CHBPR: Angiotensin AT_1a Receptors in the Subfornical Organ are Required for DOCA-Salt Hypertension

Abstract

Although elevated renin-angiotensin system activity and angiotensinergic signaling within the brain are required for hypertension, polydipsia and increased metabolic rate induced by deoxycorticosterone acetate (DOCA)-salt, the contribution of specific receptor subtypes and brain nuclei mediating these responses remains poorly defined. We hypothesized that angiotensin type 1a receptors (AT_1aR) within the subfornical organ (SFO) mediate these responses. Transgenic mice carrying a conditional allele of the endogenous AT_1aR (AT_1aR^flox) were administered an adenovirus encoding Cre-recombinase and eGFP (AdCRE) or eGFP alone (AdGFP) into the lateral cerebral ventricle. AdCRE reduced AT_1aR mRNA and induced recombination in AT_1aR^flox genomic DNA specifically in the SFO, without significant effect in the paraventricular or arcuate nuclei, and also induced SFO-specific recombination in ROSA^TdTomato reporter mice. The effect of SFO-targeted ablation of endogenous AT_1aR was evaluated in AT_1aR^flox mice at three time points: 1) baseline, 2) one week after virus injection but before DOCA-salt, and 3) after three weeks of DOCA-salt. DOCA-salt treated mice with deletion of AT_1aR in SFO exhibited a blunted increase in arterial pressure. Increased sympathetic cardiac modulation and urine copeptin, a marker of vasopressin release, were both significantly reduced in DOCA-salt mice when AT_1aR was deleted in the SFO. Additionally, deletion of AT_1aR in the SFO significantly attenuated the polydipsia, polyuria and sodium intake in response to DOCA-salt. Together, these data highlight the contribution of AT_1aR in the SFO to arterial pressure regulation potentially through changes on sympathetic cardiac modulation, vasopressin release and hydromineral balance in the DOCA-salt model of hypertension.

Introduction

Approximately 25% of hypertensive patients exhi bit low-renin hypertension that is associated with salt-sensitivity.¹ In animals, low-renin hypertension can be experimentally modeled with deoxycorticosterone acetate (DOCA)-salt treatment. Early studies showed that daily subcutaneous injections of DOCA with 1% NaCl drinking solution caused an elevation of arterial pressure in rats.² Importantly, the peripheral renin-angiotensin system (RAS), a major regulator of arterial pressure is suppressed after chronic DOCA-salt treatment³, but despite this, the brain RAS is elevated.^4,5 Hyperactivity of the brain RAS has been implicated in the development and maintenance of hypertension in several experimental and genetic animal models⁶, and can also effect fluid balance and metabolism.⁵ Angiotensin II (Ang-II), one of the major bioactive peptides of the brain RAS, mediates most of its known functions through the angiotensin II type 1 receptors (AT₁R), AT_1aR in mice.⁷ However, the exact mechanisms and specific brain nuclei involved in mediating the effects of elevated brain RAS activity remain poorly understood.

The circumventricular organs (CVO), including the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT), and the area postrema (AP), are highly vascularized brain regions that respond to circulating signals. The CVOs lack a blood-brain barrier and thus are key points of intercommunication between the blood, the brain parenchyma, and the cerebrospinal fluid. Numerous studies support their role in sodium and water balance, cardiovascular regulation and energy metabolism.⁸ The SFO has a number of axonal projections to cardiovascular control regions in the hypothalamus⁹, and some of these projections may utilize Ang-II as a neurotransmitter.¹⁰ Lesions of the anteroventral third ventricle (AV3V) region, which include the SFO, abolish the effects of DOCA-salt on arterial pressure¹¹ and fluid intake¹², suggesting that the SFO is likely involved in arterial pressure and fluid volume control. Previous studies have shown that central production and action of Ang-II through AT₁R are crucial for DOCA-salt induced hypertension.^4,13 DOCA-salt increases the expression of angiotensin receptors in the SFO¹⁴, and microinjection of Ang-II into the SFO induces drinking¹⁵ and pressor responses.¹⁶ Recent studies suggest there is an interaction between Ang-II and aldosterone in the brain and that both AT₁R and mineralocorticoid receptor are necessary for the hypertension induced by either.¹⁷ Here, we tested the hypothesis that specifically targeting the murine AT_1aR in the SFO would impact arterial pressure and fluid balance in the DOCA-salt model.

