Targeting Kinin B1R Attenuates Hypertension Through AT1R-Dependent Mechanisms

Abstract

BACKGROUND:

Neurogenic hypertension is chronically high blood pressure that is initiated and maintained through excessive sympathetic nervous system activity and has been associated with increased B1R (kinin B1 receptor) activation. We previously reported a central role for B1R in mediating inflammatory pathways in the development of deoxycorticosterone acetate salt hypertension. Additionally, we identified a causal relationship between B1R expression after Ang II (angiotensin II) stimulation, and that B1R can mediate the bidirectional interaction between neuroinflammation and oxidative stress. However, whether there are any interactions between AT1R (Ang II-type I receptor) and B1R, and if B1R can mediate the effects of Ang II–induced hypertension, has not yet been investigated.

METHODS:

We used a well-established mouse model of Ang II–induced hypertension to test the hypothesis that B1R activation contributes to increased sympathoexcitation, autonomic dysfunction, oxidative stress, and inflammation, potentially through interactions with AT1R. Wild-type and BIR knockout mice were infused with Ang II or saline via osmotic minipump for 28 days, then functional and molecular changes in response to Ang II were assessed.

RESULTS:

Ang II in wild-type mice led to significant increases in B1R expression associated with sympathoexcitation, autonomic dysfunction, impaired baroreflex sensitivity, and enhanced blood pressure, whereas these changes were attenuated in B1R gene-deficient mice. B1R was shown to directly interact with AT1R, and activation of B1R was involved with microglial activation and subsequent neuroinflammation, increased neuronal firing, and altered synaptic density. We further used pharmacological blockade of B1R to dismiss potential developmental alterations in gene-deficient mice. Specific B1R antagonist attenuated Ang II–induced increases in blood pressure, supporting the role of B1R in blood pressure regulation.

CONCLUSIONS:

Our data provide the first evidence of the role of B1R in Ang II–induced hypertension and its interactions with AT1R, highlighting B1R as a potential therapeutic target for hypertension.

Novelty and Significance.

What Is Known?

• Dysregulation of the kallikrein kinin system and its receptors, notably B1R (B1 receptor), is involved in cardiovascular diseases and other pathological conditions associated with inflammation.

• Kinin B1R expression levels in the brain are upregulated during deoxycorticosterone acetate salt hypertension and are associated with increased inflammation and oxidative stress.

What New Information Does This Article Contribute?

• This study is the first to demonstrate upregulated B1R expression in the paraventricular nucleus of postmortem brains from hypertensive patients.

• B1R and AT1R (Ang II [angiotensin II] type 1 receptor) directly interact in the paraventricular nucleus during hypertension, offering a novel mechanism that may underlie the enhanced effects of Ang II observed in the hypertension brain.

• Global deletion of B1R or selective pharmacological blockade of B1R attenuated Ang II–induced hypertension, autonomic dysfunction, and inflammation.

Previous studies have shown that neuroinflammation within the brain significantly contributes to the development and progression of hypertension. This study demonstrates that B1R expression is upregulated in key autonomic nuclei, such as the paraventricular nucleus in both animal models and postmortem hypertensive human brains. Moreover, we identified a direct interaction between B1R and AT1R and that B1R blockade offered a reduction in inflammation and sympathoexcitation, therefore attenuating neurogenic hypertension. Our findings establish B1R as a key modulator of central inflammation and neural dysfunction in Ang II–induced hypertension, suggesting that blockade of B1R may serve as a novel therapeutic target for the treatment of hypertension.

Meet the First Author, see p 931

Neurogenic hypertension is chronically high blood pressure that is initiated and maintained through excessive sympathetic nervous system activity to the cardiovascular system.^1,2 Centrally acting Ang II (angiotensin II) has been shown to be the primary initiator of this increased sympathetic activity and subsequent blood pressure elevation in the central nervous system (CNS) during the pathogenesis of hypertension, specifically in autonomic regulatory centers, such as the paraventricular nucleus of the hypothalamus (PVN).^2,3 The PVN is an important integrative center for autonomic and neuroendocrine regulation, playing a central role in maintaining blood pressure homeostasis through its control of sympathetic outflow, making it a key region to study central mechanisms underlying neurogenic hypertension.^1,2 Ang II–induced hypertension disrupts the blood-brain barrier, resulting in microglial activation and neuroinflammation.^2,4,5 This perpetual proinflammatory environment significantly impairs synaptic plasticity and is associated with reduced presynaptic density and dysregulation of synaptic transmission in the PVN, ultimately increasing neuronal firing and sympathoexcitation.⁶

Ang II acts on the AT1R (Ang II type 1 receptors) in the brain to increase sympathetic tone and vasopressin secretion, leading to a substantial increase in blood pressure.⁷ Because of this, many of the current antihypertensive therapeutic agents directly target the renin-angiotensin system (RAS), such as angiotensin receptor blockers and ACE (angiotensin-converting enzyme) inhibitors. ACE inhibitors prevent the conversion of Ang I to Ang II and prevents the degradation of bradykinin.⁸ Therefore, the inhibition of ACE prolongs the half-life of bradykinin, ultimately increasing its concentration and activity.⁸ Bradykinin is a component of the kallikrein-kinin system and is known for its vasodilatory actions mediated through the B2R (kinin B2 receptor).⁹ However, bradykinin can be metabolized into an active metabolite, DABK (des-Arg⁹-bradykinin), which endogenously activates the B1R (kinin B1 receptor).¹⁰ Unlike B2R, which is constitutively expressed in various tissues, B1R is typically absent or expressed at low levels under physiological conditions but is upregulated in response to injury and inflammation.¹⁰ Previous studies have shown that B1R activation in the CNS is linked with increased oxidative stress, inflammation, and sympathetic nervous system activity, all of which contribute to the development of hypertension.¹¹ We have shown previously that in a model of deoxycorticosterone acetate (DOCA) salt hypertension, genetic deletion and pharmacological blockade of B1R prevented the increase in oxidative stress and inflammation, supporting a central role for B1R activation in neurogenic hypertension.¹¹ Using mouse primary hypothalamic neuronal cultures, we showed that B1R and AT1R are upregulated by exogenous Ang II and notably, B1R blockade attenuated Ang II–induced inflammation and oxidative stress in these neurons.¹² We also identified a bidirectional interaction between B1R and neuroinflammation in neurons, further indicating its role in hypertension pathology.¹³ These findings suggest a potential interaction between components of the RAS and kallikrein-kinin system within the brain during hypertension. Therefore, a crosstalk between B1R and AT1R may serve as a critical mechanism in the pathogenesis of hypertension, as it may potentially amplify Ang II–induced hypertensive effects by enhancing sympathetic outflow and promoting inflammation.

