Increased (pro)renin receptor expression in the subfornical organ of hypertensive humans

Abstract

The central nervous system plays an important role in essential hypertension in humans and in animal models of hypertension through modulation of sympathetic activity and Na⁺ and body fluid homeostasis. Data from animal models of hypertension suggest that the renin-angiotensin system in the subfornical organ (SFO) of the brain is critical for hypertension development. We recently reported that the brain (pro)renin receptor (PRR) is a novel component of the brain renin-angiotensin system and could be a key initiator of the pathogenesis of hypertension. Here, we examined the expression level and cellular distribution of PRR in the SFO of postmortem human brains to assess its association with the pathogenesis of human hypertension. Postmortem SFO tissues were collected from hypertensive and normotensive human subjects. Immunolabeling for the PRR and a retrospective analysis of clinical data were performed. We found that human PRR was prominently expressed in most neurons and microglia, but not in astrocytes, in the SFO. Importantly, PRR levels in the SFO were elevated in hypertensive subjects. Moreover, PRR immunoreactivity was significantly correlated with systolic blood pressure but not body weight, age, or diastolic blood pressure. Interestingly, this correlation was independent of antihypertensive drug therapy. Our data indicate that PRR in the SFO may be a key molecular player in the pathogenesis of human hypertension and, as such, could be an important focus of efforts to understand the neurogenic origin of hypertension.

NEW & NOTEWORTHY This study provides evidence that, in the subfornical organ of the human brain, the (pro)renin receptor is expressed in neurons and microglia cells but not in astrocytes. More importantly, (pro)renin receptor immunoreactivity in the subfornical organ is increased in hypertensive humans and is significantly correlated with systolic blood pressure.

INTRODUCTION

Hypertension affects one in three adults in the United States and is the most important risk factor for cardiovascular diseases, including stroke, myocardial infarction, congestive heart failure, and chronic kidney disease (18, 48). Integrated neural, humoral, and renal mechanisms that collectively mediate increased renal Na⁺ retention and sympathetic nervous system activity have been implicated in the pathogenesis of hypertension (3, 23, 51, 63). The importance of sympathetic nervous system alterations in human and experimental models of hypertension and associated mechanisms have been thoroughly previously reviewed (20, 24, 51). Of the factors that affect sympathetic activity, activity of the brain renin-angiotensin system (RAS) in cardiovascular regulatory nuclei is pivotal in the pathogenesis of hypertension in most animal models (2, 21, 69). In humans, high doses of angiotensin (ANG) II type 1 receptor blockers attenuate sympathetic outflow in chronic heart failure, indicating the clinical significance of brain RAS activity in the regulation of sympathetic activity in humans (54). On the other hand, molecular evidence for the presence of RAS components, including angiotensin-converting enzyme and angiotensin II receptors, in the human brain has been established by a number of elegant studies (1, 10, 40).

The (pro)renin receptor (PRR), encoded by ATP6AP2, is a single-pass transmembrane receptor for both renin and prorenin. In the human brain, PRR is reported to be expressed in the pituitary and hypothalamus and is colocalized to vasopressin and oxytocin neurons in the supraoptic nucleus (65). Interestingly, a mutation in an exonic splice enhancer of the ATP6AP2 gene has been reported in a family with X-linked mental retardation and epilepsy, indicating a role for PRR in cognitive functions and brain development (53). Recent studies (44, 46) have indicated that PRR nonproteolytically activates prorenin, enabling it to cleave angiotensinogen to ANG I, which is further converted to ANG II. Our laboratory recently reported that the brain PRR is a key component of local brain ANG II formation and is important for hypertension development in mice, possibly by activating prorenin (33, 35). In addition, the PRR also mediates direct activation of ANG II-independent signaling pathways (13, 46, 52, 69).

Knockdown of the PRR in the subfornical organ (SFO) attenuates ANG II-induced hypertension in mice, indicating the importance of PRR in this brain region (32). The SFO is one of seven circumventricular organs in the brain that lack a traditional blood-brain barrier (BBB) in both humans and rodents (16, 17, 22, 37). The permeability provided by this BBB deficiency makes it possible for the SFO to sense circulating factors (e.g., ANG II or prorenin) and electrolytes (e.g., salts) as well as interact with factors generated locally in the brain, allowing it to play a key role in blood pressure (BP) regulation and body fluid homeostasis (9, 39, 49, 59, 66). The SFO is also a major site of RAS activity (15, 32, 55, 62). In addition to sensing circulating ANG II, the SFO produces ANG II locally to control BP and body fluid homeostasis through projections to downstream neural circuits in the brain (55, 61).