Methods

Please see the online-only Data Supplement for a more detailed Methods section.

Animals

Male mice (12–18 weeks) containing a conditional allele of the endogenous AT_1aR (AT_1aR^flox) gene were studied (the N is indicated in the figures/legends).¹⁸ ROSA^TdTomato mice were from the Jackson Laboratory (Stock 007914). Mice were maintained on standard laboratory chow and tap water ad libitum. All studies were approved by the University of Iowa Animal Care and Use Committee and was performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Surgery and DOCA-salt model

All measurements were performed in mice during baseline (before any treatment), 1 week after virus injection, and again 3-weeks after DOCA implantation (Figure 1A). Three separate cohorts of mice were utilized for arterial pressure, intake behavior and indirect calorimetry. Recombinant adenovirus encoding Cre-recombinase and eGFP (AdCRE) or eGFP alone (AdGFP) obtained from the University of Iowa Vector Core was microinjected (1μL, 10¹⁰ plaque-forming units/mL) into the lateral cerebral ventricle (ICV).¹⁹ After one week, the same cohort of mice underwent a subcutaneous implantation of a 50-mg pellet of DOCA (Sigma) performed under isoflurane anesthesia without uninephrectomy.²⁰ Mice were singly housed after recovery and maintained on standard chow with ad libitum access to tap water and 0.15 mol/L (0.9%) NaCl for 4 weeks. Arterial pressure was measured by radiotelemetry (TA11PA-C10, Data Sciences International).^5,20 Spectral analysis of heart rate (HR) was performed to assess autonomic modulation of cardiac function, and baroreflex sensitivity was determined using the sequence technique as detailed in the online-only Data Supplement.²¹ Resting metabolic rate was assessed by indirect calorimetry.⁵ Photomicrographs of the ROSA^TdTomato brains injected with AdCRE or AdGFP (ICV) were obtained from 16 brains (total 4 AdGFP and 12 AdCRE) using a Nikon fluorescence microscope and SFO images were merged using Adobe Photoshop CS (version 8.0).

Figure 1. Experimental Design.

A) Schematic of the protocol utilized in the study. B) Schematic map of the intact and recombined AT_1aR^flox allele. Primers for the genomic DNA PCR are represented.

Statistics

Data were analyzed by two-way ANOVA, with repeated measures as appropriate. Bonferroni post-hoc tests were utilized to dissect treatment effects. Data sets failing normality or equal variance tests were subjected to nonparametric analyses, including Friedman's ANOVA and Mann-Whitney U tests. Fold changes (mRNA) from the RT-PCR assays were analyzed using the Livak method.²² Differences were considered significant at P<0.05. All data are presented as mean±SEM.

Results

AT_1aR^flox mice underwent the experimental protocol shown in Figure 1A. Experimental end points were measured at baseline, 7 days after AdCRE or AdGFP was administered, and during the third week after DOCA-salt.

Validation of SFO-targeted AT_1aR

Brains were collected from each mouse at the end of the protocol and micropunches of SFO, paraventricular nucleus (PVN), arcuate nucleus (ARC) and cortex were used to evaluate the deletion of AT_1aR. Expression of AT_1aR mRNA was significantly decreased in the SFO of AdCRE-treated mice (Figure 2A). Decreased AT_1aR mRNA expression was observed in PVN but it was not statistically significant (Figure 2B). There was no change in AT_1aR mRNA expression in ARC (Figure 2C) or cortex (data not shown). We further evaluated the site-specificity of the AT_1aR deletion by assaying for Cre-mediated recombination in genomic DNA. A PCR product representing the intact gene was detected in the SFO of mice treated with AdGFP, whereas in the AdCRE-treated group, the recombined allele was detected in 6 of 7 samples (Figure 2D). No recombination was observed in the cortex. Weak recombination was noted in the ARC but little was observed in the PVN (Figure S1, see http://hyper.ahajournals.org).