Studies have investigated the interaction among these systems and showed that heterodimerization between AT1R and B2R may contribute to an increased sensitivity to Ang II in hypertensive women.^14–16 However, there is limited knowledge on the precise role of B1R in Ang II–induced hypertension, and the possibility of B1R amplifying hypertensive effects through interactions with AT1R remains unknown. Our findings provide novel evidence demonstrating that B1R is upregulated in the PVN of patients with hypertension. Therefore, in the current study, we investigated the interactions between B1R and AT1R in the PVN and hypothesized that B1R activation contributes to increased sympathoexcitation, oxidative stress, and inflammation, potentially through interactions with AT1R in a model of Ang II–induced hypertension. We further report that blockade of B1R prevented Ang II–induced effects, supporting its role as a central player in the pathogenesis of hypertension. By elucidating the mechanisms of B1R in neurogenic hypertension, we aim to identify a novel therapeutic target that may lead to more efficient treatment modalities for hypertension.

Methods

Data Availability

The authors declare that all supporting data are available within the article and its Supplemental Material. Detailed descriptions of experimental methods of the current study are provided in the Supplemental Material.

Results

Kinin B1R Expression is Upregulated in the PVN of Patients With Hypertension

Details of all study subject characteristics are shown in Table S1. To determine whether B1R is upregulated in the brain of hypertensive subjects, we performed immunohistochemistry for B1R expression within the human brain PVN and subfornical organ (SFO). Immunostaining with a specific B1R antibody showed a significant upregulation of B1R expression in the PVN and SFO of hypertensive subjects^17,18 (Figure 1A; Figure S1A). No primary antibody control confirmed that there is no nonspecific binding of our secondary antibody. In addition, we performed double immunolabeling of B1R and HuC/D (Hu antigen C and D), a neuronal marker, which revealed colocalization of B1R within neurons (Figure 1B). Quantification of B1R staining further illustrated elevated B1R expression in the PVN of hypertensive subjects compared with normotensive subjects (Figure 1C). This suggests that neuronal expression of B1R is upregulated during hypertension. We then performed immunolabeling of B1R and IBA1, a microglia marker, which revealed colocalization with some, but not all, microglia in the PVN (Figure S1B). To elucidate whether B1R expression in the brain is correlated with hypertension, we performed a linear regression analysis. Systolic blood pressure (SBP) was increased in patients with hypertension (Figure 1D), and this was positively correlated with B1R expression (Figure 1E). Lastly, proximity ligation assay revealed increased B1R-AT1R interactions in both the PVN and SFO of hypertensive subjects compared with normotensive (Figure S1C and S1D), supporting a potential mechanistic role for B1R-AT1R crosstalk in central blood pressure regulation. Together, these data confirm that B1R is expressed and upregulated in the human brain during hypertensive states.

Figure 1.

Increased B1R (kinin B1 receptor) immunoreactivity in the paraventricular nucleus (PVN) neurons of hypertensive human brains. A, Representative images of B1R staining in the PVN of subjects with normotension (NTN) and hypertension (HTN). B, Immunofluorescence images displaying B1R (red) colocalization with neurons (HuC/D [Hu antigen C and D], green) in a hypertensive PVN. Hypertensive patients have elevated B1R expression (C) and systolic blood pressure (D) compared with normotensive subjects (n=4–7 patients/condition, Mann-Whitney U test). E, B1R immunoreactivity is positively correlated with increased systolic blood pressure in hypertensive subjects (Pearson correlation and least-squares linear regression). Data are presented as mean±SEM.

Kinin B1 Receptor Is Upregulated in Brain Regions Critical for Blood Pressure Regulation

To determine whether B1R expression is involved in Ang II–induced hypertension in mice and if there are sex-dependent responses, we measured plasma and hypothalamic levels of bradykinin and its metabolite DABK using quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Figure S2). Our results indicate that in Ang II–induced hypertension, there is upregulation of plasma and hypothalamic bradykinin and DABK levels compared with saline controls (Figure 2A). These findings suggest that in Ang II–induced hypertension, the kallikrein-kinin system is activated both peripherally and centrally, possibly contributing to autonomic dysfunction seen in hypertension. Gene expression revealed that B1R mRNA was upregulated in both males and females within SFO, PVN, nucleus tractus solitarius, and rostral ventrolateral medulla, all of which are key brain regions critical for blood pressure regulation (Figure 2B). However, B1R gene expression was higher in the PVN than in other regions associated with the regulation of blood pressure; therefore, this region was used for the rest of the study. We then performed immunolabeling for B1R in the PVN and identified a substantial upregulation of B1R in hypertensive mice compared with control mice (Figure 2C). In addition, B1R expression is predominantly colocalized with neurons and microglia and minimally in astrocytes within the mouse hypertensive brain (Figure S3). These results suggest that enhanced B1R expression is associated with Ang II–induced hypertension at both the mRNA and protein levels in the hypothalamus, highlighting a potential role of B1R contributions in the pathophysiology of hypertension.

Figure 2.

Increased B1R (kinin B1 receptor) expression in key cardiovascular regulatory centers. A, Plasma and hypothalamic bradykinin and DABK (des-Arg⁹-bradykinin) as measured by quantitative liquid chromatography-tandem mass spectrometry determination. Values that were below detection levels were excluded from statistical analysis; (n=6 mice/group with 6 total samples measured/group, Unpaired Student t test). B, B1R mRNA is upregulated in both males and females in key cardiovascular regulatory centers (n=6, repeated measures 2-way ANOVA). C, Representative immunofluorescence images and quantification of B1R expression (red) within the paraventricular nucleus (PVN) of the hypothalamus (third ventricle [3V]; n=5, Mann-Whitney U test). Data are presented as mean±SEM. Ang II indicates angiotensin II; DAPI, 4’,6-diamidino-2-phenylindole; NTS, nucleus tractus solitaries; RVLM, rostral ventrolateral medulla; SFO, subfornical organ; and WT, wild type.

