement of arterial blood pressure, i.v. administration of isotonic saline, and/or pharmacological agents, respectively14–17.
After surgical instrumentation and a 2-h stabilization period in which rats were infused i.v. with isotonic saline (20 µl/min), baseline mean arterial blood pressure was recorded continuously over a 30-min period in conscious rats (N=6/treatment group/diet).
After measurement of baseline mean arterial pressure (MAP), animals received an i.c.v. injection of guanabenz (5µg/5µl) and peak changes in heart rate (HR) and MAP were recorded as previously described (N=6/treatment group/diet) 15.
In separate groups of rats, following baseline MAP measurement i.v. bolus atropine (1mg/kg), propranolol (1mg/kg) or hexamethonium (30mg/kg) was administered and peak changes in HR and MAP were recorded (N=6/treatment group/diet).
Plasma renin activity (PRA) was determined using a GammaCoat® Plasma Renin Activity 125I RIA Kit (DiaSorin, MN). Plasma norepinephrine (NE) content was determined using a NE enzyme-linked immunosorbent assay kit (Immuno-Biological Laboratories, Inc., MN)14. Plasma osmolality was determined using a Vapor Pressure osmometer (model 5600; Wescor Inc., South Logan, UT). Plasma sodium was determined by flame photometry (model 943; Instrumentation Laboratories, MA)
Frontal brain cortex (BC), hypothalamic paraventricular nucleus (PVN), supraoptic nucleus (SON), posterior hypothalamic nuclei (PH) and ventrolateral medulla (VLM) samples (N=6/brain site/treatment group/diet) were extracted from frozen brains cut on a cryostat using a brain punch tool (Stoelting, IL)14, 15. Gα-subunit antibodies purchased from Santa Cruz Biotechnologies (Santa Cruz, CA), anti-Gαi1 (1:100, sc-391), anti-Gαi2 (1:200, sc-13534), anti-Gαi3 (1:1000, sc-262) and anti-Gαo (1:200, sc-382) were used to detect respective proteins; anti-GAPDH was used as a loading control (1:1000, ab-9483, Abcam, MA)14, 15.
Data are expressed as mean ± SEM. Differences occurring between treatment groups (e.g., SCR/Gαi2 ODN treatment vs. normal/high salt diet) were assessed by a two-way ANOVA, followed by a Newman-Keuls post hoc test, to compare variations among the groups. Where appropriate, an unpaired Student’s t test was also used to compare means between two groups. Statistical analysis was carried out using a software program (GraphPad Prism version 5; GraphPad Software, CA). Statistical significance was defined as probability (P) < 0.05.
When faced with a 21-day dietary high-salt challenge, Sprague-Dawley rats exhibited a salt-resistant phenotype maintaining normotension, fluid and electrolyte balance, and suppression of neural (plasma NE) and humoral (plasma renin activity) sodium retaining mechanisms (Table 1).
In the same animals for which physiological parameters are shown in Table 1, a 21-day high-salt intake did not alter brain expression levels for Gαi1, Gαi3 or Gαo-subunit proteins (Figs. 1A, 1B, S1). In contrast. elevated dietary sodium intake produced a site-specific and significant, approximately 4.8-fold increase in PVN Gαi2 protein expression (PVN Gαi2 protein levels [ODU/mm2 normalized to GAPDH]; standard NaCl diet, 3.9±0.4 vs. high NaCl diet 22.8±3.1, P<0.05) (Figs. 1A, 1B).
The salt-resistant phenotype exhibited by Sprague-Dawley rats was not altered by the continuous central infusion of a control SCR ODN during high salt-intake (Table 2). In these SCR ODN-treated animals high salt treatment produced a significant up-regulation of hypothalamic PVN Gαi2 proteins, but no change in SON, PH or VLM Gαi2 protein levels (Figs. 2A, 2B); further high salt treatment failed to produce a detectable change in Gαi1, Gαi3 or Gαo protein levels (Figs. 2A, 2B, S2) as reported for naïve rats (Fig. 1). In rats fed a high, but not a normal sodium content diet, 21-day central Gαi2 ODN-infusion resulted in the development of salt-sensitive hypertension (MAP [mmHg] SCR ODN high NaCl, 127±4, Gαi2 ODN normal NaCl, 128±3, vs. Gαi2 ODN high NaCl, 147±3, P<0.05) that was accompanied by elevated 24-h water and sodium balance (P<0.05, Table 2). In these hypertensive Gαi2 ODN-infused animals, PRA was suppressed whereas plasma NE levels were significantly elevated; this is in opposition to the suppression of NE observed in naïve (Table 1) and SCR ODN-infused rats that consumed a high salt diet (P<0.05, Table 2). At the end of the 21-day study, central Gαi2 ODN infusion selectively blocked the hypotensive, but not bradycardic, responses to i.c.v. injection of the α2-agonist guanabenz (Table 2); this is a finding similar to that which we observed following acute central Gαi2 protein down-regulation15. There was no difference in plasma sodium concentration or osmolality between experimental treatment groups (Table S1). In the animals for which physiological data are presented in Table 2, targeted ODN-treatment produced selective and efficacious (approximate 85%) down-regulation of Gαi2 proteins in the BC, PVN and VLM, with no change in the regional expression of Gαi1, Gαi3 and Gαo-subunit proteins (Figs. 3A, 3B). Finally, in subgroups of SCR or Gαi2 ODN-infused high salt treated rats, there were no differences in the peak tachycardia or bradycardia responses elicited by atropine or propranolol, respectively, across treatment groups (Fig. 4A. 4B). In contrast, hexamethonium-mediated ganglionic blockade resulted in a significantly greater reduction in MAP in hypertensive Gαi2-ODN as compared to SCR-ODN infused rats (Fig. 4C).