Figure 2. Validation of AT_1aR Deletion.

A–C) Expression of AT_1aR mRNA was measured in AT_1aR^flox mice by qPCR from RNA isolated from micropunches of SFO (A), PVN (B) and ARC (C). D) Evidence of Cre-mediated recombination in the SFO measured by PCR of genomic DNA from micropunches. M=PCR marker (1Kb Plus DNA ladder, Invitrogen). N=negative control. Validation was performed at the end of DOCA-salt treatment. N=11 for AdGFP; N= 14 for AdCRE. *P<0.05 vs. control (AdGFP). All data are mean ± SEM.

ROSA^TdTomato reporter mice were injected with AdCRE and brains were collected 1 to 4 weeks after injection to further validate specificity. In this model, red fluorescence becomes apparent only after Cre-mediated recombination. Abundant TdTomato staining is evident that overlaps with eGFP (also encoded on AdCRE) and the DAPI stain for nuclei (Figure 3A). Three other representative images of the SFO from different mice indicate robust red fluorescence in neuronal cell bodies and processes (Figure 3B). We also examined the OVLT, median preoptic nucleus (MnPO), supraoptic nucleus (SON), ARC, AP, and the PVN weekly for 4 weeks. There was no red fluorescence in any region in mice injected with AdGFP. One week after AdCRE, red fluorescence was detected in a few ependymal cells of the OVLT, MnPO, SON, ARC, AP and PVN (Figure S2). This may account for the recombination noted at the genomic DNA level in the ARC. By 2-weeks, there was no signal in the OVLT, and by 3-weeks the ependymal signal in the PVN was gone (Figure S3). However, there was a preservation of a robust red fluorescent signal in the SFO even 4 weeks after AdCRE. Together, our data measuring AT_1aR mRNA, recombination of AT_1aR^flox genomic DNA, and the ROSA^TdTomato reporter mouse indicate that our ICV injection is efficient at specifically-targeting the SFO.

Figure 3. Validation of SFO-specific Targeting.

A) Images from the SFO of ROSA^TdTomato mice injected with AdCRE. B) Additional merged images from the SFO of independent ROSA^TdTomato mice injected with AdCRE.

SFO AT_1aR are necessary for DOCA-salt hypertension

Arterial pressure was unchanged at baseline in groups destined to receive either AdCRE or AdGFP, and virus administration did not change arterial pressure (Figure 4A). DOCA-salt treatment increased arterial pressure by approximately 30 mmHg in the AdGFP mice (Figure 4B), whereas deletion of AT_1aR in the SFO blunted the increase in arterial pressure by DOCA-salt (Figure 4A–B). Interestingly, heart rate (HR) decreased to a similar degree in both AdGFP and AdCRE mice in response to DOCA-salt (Figure 4C).

Figure 4. Effects of AT_1aR deletion in the SFO on DOCA-salt hypertension.

A–C) 24 hour mean arterial pressure (MAP, A), changes in MAP compared to baseline (B) and heart rate (C). D) Spectral analysis of heart rate (HR) in low-frequency (sympathetic and parasympathetic cardiac modulation) and (E) high frequency (parasympathetic cardiac modulation). F) Measurements of urine copeptin. The N is indicated in each bar. *P<0.05 vs. AdGFP; ^†P<0.05 vs. baseline; ^‡P<0.05 vs. virus. All data are mean ± SEM.

Sympathetic activation and impaired baroreflex function are closely related to increased BP in the DOCA-salt model of hypertension.²³ Therefore, we studied autonomic modulation of cardiac function using spectral analysis of HR and baroreflex function using the spontaneous sequence technique. Interestingly, the increases in low frequency HR variability (which reflects both sympathetic and parasympathetic modulation) seen with DOCA-salt were blunted by AdCRE (Figure 4D). High frequency HR variability (which reflects only parasympathetic cardiac modulation) did not differ between the two groups (Figure 4E), suggesting that the blunted low frequency HR variability in the AdCRE-treated mice reflects reduced sympathetic cardiac modulation. Baroreceptor-heart rate reflex sensitivity tended to be larger in the AdCRE group compared to the AdGFP control group after DOCA-salt (Figure S4).