To delineate the role of direct B1R activation in neurogenic hypertension, we examined the effects of intracerebroventricular administration of the B1R agonist DABK or artificial cerebrospinal fluid on SBP. The experimental timeline is shown in Figure S4A. DABK injection elicited an immediate and sustained increase in SBP compared with artificial cerebrospinal fluid-treated controls (Figure S4B). Quantitative analysis demonstrated that SBP was elevated in DABK-treated mice compared with artificial cerebrospinal fluid-injected mice (Figure S4C). To localize B1R expression in response to DABK, we used immunofluorescence staining. We found that B1R colocalizes with neurons in the PVN following DABK injection, confirming that B1R activation is upregulated in neurons acutely (Figure S4D). B1R expression and colocalization were absent in artificial cerebrospinal fluid-injected controls. These results demonstrate that B1R activation in the brain contributes to increased SBP, confirming the central activation of this pathway in hypertension.

Kinin B1R Deletion Attenuated Neurogenic Hypertension and Autonomic Dysfunction

To further identify the role of B1R in neurogenic hypertension, we used global B1R gene-deficient mice (kinin B1 receptor knockout [B1RKO]) and wild-type (WT) controls that underwent 4 weeks of Ang II (600 ng/kg per minute) or saline infusion. Within the brain, Ang II induces oxidative stress, inflammation, and transcription factor activation, making it a suitable model to study B1R’s role in hypertension and its associated neuroinflammatory mechanisms.¹⁹ Mean arterial pressure (MAP) was similar in both WT and B1RKO mice at baseline; however, Ang II treatment increased MAP in WT mice by day 14 (Figure 3A) and 28 (145±13 mm Hg versus 103±6 mm Hg; Figure 3B and 3C). This increase in MAP was attenuated in B1RKO mice, suggesting that B1R is a central player in the development of Ang II–induced hypertension. To assess autonomic function, spontaneous baroreceptor reflex sensitivity was determined using the sequence method. WT+Ang II mice displayed reduced spontaneous baroreflex sensitivity, whereas B1RKO mice treated with Ang II showed no impairment of spontaneous baroreceptor reflex sensitivity, suggesting B1R’s contributions to baroreflex dysfunction during hypertension (Figure 3D). Sympathoexcitation in WT mice was increased with Ang II, as indicated by increases in urinary norepinephrine levels (Figure 3E). Furthermore, WT+Ang II mice had increased urinary arginine vasopressin, measured by its surrogate marker, copeptin (Figure 3F). The increase of copeptin and norepinephrine was blunted in B1RKO. Ang II administration resulted in no alterations in vagal tone (reduced tachycardia with atropine); however, there were increases in vascular (decreased MAP with chlorisondamine) and cardiac (decreased MAP with propranolol) sympathetic drive, indicating excessive sympathetic activity (Figure 3G through 3I). These Ang II–induced changes in sympathetic drive were attenuated in B1RKO mice. There were no observed changes in intrinsic heart rate in both genotypes of mice infused with saline (WT, 393±33; B1RKO, 371±24) and Ang II (WT, 395±22; B1RKO, 388±32), suggesting that the increased sympathetic drive is mediated by autonomic nervous system modulation and not intrinsic cardiac dysfunction. Taken together, these data suggest that B1R gene deletion attenuates Ang II–induced hypertension and protects the mice from Ang II–induced impaired spontaneous baroreceptor reflex sensitivity and autonomic dysfunction.

Figure 3.

Kinin B1 receptor (B1R) gene deletion prevents the development of neurogenic hypertension. A, Chronic Ang II (angiotensin II) induced a progressive rise of mean arterial pressure (MAP) in wild-type (WT) control mice that was attenuated in B1R knockout (B1RKO) mice by day 14. B, There were no significant sex differences in MAP after 28 days of Ang II infusion in B1RKO mice. C, Ang II infusion increases MAP in male and female WT and B1RKO mice. However, blockade of B1R is able to reduce MAP compared with saline controls. D, Spontaneous baroreceptor reflex sensitivity (SBRS) was determined using the sequence method and improved in B1RKO mice treated with Ang II compared with WT mice. E, Indirect sympathoexcitation levels were determined through urine norepinephrine levels by ELISA and are increased by Ang II in WT mice but not B1RKO mice. F, Urine copeptin levels were increased by Ang II in WT mice but not in B1RKO mice. Autonomic function was assessed pharmacologically by determining the changes in MAP (ΔMAP) and heart rate (ΔHR) after ip injections of a (G) muscarinic antagonist (atropine 1 mg/kg, cardiac parasympathetic tone), (H) β-blocker (propranolol 4 mg/kg, cardiac sympathetic tone), and (I) ganglionic blocker (chlorisondamine 5 mg/kg, vascular sympathetic tone). n=4 to 6, 2-way ANOVA with Tukey multiple comparison tests. Data are presented as mean±SEM.

Blockade of B1R Attenuated Ang II–Induced Microglial Activation and Neuroinflammation

It is widely accepted that neuroinflammation is a key factor that drives sympathetic nerve activation and exacerbates hypertension.^20–22 Studies have shown that during hypertension, stimuli such as Ang II can activate microglia to promote the release of cytokines and chemokines, suggesting its role in neuroinflammation and blood pressure regulation.^23,24 To determine the role of B1R in microglia activation, immunostaining was performed for TMEM119 (transmembrane protein 119; microglia marker), and morphology was quantified using ImageJ AnalyzeSkeleton (2-dimensional/3-dimensional) plugin.²⁵ Ang II resulted in an increase of activated microglia in the PVN of WT mice but not B1RKO mice (Figure 4A and 4C). Photomicrographs were made binary, skeletonized, then tagged for end point voxels (<2 neighbors), slab voxels (exactly 2 neighbors), and junction voxels (>2 neighbors; Figure 4B). Quantification revealed that microglia from Ang II–treated WT mice had enlarged somas (Figure 4D) and a retraction of processes as indicated by decreased branching (Figure 4E) and number of end points (Figure 4F) per cell compared with B1RKO mice, which displayed a ramified appearance indicative of naive microglia.