Bilateral RDNX abolished the chronic high salt-induced hypertension produced by continuous central Gαi2 ODN-infusion (MAP [mmHg] Gαi2 ODN sham RDNX high NaCl, 146±2, vs. Gαi2 ODN RDNX high NaCl, 132±2, P<0.05, Table 3). Further, these RDNX animals which remained normotensive, maintained fluid and electrolyte homeostasis following the 21-day high sodium intake when compared to sham operated animals (Table 3). These animals also did not exhibit elevated global sympathetic activity as was observed in intact and sham operated animals (P<0.05, Table 3). There were no differences in plasma sodium or plasma osmolality between experimental treatment groups in sham or RDNX animals (Table S2). In Gαi2 ODN treated rats, the hypotension, but not bradycardia produced by i.c.v. guanabenz was prevented (Table 3). In these studies, RDNX was verified by a significant reduction in kidney norepinephrine content in denervated vs. sham operated animals. When calculated as an index of salt-sensitivity, SCR ODN-infused Sprague-Dawley rats exhibited a classical salt-resistant phenotype (Fig. 5). In contrast Gαi2 ODN-infusion caused a significant rightward-shift of the pressure-natriuresis relationship, which is typical of a salt-sensitive phenotype; this rightward shift was prevented in bilaterally RDNX animals which maintained a salt-resistant phenotype (Fig 5).
Reduced central sympathetic outflow, particularly to the kidneys, is critical to maintain fluid and electrolyte homeostasis and the long-term regulation of blood pressure during chronic high salt challenge12, 18. At present the central molecular mechanisms that mediate sympathoinhibition in response to excess dietary sodium intake, as is found in the typical western diet19, remain to be fully elucidated. The key finding of the present study is that during the stress of a high dietary salt intake, brain Gαi2 protein signaling pathways are activated to inhibit central sympathetic outflow and thereby maintain sodium and water balance and prevent the development of salt-sensitive hypertension.
In the present study we demonstrated that a chronic high dietary sodium intake evoked a significant, PVN specific up-regulation of Gαi2 protein levels in naïve and SCR ODN infused rats, which was not detected in neighboring hypothalamic regions (i.e., SON, PH) or other neural control sites examined (VLM). In naïve or control SCR ODN infused Sprague-Dawley rats, which maintained a salt-resistant phenotype, chronic high dietary sodium intake did not alter the expression of Gαi1, Gαi3 or Gαo-subunit proteins in any brain site examined. The endogenous increase in PVN Gαi2 protein expression in animals exhibiting a salt-resistant phenotype is of high physiological interest owing to the fact the PVN plays a key role in the central control of sympathetic outflow and the long-term regulation of arterial blood pressure11, 13, 20.
To investigate the functional significance of the observed sodium-evoked PVN specific increase in Gαi2 proteins, we examined how chronic ODN-mediated down-regulation of brain Gαi2 protein levels impacts fluid and electrolyte balance, blood pressure and neural/humoral sodium retaining mechanisms in rats maintained on a normal or high salt diet for 21-days. In rats fed a normal sodium diet, ODN-mediated knockdown of brain Gαi2 proteins did not alter daily sodium or water balance or mean arterial blood pressure from levels observed in SCR ODN-treated animals. Further, chronic down-regulation of brain Gαi2 proteins did not alter baseline levels of PRA or plasma NE during normal sodium intake. In contrast, in rats facing a chronic high sodium diet challenge, ODN-mediated down-regulation of central Gαi2 proteins evoked the development of salt-sensitive hypertension. Evidence supporting this is marked sodium and water retention, a significant elevation in systemic arterial blood pressure, and a profound rightward shift of the pressure-natriuresis curve.