Increased vasopressin (AVP) release has been reported to be required for DOCA-salt hypertension.²⁴ Consequently, we estimated AVP release by measuring urine copeptin, a stable product from the preproAVP produced in a 1:1 ratio with AVP.²⁵ Like arterial pressure, urine copeptin was equivalent at baseline and after virus, and the increase caused by DOCA-salt was significantly blunted by AdCRE (Figure 4F).

SFO AT_1aR are necessary for DOCA-salt induced changes in fluid balance

Fluid intake was equal at baseline and after virus, but markedly increased in response to DOCA-salt (Figure 5A). A marked increase in urine output (Figure 5B), sodium intake (Figure 5C) and sodium excretion (Figure 5D) was also observed after DOCA-salt. The induction of each of these phenotypes was blunted in the AdCRE treated mice. Male DOCA-salt mice exhibited a mild hypernatremia that was blunted by AdCRE (Table 1). Hypokalemia was not altered by AdCRE.

Figure 5. Effects of AT_1aR deletion in the SFO on Fluid Turnover.

Daily fluid intake (A), urine output (B), sodium intake (C), and sodium excretion (D) measured in metabolism cages. N=11 for AdGFP; N= 14 for AdCRE. *P<0.05 vs. AdGFP; ^†P<0.05 vs. baseline; ^‡P<0.05 vs. virus. All data are mean ± SEM.

Table 1.

Body weight and blood analyzes in non-treated mice (control) and DOCA-salt treated AdGFP and AdCRE

Group	Body Weight (g)	Blood Na (mM)	Blood K (mM)	Blood Glucose (mM)
Control	28.8 ± 0.5	148±0.5	6.9±0.2	204±10
AdGFP	28.3 ± 0.6	155±1.2*	4.3±0.3*	172±8*
AdCRE	29.7 ± 0.6	149± 2.8	4.7± 0.6*	166±8*

^*P<0.05 vs. control

SFO AT_1aR are not required for DOCA-salt induced changes in metabolic rate

Body weight was unchanged, whereas blood glucose was decreased by DOCA-salt but was unaffected by deletion of SFO AT_1aR (Table 1). Oxygen consumption during the baseline and virus time periods was not different between groups (Figure 6). DOCA-salt increased oxygen consumption, but this increase was not affected by the deletion of AT_1aR in the SFO.

Figure 6. Effects of AT_1aR deletion in the SFO on Metabolic Rate.

Resting metabolic rate measured by indirect calorimetry. N=14; ^†P<0.05 vs. baseline; ^‡P<0.05 vs. virus. All data are mean ± SEM.

Discussion

The main findings from these studies are that AT_1aR in the SFO, and possibly other nuclei such as the PVN, contribute to the effects of DOCA-salt on arterial pressure and fluid and sodium intake, but are not required to mediate the DOCA-salt-induced increase in oxygen consumption. These data support the hypothesis that AT_1aR signaling in the SFO is important for DOCA-salt hypertension, and further suggest that sympathetic activation and vasopressin release are possible mechanisms.

Human hypertensive patients more frequently exhibit depressed than elevated circulating RAS activity.¹ Despite this, very few models of low-renin hypertension are commonly studied. DOCA-salt treatment, first reported to have hypertensive effects in rodents², is a model of low-circulating-RAS activity that correlates with (and is dependent upon) elevated brain RAS activity.^4,13 Previous pharmacological studies have demonstrated that RAS peptide production and action in or near the cerebral ventricles and within the hypothalamus are required for DOCA-salt hypertension.^4,13 Similarly, early work supported the concept of cross-talk between mineralocorticoids and the RAS within the brain in the control of fluid and sodium balance.²⁶ Whereas, the ability to chronically target individual brain nuclei pharmacologically has limited utility, genetic techniques such as those employed in this study provide powerful tools that increase selectivity and precision and can be maintained chronically.