Figure 4.

B1R (kinin B1 receptor) blockade prevents microglial activation in Ang II (angiotensin II)–induced hypertension. A, Immunofluorescence staining showed increased microglia expression (TMEM119 [transmembrane protein 119] staining) in the paraventricular nucleus (PVN) of Ang II–treated wild-type (WT) mice compared with saline-treated mice. B, Representative illustration of the skeleton analysis protocol applied to photomicrographs with a single cell cropped. Photomicrograph converted to binary, skeletonized, and then tagged following analyze skeleton plugin. C, Quantification showed increased microglia immunoreactivity in WT mice following Ang II treatment compared with B1R knockout (B1RKO) mice with Ang II. Summary data of microglia soma size (D), number of branches/cell (E), and number of microglia end points/cell (F). n=5 images/mouse PVN, 4 mice/group, 2-way ANOVA with Tukey multiple comparisons test. Data are shown as mean±SEM.

Chronic microglial activation has been shown to alter neuronal plasticity and brain homeostasis, worsening neuroinflammation, and hypertension.²⁶ To understand the contribution of B1R to neuroinflammation, we measured inflammatory cytokines and chemokines in the PVN following Ang II–induced hypertension using a MILLIPLEX MAP mouse cytokine/chemokine array. In Ang II treated WT mice, protein levels of TNF (tumor necrosis factor), IFN-γ (interferon γ), IL-1β (interleukin 1β), MCP-1 (monocyte chemoattractant protein 1), eotaxin, G-CSF (granulocyte colony-stimulating factor), IL-17 (interleukin-17), IL-6 (interleukin-6), VEGF (vascular endothelial growth factor), and RANTES (regulated on activation, normal T cell expressed and secreted protein) were upregulated, suggesting neuroinflammation. Additionally, IL-10 (interleukin-10) and IL-4 (interleukin-4) were decreased in Ang II–treated WT mice, indicating a reduction in anti-inflammatory signaling, further suggesting a shift towards a proinflammatory state (Figure S5). However, these increases were attenuated in B1RKO mice treated with Ang II, suggesting that blockade of B1R can reduce inflammation in the brain during hypertensive states. In line with this, we observed excessive neuronal activation and heightened sympathetic drive indicated by increased cFos and TH (tyrosine hydroxylase) immunostaining in WT mice treated with Ang II but not B1RKO (Figure S6). Taken together, these data indicate that B1R upregulation can induce microglia activation, creating a perpetual neuroinflammatory environment that could enhance neuronal activation.

Upregulation of B1R Can Induce Neuronal Hyperactivity

Neurogenic hypertension is driven by excessive sympathetic nerve activity, possibly stemming from increased sympathetic nerve firing rates.²⁷ We have previously shown that glutamate, which is involved in enhanced sympathetic drive, neuronal firing, and inflammation in hypertension, can induce B1R expression in primary hypothalamic neurons.²⁸ However, whether B1R activation can directly induce neuronal firing associated with elevated sympathetic drive remains unknown. To address whether B1R is associated with excessive neuronal firing, cortical neurons were isolated from embryonic day 18.5 mice and cultured in multielectrode array plates for 21 days to re-establish connections before being treated with a B1R selective agonist Lys-des-Arg⁹-bradykinin (LDABK; 30 nmol/L) with or without a specific B1R antagonist R715 (10 µmol/L). Cortical neurons were used for multielectrode array recordings as they form robust and spontaneous network activity needed to assess electrophysiological properties in vitro.^29,30 On 21 days in vitro, neuronal metrics were recorded on the Maestro Edge multielectrode array system (Axion Biosystems) before being stimulated with LDABK to obtain baseline measurements, then agent was given, and recordings occurred at 30 minutes, 3 hours, 6 hours, and 24 hours (Figure 5A). B1R activation was able to increase neuronal weighted mean firing rate at 30 minutes, 3 hours, and 6 hours; however, by 24 hours, weighted mean firing rate (WMFR) returned to baseline (Figure 5B). These findings are consistent with elevated sympathetic drive associated with hypertension, suggesting that B1R contributes to heightened neuronal activity seen in neurogenic hypertension. Network burst duration showed similar trends to the weighted mean firing rate, where bursting duration was increased following B1R activation, indicating sustained excitatory activity that is most likely exacerbating autonomic dysfunction through amplification of sympathetic output for extended periods of time (Figure 5C). More notably, the blockade of B1R with R715 was able to reduce both weighted mean firing rate and network burst duration relative to levels of vehicle, suggesting a critical role for B1R in mediating these neurophysiological changes. Interestingly, there were no observed changes in neuronal synchrony at any time point, indicating that B1R-induced alterations in neuronal activity occur at the level of individual neuronal excitability instead of a coordinated network-wide activity, further confirming B1R’s role in neuronal and autonomic dysfunction (Figure 5D).

Figure 5.

B1R (kinin B1 receptor) stimulation induces neuronal firing and burst duration. A, Schematic illustrating the process of cortical neuron dissociation from embryonic day 18.5 mice, treatment with B1R agonist Lys-des-Arg⁹-bradykinin (LDABK; 30 nmol/L) with or without B1R selective antagonist R715 (10 umol/L) after 21 days, and multielectrode array (MEA) recording. B, Weighted mean firing rate (WMFR) increased following B1R activation at 30 minutes, 3 hours, and 6 hours. Pretreatment with R715 before LDABK prevented this increase in firing. No change in firing was found at 24 hours. C, LDABK-induced increase in network burst duration was prevented by B1R antagonism at 30 minutes, 3 hours, and 6 hours. D, No changes in neuronal synchrony were found at any time point. n=2 to 3 wells/group/culture, 4 independent cultures/group. One-way ANOVA followed by Tukey multiple comparisons. Data are expressed as mean±SEM.