Mechanistically, the development of salt-sensitive hypertension produced by sustained (21-day) Gαi2 protein down-regulation appears to involve global sympathoexcitation, as evidenced by increased plasma NE levels in central Gαi2 ODN-treated rats. Elevation in circulating levels of NE may contribute to an increase in systemic arterial pressure over time through enhanced renal tubular sodium reabsorption5, 21. This is supported by the observed increase in 24-h sodium (and water) balance (Tables 2, ,3)3) and the impaired pressure-natriuresis (Fig. 5) observed in rats made hypertensive by central Gαi2 ODN-treatment and a high salt intake. However, despite global sympathoexcitation, the sympathetic (propranolol challenge) and parasympathetic (atropine challenge) control of heart rate remained unaltered in animals following down-regulation of brain Gαi2 proteins. This is of considerable interest since an enhanced hypotensive response to ganglionic blockade was observed in these same animals. These findings suggest that the elevated plasma levels of NE also contribute to the elevation in MAP in Gαi2 ODN-treated rats on high salt intake by increasing vascular resistance to peripheral organ beds (e.g., potentially splanchnic or renal vasculature). Interestingly, in contrast to an inhibitory influence on sympathetic activity and NE release, our data demonstrate that central Gαi2 protein signal transduction pathways do not play a role in the central pathways that suppress the activity of the renin-angiotensin system (as assessed by reduced plasma renin activity [PRA]) during chronic increases in dietary sodium intake. This non-Gαi2 protein dependent reduction in PRA, and presumably aldosterone secretion, will contribute to the increased renal excretion of sodium and highlights the importance of non-neural mechanisms in maintaining fluid and electrolyte homeostasis. The current studies do not elucidate the mechanism underlying the observed suppression of PRA, however it is possible that suppressed PRA during high salt intake is mediated by a renal baroreceptor pathway or reflects a salt-evoked decrease in the number or renin secreting cells present in the afferent arteriole.
Based on the findings above, studies were conducted to determine the role of intact renal nerves in producing the observed increase in plasma NE levels and hypertension in central Gαi2 ODN-treated Sprague-Dawley rats fed a high salt diet. In these studies, chronic bilateral renal denervation, a technique used to remove influence of the renal sympathetic nerves on kidney function, prevented the central Gαi2 ODN-induced salt-sensitive hypertension. In renal denervation studies, global sympathoexcitation did not occur in response to an elevation in dietary sodium intake. Along with these findings, chronic renal denervation reset the pressure natriuresis curve of animals treated with the Gαi2 ODN and high salt to a level comparable to those of a salt-resistant phenotype (e.g., SCR ODN infused Sprague-Dawley rats). Collectively our data demonstrate that central Gαi2-subunit protein-gated pathways, likely PVN specific, are involved in opposing salt-induced increases in central sympathetic outflow to maintain fluid and electrolyte homeostasis and the long-term regulation of systemic arterial blood pressure.
The pathophysiological importance of brain Gαi2 proteins as a central molecular signal transduction pathway is demonstrated by the observation that in salt-resistant rats, down-regulation of brain Gαi2 protein expression leads to elevated sympathetic drive, renal sodium retention, and the development of renal nerve-dependent salt-sensitive hypertension. These findings support Guyton’s circulatory model, which highlight the importance of renal salt and water balance in setting the long-term level of blood pressure and the phenomenon of pressure-natriuresis7, 22. Further, as proposed by Rodriguez-Iturbe and Vaziri3, our studies support the addition of a role of enhanced central sympathetic outflow in the regulatory processes first proposed by Guyton. Recently debate has arisen regarding the underlying cause of hypertension, which could either result from over-activity of the sympathetic nervous system23 or excessive salt reabsorption at the level of the kidneys24. Our studies, as with those of Mu et al 2011, suggest the underlying pathogenesis of hypertension involves integration of both the central nervous system and the kidneys and that both organ systems directly contribute to the development of hypertension. Highlighting the translational significance of the present findings is evidence that a single nucleotide polymorphism in the Gαi2 gene results in a 2.2-fold increased risk for hypertension in Caucasian Italians25 and the Millennium Genome Project for Hypertension in Japan report that the Gαi2 gene is associated with hypertension-susceptibility26. Extending the clinical relevance of our findings and the role of the renal nerves in the pathophysiology of hypertension are the recent renal denervation studies in humans that have resulted in a persistent reduction in blood pressure27. It has been speculated that the success of this clinical procedure reflects, in part, a reduction in hypothalamic-mediated stimulation of central sympathetic outflow28, a hypothesis in line with our current data.
We hypothesize that these findings provide evidence for a novel endogenous PVN specific Gαi2 “anti-hypertensive” pathway, which when activated during the stress of a high salt-intake produces renal sympathoinhibition to maintain fluid and electrolyte homeostasis and normotension in a salt-resistant phenotype. Our findings provide further support for the inclusion of a role of central sympathetic outflow in the current modeling of the pathophysiology of hypertension that is based on classical Guytonian theory and highlight the intimate connection between the central nervous system and kidneys in hypertension. Collectively, these data suggest a new integrated central molecular target (brain Gαi2 subunit signal transduction proteins) for which new therapeutics can be targeted to alter central sympathetic outflow and renal excretory function to treat the multiple disease states that feature sympathoexcitation and the impaired renal handling of sodium, e.g., salt-sensitive hypertension, congestive heart failure.
Sources of Funding
This work was supported by the National Institutes of Health grants HL-107330 (RDW) and 8P20 GM103514 and P20 RR018766 (RDW and DRK) and the American Heart Association grant 12GRNT12060613 (DRK).
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