Previous studies have demonstrated a surprising specificity of SFO targeting with injection of adenoviruses into the lateral cerebral ventricles.¹⁹ We previously demonstrated a similar level of specificity with direct injection of viruses into the SFO²⁷, though with its small size, direct targeting is extremely difficult, occurs with a very low yield, and has the potential to damage the SFO. ICV injection of AdCRE resulted in SFO-specific genetic recombination, evidenced by robust red fluorescence in neuronal cell bodies and SFO neuronal processes in the ROSA^TdTomato reporter strain, and attenuated expression of the AT_1aR in AdCRE-treated AT_1aR^flox mice. Evidence of recombination was initially observed in ependymal cells throughout the ventricles, but this disappeared after 1–2 weeks, suggesting these cells turnover rapidly. Similarly, although we observed some evidence for AT_1aR recombination in the ARC, there was no decrease in expression of AT_1aR mRNA, and no evidence of recombination in the ARC in ROSA^TdTomato mice. Likewise, although there was a trend toward a decrease in AT_1aR mRNA expression in the PVN, this decrease was not statistically significant, and like the ARC, could not be replicated in ROSA^TdTomato mice. Despite consistent and efficient targeting of the SFO by AdCRE, we recognize that we cannot completely rule out the possibility that recombination and AT_1aR gene ablation occurred in regions that we did not examine in detail. Nevertheless, we are confident that our data demonstrate undetectable recombination in non-ependymal cells in regions that are known contributors to brain RAS signaling, including the MnPO, OVLT, SON, ARC, PVN and AP. This is important because DOCA-salt has been reported to increase angiotensin receptor density in some of these structures.²⁸ Thus, we conclude that the physiological consequences of AT_1aR recombination in this model are a result of a primary defect in AT_1aR activity likely due to SFO-specific deletion of AT_1aR.

What remains unclear are the post-receptor mechanisms that are most affected by the attenuation of AT_1aR activity in the SFO and ultimately cause a decrease in arterial pressure and fluid intake. For example, oxidative stress in the SFO has been shown to mediate the arterial pressure effects of Ang-II.²⁹ Similarly, we cannot exclude the possibility that the effects observed in AdCRE animals could be caused by reduced neuronal activation to the PVN due to a loss of angiotensinergic signaling between the SFO and PVN.¹⁰ Ongoing and future studies will utilize PVN-specific microinjection of AdCRE to examine the contribution of this nucleus to the phenotypes observed in the current study. DOCA-salt also increases aldosterone levels and central expression of mineralocorticoid receptors, and an interaction between the actions of Ang-II and aldosterone in the brain in hypertension has been reported.¹⁷ Future studies will be necessary to unravel the importance of this crosstalk in the SFO.

It is notable that SFO-targeted ablation of AT_1aR had a minimal impact on baseline arterial pressure or fluid intake, whereas it effectively attenuated the fluid turnover and arterial pressure effects of DOCA-salt. Our data and other studies suggest that AT_1aR in the SFO may be dispensable under normal conditions, but are essential to mediate arterial pressure and fluid intake responses after Ang-II and DOCA-salt.^15,29 Further, roles for both vasopressin and the sympathetic nervous system in DOCA-salt hypertension have been previously established³⁰, and our data are consistent with contributions of the SFO in the control of both of these mechanisms. It is notable that lesioning the SFO significantly reduces vasopressin secretion after central Ang-II.³¹ Whereas the PVN and SON are known to produce AVP, the SFO does not. Our data suggest that AVP release in DOCA-salt is associated with AT_1aR signaling in the SFO. Consequently, the deletion of AT_1aR in the SFO could influence AVP production in these nuclei by virtue of the efferent projections from the SFO to the PVN and SON, some of which may be angiotensinergic.