In the present study, we examined the PRR level in the SFO and assessed its significance in hypertensive patients. PRR immunoreactivity was prominently detected in SFO neurons of the adult human brain. Importantly, the intensity of PRR immunoreactivity was significantly correlated with systolic BP (SBP) in humans and was elevated in hypertensive subjects.

METHODS

Human subjects.

A total of 23 postmortem SFO tissues from hypertensive and normotensive human subjects were collected at Tulane University Medical Center between May 2011 and August 2015. Clinical data, including patient history, diagnosis, and medications, were used in this retrospective analysis. Office BPs recorded during standard care, obtained from patients’ charts, were used for analyses. The designation of hypertension or normotension used in this study was based on clinical diagnoses in patients’ histories. Patients’ personal information was deidentified. The Research Integrity Offices at the University of Nevada (Reno, NV) and Tulane University have determined that this project complies with human research protection oversight by the Institutional Review Board. All study subjects were admitted to Tulane University Medical Center Morgue for immediate autopsy, and SFO tissue was collected within 5 h of death.

Localization of PRR in specific cell types of the human brain by immunofluorescence labeling.

SFO tissues were dissected and fixed in 10% formalin overnight. After being embedded in paraffin, tissues were sectioned at 5 μm thickness and mounted onto slides. Tissue was deparaffinized by heating slides in an oven at 60°C for 1 h. Slides were rehydrated by two washes with xylene for 10 min each followed by a graded ethanol series (100% to 95% to 70% to 50% to 30%, 2 min each) and a final wash in double-distilled water (ddH₂O) for 2 min. Antigen retrieval was performed by boiling slides in Na-citrate buffer (10 mM, pH 6.0) for 45 min. After nonspecific binding was blocked with 10% goat serum for 30 min, sections were incubated for 24 h at 4°C with a rabbit anti-PRR antibody characterized in our laboratory [diluted 1:200 in PBS containing 0.2% Triton X-100 (PBST)]. This PRR antibody was raised against a synthetic peptide (GLDELGKRYGEDSEQFRD) that recognizes the extracellular domain of both the mouse (32, 33) and human full-length PRR (35, 52) but not the truncated form of PRR (32). Sections were additionally incubated with rabbit anti-microtubule-associated protein 2 (MAP2; Ab32454, diluted 1:200 in PBST, Abcam, Cambridge, UK), chicken anti-glial fibrillary acidic protein (GFAP; AB5541, diluted 1:200 in PBST, Millipore, Billerica, MA), or goat anti-Iba1 (NB100-1028, diluted 1:500 in PBST, Novus, Littleton, CO) under the same conditions. This was followed by an incubation at room temperature for 1 h with Alexa 594-conjugated donkey anti-rabbit (1:1,000) or Alexa 488-conjugated donkey anti-goat (Invitrogen, Carlsbad, CA) secondary antibodies, as appropriate. Of the numerous neuronal marker antibodies tested, only the anti-MAP2 antibody was suitable for immunofluorescence applications in our human tissue. Because the antibodies against MAP2 and PRR are both raised in rabbits, to best colocalize PRR with the neuronal marker (MAP2), we separately labeled two consecutive sections 5 μm apart for PRR and MAP2 using Alexa 594-conjugated donkey anti-rabbit secondary antibodies. Colocalization of GFAP and Iba1 with PRR was assessed by performing immunolabeling in the same section. Images were captured using a Leica confocal fluorescence microscope (Leica SP8). Specificity was confirmed by incubating control sections with preimmune serum or processing without inclusion of primary antibodies.

Immunohistochemical labeling to evaluate PRR expression levels.