B1R is Localized at Presynaptic Sites in the PVN and Contributes to Synapse Loss in Hypertension

Previous studies have shown that Ang II can directly activate presynaptic AT1R in the brain during Ang II–induced hypertension and is associated with excessive synapse loss.^6,31,32 However, whether B1R is found on presynaptic or postsynaptic nerve terminals and its role in synaptogenesis remain unexplored. To determine the effects of B1R on synaptogenesis, we independently measured presynaptic and postsynaptic areas using antibodies against the presynaptic markers, VGLUT2 (vesicular glutamate transporter 2), and postsynaptic marker, PSD-95 (postsynaptic density-95), in the PVN according to previous literature (Figure 6A).^33,34 The area of overlap between presynaptic and postsynaptic markers was considered a synapse (Figure S7). Ang II–treated WT mice displayed elevated presynaptic density as indicated by increased expression of VGLUT2; however, this effect was blunted in B1RKO mice. Additionally, PSD-95 was not altered; however, Ang II treatment decreased colocalization of VGLUT2 and PSD-95, suggesting that B1R activation is associated with an imbalance and loss of synaptic density following Ang II in WT mice that is reduced by B1RKO (Figure 6B). Traditional microscopy methods are limited by resolution; therefore, we used super-resolution stochastic optical reconstruction microscopy to determine if B1R is localized in presynaptic or postsynaptic terminals. To determine this, we analyzed 15 to 20 individual synapses per PVN section and found that in the PVN of WT mice treated with Ang II, B1R is localized within presynaptic terminals. The median distance between B1R and presynaptic marker (VGLUT2) was 0.11 µm, which is less than the distance between B1R and postsynaptic marker PSD-95, which was 0.31 µm (Figure 6C). To confirm these findings, we performed this analysis once more using synapsin as a presynaptic marker and Homer1 as a postsynaptic marker. Our results match our previous findings, with B1R being more closely localized to presynaptic proteins over postsynaptic markers (Figure 6D).

Figure 6.

B1R (kinin B1 receptor) blockade reduces Ang II (angiotensin II)–induced synapse loss. A, Representative images of a male mouse paraventricular nucleus of hypothalamus (PVN) stained for presynaptic marker VGLUT2 (vesicular glutamate transporter 2; green) and postsynaptic marker PSD-95 (postsynaptic density-95; red). Inset shows complete synapse in higher magnification. Images shown in Figure 6A are also presented in Figure S7, where additional image details and expanded panels are provided for clarity. B, Ang II–treated WT mice displayed elevated presynaptic marker density relative to WT+Saline. Control and postsynaptic density were not significantly altered across the groups, signifying loss of synaptic density. B1R knockdown rescued this effect; (N=6, 2-way ANOVA followed by Tukey multiple comparisons. C, Example of a synapse from WT mice treated with Ang II and captured using super-resolution stochastic optical reconstruction microscopy (STORM) microscopy with VGLUT2, PSD-95, and B1R. The median distance between B1R and presynaptic marker VGLUT2 is less than the distance between B1R and postsynaptic marker PSD-95, indicating presynaptic localization of B1R in hypertensive states (n=15–20 synapses/mouse, 6 mice/group, 1-way ANOVA followed by Tukey multiple comparisons). D, Presynaptic localization of B1R was further confirmed with presynaptic marker Synapsin, and postsynaptic marker Homer1 (n=15–20 synapses/mouse, 4 mice/group, Kruskal-Wallis test with posthoc Dunn multiple comparisons. Data are expressed as mean±SEM.

To determine whether changes in synaptic density were confounded by neuronal loss, we performed terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining co-labeled with NeuN to assess apoptosis specifically in neurons (Figure S8). In Ang II–treated WT mice, TUNEL-positive cells were infrequent and showed minimal colocalization with NeuN, indicating that neuronal apoptosis is not a prominent feature of this model. Furthermore, B1R knockout did not alter the incidence of TUNEL-positive cells, supporting the conclusion that B1R blockade does not affect neuronal survival. This suggests that the effects of hypertension and B1R inhibition on synaptic density occur independently of neuronal cell death. Together, these data highlight a direct role for B1R in influencing neuronal excitability, synaptic communication, and autonomic dysfunction in hypertension.

B1R and AT1R Interactions are Amplified During Hypertension

Previous work has highlighted that Ang II can activate B1R signaling, but whether B1R and AT1R directly or indirectly interact to mediate hypertensive responses remains unclear. To investigate this, we used a proximity ligation assay (PLA), which allows us to detect and quantify B1R and AT1R interactions with high selectivity and specificity. PLA, through the use of secondary antibodies coupled to oligonucleotides and DNA amplification, allows for precise spatial resolution to detect dynamic receptor interactions that may drive cellular signaling (Figure 7A).³⁵ We validated the specificity of our PLA signal by omitting the probe and primary antibody, as well as by using tissue from AT1R knockout mice, all of which resulted in no detectable signal (Figure S9). In the PVN, we observed that Ang II treatment increased the interactions between B1R and AT1R compared with saline-treated mice, as indicated by an increased density of PLA fluorescent puncta (Figure 7B). Similarly, primary hypothalamic neurons treated with 300 nmol/L Ang II for 24 hours also exhibited an increase in B1R-AT1R interactions, further supporting the hypothesis that B1R and AT1R directly interact during hypertension (Figure 7C). To validate these findings, we used the brains from DOCA-salt hypertension mice and once again found that B1R-AT1R interactions were elevated in the PVN compared with Sham mice. Importantly, blockade of AT1R with losartan or B1R with R715 showed a reduction of PLA puncta in the DOCA model of hypertension, confirming the specificity of these interactions and their dependence on both receptors being present and active (Figure 7D). These results suggest that B1R-AT1R receptor crosstalk in the PVN may enhance neurogenic mechanisms of hypertension.

Figure 7.