Although the importance of the SFO in regulating fluid intake is undisputed, its role in regulating arterial pressure is less clear, as in rats lesions of the SFO attenuate Ang-II-induced hypertension³², but do not attenuate DOCA-salt-induced hypertension.³³ On the contrary, lesions of the anteroventral third ventricle (AV3V), which may ablate efferent projects from the SFO to sites in the AV3V region, prevent DOCA-salt-induced hypertension.¹¹ Studies in mice, where angiotensinogen expression is specifically ablated in the SFO support the importance of this region in mediating responses to Ang-II.²⁷ It remains unclear if the finding here of a role for the SFO in mediating arterial pressure responses to DOCA-salt are due to species-specific differences (lesion studies in rats vs genetic studies in mice), or are related to the different methodologies employed. Physical lesions of the SFO cause a complete loss of neuronal connectivity, a loss of both excitatory and inhibitory outputs. This could cause widespread changes in functional activity in areas targeted by the SFO and/or release compensatory mechanisms that may mask the role of the SFO. In contrast, highly specific deletion of a single receptor, in this case the AT_1aR, may affect only a single SFO output that is critical to arterial pressure regulation, a notion consistent with evidence for multiple brain RAS mechanisms.

We previously demonstrated that DOCA-salt treatment of wildtype mice results in elevated resting metabolic rate, and that ICV infusion of the AT₁R antagonist losartan significantly attenuated this effect.²⁰ Interestingly, lesion studies suggest that the SFO may mediate some thermogenic effects of Ang-II in rats.³⁴ Moreover, sRA double transgenic mice which exhibit abundant Ang-II in the SFO exhibit increased metabolic rate.⁵ It was therefore surprising that SFO-targeted deletion of the AT_1aR did not attenuate the induction of resting metabolism by DOCA-salt. These findings may be explained by the metabolic protective effects of ICV losartan being mediated by a different receptor subtype (e.g. AT_1bR), that the bio-distribution of losartan and AdCRE administered ICV may be different, and/or that AT_1aR receptors within the SFO are not involved in the control of resting metabolic rate. The latter would suggest that AT₁R in other nuclei which can be accessed by ICV losartan may play a role in regulating the metabolic responses to DOCA-salt. AT_1aR are present at high density in the MnPO, an area often associated with osmo- and thermo-regulation³⁵, and AT_1aR-containing and heat-sensitive thermoregulatory neurons are localized in the MnPO.³⁶ Angiotensinergic projections between the SFO and MnPO have been electrophysiologically and functionally characterized.¹² Similarly, AT_1aR have also been identified in the ARC, a site most often identified in the regulation of food intake, and the classical site of leptin action.³⁷ Ang-II-induced neuronal activity in the PVN can be augmented by changes in temperature suggesting that the PVN may be thermoregulatory in response to Ang-II.³⁶ Indeed, the SFO has angiotensinergic projections to the PVN, and Ang-II causes neuronal activation of PVN neurons.¹⁰ Further studies will be needed to assess if any of these regions, perhaps in cooperation with AT_1aR in the SFO play a role in mediating the metabolic effects of DOCA-salt.

Perspectives

Our recent studies in DOCA-salt mice and previously in double transgenic sRA, both models of hyperactivity of brain RAS, advance the concept that the brain RAS plays an important role in BP control, fluid turnover and energy expenditure. In the present study, we have reported that AT_1aR signaling in the SFO is important for DOCA-salt induced hypertension and influences sympathetic cardiac modulation, increases vasopressin release, and robustly stimulates fluid intake. On the contrary, ablating AT_1aR signaling within the SFO is alone insufficient to attenuate increased resting metabolic rate in the DOCA-salt model. We have previously provided compelling evidence supporting the hypothesis that there are differential efferent mechanisms regulating arterial blood pressure, fluid turnover and metabolic rate by the brain RAS.⁵ These new data suggest divergent region-specific signaling, some angiotensinergic, may also be mediating these responses.

Novelty and Significance.

What Is New?

• AT_1aR in the SFO are important for mediating increases in arterial pressure in DOCA-salt mice.

• DOCA-salt induced changes on vasopressin release, sympathetic cardiac modulation and fluid turnover were attenuated when AT_1aR were specifically deleted in the SFO.

• Ablation of angiotensin signaling through AT_1aR within the SFO did not affect DOCA-salt induced increases in resting metabolic rate.

What Is Relevant?

• The SFO is a circumventricular organ accessible to many circulating endogenous factors and pharmacological compounds and AT_1aR in this important area of the brain are required to fully mediate the arterial pressure increases in the DOCA-salt model of experimental hypertension.