SFO tissues were fixed, paraffin embedded, sectioned, and rehydrated followed by antigen retrieval using Na-citrate buffer as described above. Slides were then washed with 1× PBS (Fisher Scientific, Fair Lawn, NJ) for 5 min, placed in 3% H₂O₂ for 5 min, and washed in 1× PBS for 5 min. Blockade of nonspecific binding and permeabilization were performed simultaneously by incubating with 10% goat serum in 1× PBS containing 0.3% Trixton X-100 for 1 h. Sections were incubated for 24 h at 4°C with the same rabbit anti-PRR antibody (1:200 dilution in PBST containing 10% goat serum) as described above for immunofluorescence experiments. After being washed with 1× PBS for 5 min, sections were incubated with diluted, biotinylated rabbit IgG secondary antibody from the Vectastain ABC HRP Peroxidase Kit (PK-4001, Vector Laboratories, Burlingame, CA) for 30 min followed by a 5-min wash in PBS. All slides were incubated at the same time for 2 min in a working solution of 3,3-diaminobenzidine (DAB) Peroxidase Substrate (SK-4100, Vector Laboratories) containing 250 ml of ddH₂O, 4.2 ml of buffer stock solution, 4 ml of H₂O₂ solution, and 5 ml of DAB stock solution. Slides were then rinsed with tap water for 5 min to stop the reaction, after which they were mounted with a xylene-based hard-set mounting medium (Cytoseal XYL, Richard-Allen Scientific, Kalamazoo, MI). All immunoreactions with DAB substrate, including no-primary antibody and preimmune serum negative controls, were performed at the same time in the same DAB substrate developing solution. Images were captured using a Keyence Microscope (model Bz-X710, Elmwood Park, NJ) under bright field. At least five to six randomly selected images were captured from each slide.

The relative intensity of the entire image was analyzed using ImageJ software (version 2.0.0-rc-49/1.51a) and presented as mean intensity in arbitrary units. All experiments and data analyses were performed in a blinded fashion.

Statistical analysis.

The significance of differences in SBP and diastolic BP (DBP), age, body mass index, and PRR immunoreactivity between hypertensive and normotensive subjects was assessed with an unpaired Student’s t-test using Graphpad Prism 7 software. Pearson correlation and least-squares linear regression were used to determine correlation coefficients between variables. Ordinary one-way ANOVA with Bonferroni post hoc analysis was used to test for differences among normotensive and hypertensive subjects treated with a RAS blocker (RASB) or other medications. The multiple linear regressions were performed using SAS 9.4. All statistical tests were two-tailed, and α was set at 0.05. Data are reported as means ± SE.

RESULTS

Study subject characteristics.

Details of all study subject characteristics are shown in Table 1. Normotensive (n = 7) and hypertensive (n = 12) subjects were similar in terms of age (P = 0.113) and body mass index (P = 0.503), although there was a tendency toward higher body weight in the hypertensive group (P = 0.272), possibly because of the greater number of men in this latter group. All hypertensive subjects were clinically diagnosed with hypertension and had a therapeutic history of zero to two antihypertensive drugs, whereas no diagnosis of hypertension was recorded in the patient histories of normotensive subjects.

Table 1.

Characteristics of study subjects

	Normotensive	Hypertensive
Number of subjects/group	7	12
Demographics
Sex, men/women	3/4	9/3
Race, Caucasian/African-American/other	3/4/0	6/5/1
Age, yr	55 ± 4	66 ± 4
Weight, kg	78 ± 7	91 ± 8
Height, cm	170 ± 3	175 ± 3
Body mass index, kg/m2	27.3 ± 2.8	29.7 ± 2.3
Last office measurements
Systolic blood pressure, mmHg	118 ± 8	143 ± 5*
Diastolic blood pressure, mmHg	74 ± 6	80 ± 4
Heart rate, beats/min	99 ± 12	101 ± 11
Antihypertensive medications	0	0–2
Angiotensin-converting enzyme inhibitor, %	0	25
Angiotensin receptor blocker, %	0	8
Diuretic, %	0	16
β-Blocker, %	0	25
Direct vasodilator, %	0	33
No medication listed, %	0	33
Combination therapy, %	0	33

Values are means ± SE where appropriate.

^*P = 0.01 vs. normotensive patients.

Localization of PRR in neurons of the human SFO.

The SFO is one of the circumventricular organs that interacts with circulating hormones or factors and sends projections to hypothalamus and brain stem regions to regulate autonomic function. Using an anti-PRR antibody and antibodies specific for neuron, astrocyte, or microglial markers, we performed immunofluorescence labeling of the SFO. Within this region, we found that PRR was expressed in most neurons and microglia, as evidenced by its colocalization with the neuronal marker MAP2 and the microglial marker Iba1, respectively. In contrast, no colocalization of PRR with the astrocyte marker GFAP was observed (Fig. 1), suggesting that PRR is not expressed in astrocytes. A very small number of neurons and microglia were PRR negative, indicating that these cells might be a different type of neurons or microglia in a different state.