Interactions between AT1R-B1R (Ang II [angiotensin II] type 1 receptor and kinin B1 receptor) are upregulated in hypertension. A, Proximity ligation assay (PLA) uses specific primary antibodies and secondary antibodies coupled with oligonucleotides that undergo DNA amplification to visualize receptor interactions that are in close proximity (40 nm). B, B1R and AT1R interactions were increased in the paraventricular nucleus of hypothalamus (PVN) following Ang II treatment in male WT mice compared with saline controls, as indicated by the increase in green fluorescence. C, Primary hypothalamic neurons stimulated with Ang II (300 nmol/L, 24 h) displayed upregulated AT1R-B1R interactions. D, We further report increased AT1R-B1R interactions in a model of deoxycorticosterone acetate (DOCA) salt hypertension. These interactions were attenuated with blockade of B1R (R715) or AT1R (losartan), confirming the specificity and selectivity of PLA. Representative images in Figure 7B and Figure S8 are intentionally the same to demonstrate staining specificity and negative controls.

To understand whether the effects seen are specifically driven by central B1R-AT1R interactions rather than peripheral receptor activation, we compared the effects of central versus peripheral B1R blockade in the DOCA-salt model of hypertension. R715, a peptide B1R selective antagonist, was administered via intracerebroventricular to selectively block central B1R, although the same dose of R715 was administered subcutaneously to restrict its action to the periphery, as R715 does not cross the blood-brain barrier. Central B1R blockade attenuated DOCA salt–induced blood pressure response,¹¹ B1R-AT1R interactions (Figure S10A), and oxidative stress levels (Figure S10B) in the PVN. In contrast, peripheral administration of R715 did not reduce blood pressure¹¹ and was unable to reduce oxidative stress and B1R-AT1R interactions in DOCA-salt hypertension. These findings suggest that the neuroprotective effects of B1R inhibition in hypertension are predominantly mediated by central rather than peripheral receptor blockade.

To further confirm and characterize the interactions between B1R and AT1R that were observed, molecular docking studies were performed to model their potential binding interface. Molecular docking was completed for AT1R (protein data bank [PDB] id: 6DO1) and B1R (PDB id: 7EIB) using ClusPro2.0 according to previous studies (Figure S11A).^36–39 The results support the interaction between AT1R and B1R and showed 5 hydrogen bond interactions between the residues of Thr190 of AT1R and Ser343 of BIR, Asp9 with Gln263, Lys135 with Gly205 and 2 interactions with Arg139 and Asn120 of B1R, visualized by using PDBsum (Figure S11B). Docking studies were then performed to elucidate the binding mode and interaction type of DABK with the AT1R-B1R complex using the GLIDE docking module of the Schrodinger suite. This molecular docking study suggested that the top-ranked conformation of DABK was well accommodated inside the active site of the AT1R-B1R complex (Figure S11C). DABK displayed hydrogen bond interactions with the residues Phe55 (chain A) and Tyr56 (chain A). DABK also exhibited a number of hydrophobic interactions with the active site residues Tyr54 (chain A), Phe55 (chain A), Lys146 (chain A), Phe55 (chain B), Phe56 (chain B), and Lys58 (chain B). This was further confirmed using Ang II as a ligand in Figure S11D, where key binding regions were located within the intracellular loops and transmembrane domains of both receptors. These predicted binding sites align with regions known to facilitate receptor heterodimerization and downstream signaling. This approach complements the in-situ PLA by offering molecular-level evidence for direct B1R-AT1R interactions and provides mechanistic insights into how Ang II promotes these interactions in neurogenic hypertension.

B1R Antagonism Reduces Ang II–Induced Hypertension

To further validate the central role of B1R, we used the blood-brain barrier crossing, nonpeptide B1R selective antagonist, SSR240612, in an Ang II–induced hypertension model. Similar to B1RKO mice data, SSR240612 attenuated the increase of blood pressure in Ang II–infused mice compared with saline-treated controls (Figure 8A and 8B). SSR240612 reduced markers of oxidative stress, dihydroethidium staining, confirming that B1R blockade mitigates Ang II–induced oxidative damage (Figure 8C and 8D). Furthermore, PLA confirmed that Ang II upregulates B1R-AT1R interactions in the PVN, which were attenuated by SSR240612 (Figure 8E). These interactions were further confirmed in primary hypothalamic neurons, where SSR240612 pretreatment blunted Ang II–induced B1R-AT1R interactions (Figure 8F). This reduction highlights the role of B1R in facilitating receptor interactions during hypertension. These findings further confirm that B1R blockade, whether genetic knockout or pharmacological blockade, can effectively lower blood pressure, reduce oxidative stress, and disrupt B1R-AT1R interactions in Ang II–induced hypertension.

Figure 8.

Pharmacological blockade of B1R (kinin B1 receptor) prevents Ang II (angiotensin II)–induced hypertension. A, SSR240612 (SSR) attenuates Ang II–induced mean arterial pressure (MAP) in males. B, At 28 days, Ang II–induced hypertension is reduced in mice given B1R antagonist SSR (n=6, 1-way ANOVA followed by Tukey multiple comparisons). General oxidative stress levels were upregulated during Ang II–induced hypertension as indicated by dihydroethidium (DHE) staining (D) and quantification (C; n=3, Kruskal-Wallis test with posthoc Dunn multiple comparisons). E, B1R blockade with SSR reduced B1R-AT1R (angiotensin II type 1 receptor) interactions detected by proximity ligation assay (PLA), and (F) this was further confirmed using primary hypothalamic neurons from wild type (WT) mice pretreated with SSR before the addition of Ang II.

Finally, to determine if Ang II activation is upstream of B1R induction or whether the receptors engage in bidirectional signaling, we cultured primary hypothalamic neurons from neonatal WT mice and treated them for 24 hours with AT1R specific agonist Ang II, and B1R specific agonist LDABK, alone or in combination with the selective AT1R antagonist telmisartan or B1R antagonist SSR240612. Ang II treatment increased B1R expression compared with vehicle-treated neurons, suggesting that AT1R activation may upregulate B1R expression (Figure S12A). This increase was attenuated by cotreatment with SSR240612 or telmisartan. Similarly, LDABK treatment elevated B1R expression, and pretreatment with SSR240612 was able to block the increase in B1R to baseline levels, whereas telmisartan attenuated this response (Figure S12A). Furthermore, Ang II upregulated AT1R expression, and this effect was blocked by telmisartan and reduced by B1R antagonism, suggesting that B1R signaling may contribute, in part, to AT1R expression (Figure S12B). LDABK also led to an increase in AT1R, and cotreatment with SSR240612 attenuated this LDABK-mediated AT1R expression, whereas telmisartan prevented the increase in AT1R expression (Figure S12B). These findings imply that while AT1R is directly regulated by Ang II, B1R activation also influences AT1R expression, likely through a feedforward mechanism. Taken together, these results support a model where AT1R activation drives B1R expression, although B1R activation can further enhance AT1R expression, establishing a bidirectional regulatory loop in neurons that may be relevant to hypertensive mechanisms.

Discussion

In the present study, we investigated the role of B1R blockade in mitigating Ang II–induced hypertension, sympathoexcitation, and neuroinflammation. The major findings of our study are that (1) B1R expression is upregulated in human PVN in hypertension; (2) Ang II–induced hypertension upregulated B1R expression in brain cardiovascular regulatory nuclei; (3) genetic knockout of B1R attenuated hypertension and associated autonomic dysfunction; (4) B1R blockade prevents microglial activation and neuroinflammation; (5) B1R can mediate neuronal hyperactivity and synapse loss during hypertension; and (6) B1R and AT1R directly interact during Ang II–induced hypertension. This study offers a novel and comprehensive understanding of B1R’s impact on the pathophysiology of hypertension.

Under physiological conditions, B1R is not usually expressed; however, it is quickly upregulated following injury or inflammatory stimuli.^10,40 B1R stimulation can modulate various processes, including immune cell infiltration, insulin resistance, bronchoconstriction, sympathoexcitation, and vasoconstriction.¹⁰ Previous studies have shown that activation of B1R can regulate T lymphocyte recruitment in the CNS,⁴¹ disrupt the blood-brain barrier, and promote leukocyte trafficking.^42,43 We have previously shown that B1R mediates inflammation in the CNS during DOCA-salt hypertension, and that blockade of B1R can reduce this effect and prevent the development of hypertension.¹¹ Furthermore, we identified that global B1R deletion does not significantly alter homeostatic cardiovascular function.¹¹ However, whether the protective effects of B1R blockade are present in other models of neurogenic hypertension or if they are specific to the DOCA-salt model remains unknown. Using primary hypothalamic neurons, we previously identified the involvement of B1R in Ang II–mediated neuroinflammation and oxidative stress.¹² Therefore, in the current study, we used Ang II–induced hypertension to model neurogenic hypertension. Ang II is the most widely used model of neurogenic hypertension because it can directly activate the CNS, resulting in increased sympathetic nerve activity and subsequent blood pressure elevation, thereby mimicking neurogenic hypertension.^19,44

Previous studies have outlined the contribution of the RAS in blood pressure regulation and autonomic function, with Ang II serving as the primary effector via AT1R-mediated signaling.^19,45,46 The findings in this study build on current literature by showing that B1R plays a significant role, possibly through its interactions with AT1R. In the current study, we showed for the first time that B1R expression is upregulated in the PVN and SFO of patients with hypertension. SBP was increased in these patients with hypertension, and this was positively correlated with B1R expression, highlighting the potential for B1R as a central player in the brain during hypertension. Additionally, in a model of Ang II–induced hypertension, elevation in blood pressure and autonomic dysfunction was attenuated with B1R blockade, more so highlighting a novel role for B1R in blood pressure regulation. Moreover, a single DABK injection was able to rapidly upregulate B1R expression and increase SBP, likely through an acute neuroinflammatory cascade. Previous studies have shown that neurons in the PVN are key contributors to sympathetic outflow in hypertensive states⁴⁷; therefore, we investigated the spatial mapping of B1R in the PVN. We identified that B1R is colocalized within neuronal and microglial populations, but to a lesser extent in astrocyte populations within the PVN. These findings support the link between receptor expression and autonomic dysfunction and support earlier observations that central RAS components and key regulators of blood pressure, like B1R, are highly localized in autonomic control nuclei. These results validate earlier work showing that B1R is upregulated in inflammatory conditions and enhance the understanding of the molecular mechanisms involved in PVN-mediated blood pressure regulation.

Our data provide direct evidence of B1R-AT1R interactions through PLA, supported by molecular docking analysis. These findings enhance existing literature that has largely focused on AT1R as the primary mediator of Ang II–induced effects.^48,49 Studies from our lab and others have recently suggested that B1R contributes to RAS signaling during pathological conditions, such as in times of injury and inflammation.^12,50,51 However, the crosstalk between B1R and AT1R in the brain during hypertension has not been explored. We identified direct interactions between B1R and AT1R in the PVN in an Ang II and a DOCA-salt model of hypertension, providing mechanistic explanation for the amplification of Ang II effects that are observed in hypertension. Despite the absence of elevated systemic Ang II in the DOCA-salt model, our findings still support a role for central AT1R in mediating B1R-dependent effects. Notably, pharmacological blockade and genetic knockout of B1R were able to prevent these interactions in both models, confirming their dependence on both receptors being active to exert their effects. To determine if these interactions had any clinical and translational relevance, we confirmed that B1R-AT1R interactions are increased in patients with hypertension compared with normotension. This novel interaction builds a foundation for understanding the mechanistic insights of neurogenic hypertension.

Previous studies have suggested that Ang II–induced hypertension results in neuron hyperexcitability, resulting in sympathetic overdrive.¹ The novel finding that B1R activation impacts neuronal firing rates and network burst duration provides functional evidence and validation of B1R’s significance in mediating sympathetic outflow and altering neuronal excitability in hypertension. A major consequence of neuronal dysregulation is excessive synapse loss.⁶ We found that Ang II–induced hypertension results in synapse loss, indicated by elevated presynaptic markers and no alteration in postsynaptic markers, suggesting an imbalance of presynaptic and postsynaptic density. Notably, B1R blockade lessened this synapse loss, suggesting that B1R activation can impair synaptic density. Additionally, we identified that neuronal apoptosis is not a prominent feature of Ang II–induced hypertension, consistent with previous reports that synaptic dysfunction can occur in the absence of overt neurodegeneration. The low number of apoptotic neuronal cells suggests that changes in synaptic density observed in the hypertensive brains are more likely due to functional or structural remodeling rather than cell loss. Furthermore, B1R inhibition did not alter the rates of apoptosis, reinforcing the conclusion that its protective effects on synaptic integrity are not mediated by enhanced neuronal survival. Literature suggests that Ang II can directly activate presynaptic AT1R in hypertension³¹; however, whether B1R is found on presynaptic or postsynaptic nerve terminals was not previously studied. Using super-resolution stochastic optical reconstruction microscopy, we were the first to identify that B1R is localized within presynaptic terminals of the PVN, further confirming the potential role of B1R and AT1R interactions in hypertension. These data establish a direct role for B1R in mediating neuronal functions during hypertension.

It is well established that neuroinflammation is a critical component of neurogenic hypertension, with microglia playing a central role. Studies have shown that microglia, when activated, mediate an inflammatory response, neuronal excitation, and overall contribute to blood pressure elevation.²⁴ AT1R has been implicated in microglial activation and promoting cytokine release,^52,53 but the contribution of B1R in microglia remains unknown. Our findings demonstrate that B1R inhibition reduces microglial activation and inflammatory cytokine expression, suggesting that B1R is a key driver of neuroinflammation. This validates prior work showing that B1R is upregulated under inflammatory conditions, particularly in the brain,^11,13 and highlights its novel role in linking hypertension to central inflammation.

The central role of B1R in Ang II–induced hypertension was identified throughout this study and provides novel evidence supporting and expanding on our prior observations in a DOCA-salt model of hypertension.¹¹ Throughout these studies, genetic knockout of B1R was used to delineate its role. To further confirm these results in Ang II–treated mice, we used SSR240612, a B1R selective antagonist capable of crossing the blood-brain barrier. We found similar results to those in B1RKO mice, where blockade of B1R was able to offer a reduction in blood pressure, autonomic dysfunction, inflammation, and oxidative stress. To further confirm if the effects are driven by central B1R-AT1R interactions, rather than peripheral receptor activation, we administered R715 to DOCA-salt mice both centrally and peripherally. Although R715 is a selective antagonist of B1R, only R715 administered centrally could effectively disrupt B1R-AT1R interactions and oxidative stress. Peripheral B1R blockade alone appeared insufficient to modulate these central mechanisms. This supports our previous findings, where central B1R blockade attenuated DOCA-salt hypertension and peripheral blockade could not prevent the development of hypertension.¹¹ These findings validate that central B1R blockade, whether genetic or pharmacological, can mediate the effects seen in neurogenic hypertension.

Building on our in vivo findings, we used primary neurons to directly delineate if AT1R activation is upstream of B1R or if they engage in a bidirectional signaling mechanism. Our data with primary neuronal cultures showed that stimulation with Ang II–increased B1R expression, and this effect was attenuated by both B1R and AT1R antagonism, suggesting that AT1R activation is upstream of B1R induction when there is direct ligand binding. Conversely, B1R stimulation with LDABK enhanced AT1R expression, which was reduced by B1R receptor blockade. These results support the existence of a feedforward loop, revealing a bidirectional relationship between B1R and AT1R. This mechanism may contribute to sustained neuroinflammatory and excitatory signaling in the hypothalamus and highlights the therapeutic potential of disrupting this receptor crosstalk to attenuate central drivers of hypertension.

Although this study provides compelling evidence for the central role of B1R in Ang II–induced hypertension, some limitations should be acknowledged. First, the mechanistic pathways downstream of B1R activation, including specific signaling cascades and cellular interactions within the brain, remain to be fully elucidated. Furthermore, initiating B1R blockade after the onset of hypertension would offer a more clinically relevant approach and is a limitation of the current study that we aim to address in the future. Based on our current findings, we believe that B1R antagonism would attenuate preexisting hypertension by lessening neuroinflammatory signaling and reducing sympathetic drive. Lastly, although we focused on central mechanisms, the potential contributions of peripheral B1R signaling in hypertension warrant further investigation.

The novel findings in our study highlight B1R as a potential therapeutic target in hypertension. Current treatment modalities predominately target RAS and AT1R, but our data suggests that B1R inhibition could provide additional benefits. The observed interactions between B1R and AT1R also highlight the possibility of combination therapies to target both receptors for enhanced efficacy to combat resistant hypertension. Overall, this study advances the understanding of the role of B1R in Ang II–induced hypertension, integrating systemic inflammatory, and neuronal mechanisms. These findings validate and extend the current literature, offering novel insights into the molecular processes underlying hypertension. Future studies are needed to explore the long-term effects of B1R blockade and its potential application in other cardiovascular and neuroinflammatory disorders.

ARTICLE INFORMATION

Acknowledgments

The figures were designed in part using BioRender (http://www.biorender.com).

Author Contributions

S. Sriramula conceptualized and designed research; S. Sriramula, D. Theobald, R.N. Bessetti, Y. Feng Earley, E. Lazartigues, and K. Litwa performed experiments; S. Sriramula, D. Theobald, R.N. Bessetti, E. Lazartigues, and K. Litwa analyzed data; S. Sriramula, D. Theobald, R.N. Bessetti, Y. Feng Earley, E. Lazartigues, and K. Litwa interpreted results of experiments; S. Sriramula and D. Theobald prepared figures; S. Sriramula and D. Theobald drafted article; S. Sriramula, D. Theobald, R.N. Bessetti, Y. Feng Earley, E. Lazartigues, and K. Litwa edited and revised article; and all authors approved the final version of the article.

Sources of Funding

This work was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under award numbers 5R01HL153115 to S. Sriramula; R01HL122770, R01DK135621 to Y. Feng Earley; and R01HL150592, R01HL163588 to E. Lazartigues.

Disclosures

None.

Supplemental Material

Supplemental Materials and Methods

Tables S1–S2

Figures S1–S12

Major Resources Table

References 54–63

Supplementary Material

res-137-950-s001.pdf ^{(2.5MB, pdf)}