Fig. 1.

Expression of the (pro)renin receptor (PRR) in subfornical organ (SFO) neurons of the human brain. A and B: SFO tissues were double immunolabeled for PRR (red) and neuronal marker microtubule-associated protein 2 (MAP2; green), astrocyte marker glial fibrillary acidic protein (GFAP; green), or microglial marker Iba1 (green). In B, white arrows indicate colocalization of PRR with MAP2 and Iba1. C: control sections without primary antibody and Alexa 488 (green)- or Alexa 594 (red)-labeled secondary antibody.

Elevated BP in hypertensive subjects.

As shown in Fig. 2, SBP was significantly higher in hypertensive compared with normotensive subjects (P = 0.01) regardless of antihypertensive therapy in the former group, whereas DBP (P = 0.355) and heart rate (P = 0.406) were not different between groups. There was no significant correlation between SBP and age (r = 0.245, P = 0.312) or body mass index (r = 0.028, P = 0.910), as analyzed by Pearson correlation coefficient. Least-squares linear regression analyses revealed that neither age nor body mass index was significantly correlated with SBP in our study subjects (Fig. 2, E and F), although we observed a tendency of older subjects to have higher SBP.

Fig. 2.

Elevated systolic blood pressure (SBP) in hypertensive subjects that had undergone antihypertensive drug treatment. Data from normotensive (NTN) subjects are in blue, and those from hypertensive (HTN) patients are in red. A: SBP in NTN and HTN groups. B: diastolic blood pressure (DBP) in NTN and HTN groups. C: age of study subjects. D: body mass index (BMI). E: correlation of age with SBP. F: correlation of BMI with SBP.

Increased PRR immunoreactivity in the SFO of hypertensive subjects.

Some human brain tissues exhibit strong autofluorescence owing to lipofuscin accumulation (19), as we also observed in our samples. We thus used immunochemistry (with DAB as substrate) to semiquantitatively determine PRR expression levels, presented as the relative intensity in arbitrary units (AU). Figure 3 shows representative images of immunolabeling of the SFO in normotensive (Fig. 3B) and hypertensive (Fig. 3C) subjects. The SFO from hypertensive subjects exhibited a significantly greater intensity of PRR immunoreactivity (145.3 ± 1.4 AU) than that of normotensive subjects (136.5 ± 1.2 AU, P = 0.0005), as shown in Fig. 3D. PRR immunoreactivity (Fig. 3E) remained significantly higher in hypertensive subjects after we subdivided our subjects into groups that received RASB drugs (145.1 ± 3.0 AU, P = 0.008) or other antihypertensive agents (144.6 ± 1.3 AU, P = 0.001) compared with normotensive subjects. Interestingly, SBP remained significantly higher in hypertensive subjects (146.7 ± 5.7, P = 0.011) that received non-RASB antihypertensive agents compared with normotensive subjects, whereas SBP in our limited number of hypertensive subjects that received at least one RASB as an antihypertensive agent (134.6 ± 4.7) was not significantly different (P = 0.354) from that in normotensive subjects (121.1 ± 8.2), despite an upward trend.

Fig. 3.

Increased (pro)renin receptor (PRR) expression levels in the subfornical organ (SFO) of the hypertensive human brain. A: representative image of SFO tissue immunolabeled with rabbit preimmune serum. B: representative image of a normotensive (NTN) human SFO immunolabeled for PRR. C: representative image of a hypertensive (HTN) SFO immunolabeled for PRR. D: relative intensity of PRR immunoreactivity in NTN and HTN groups. E: relative intensity of PRR immunoreactivity in NTN and HTN subjects, subdivided into groups that received renin-angiotensin system blockers (RASBs) or other antihypertensive drugs (HTN Others). F: SBP of NTN and HTN subjects, subdivided into groups that received RASBs or HTN Others. AU, arbitrary units.

SFO PRR immunoreactivity is correlated with SBP in humans.

To determine whether PRR levels in the SFO are significantly correlated with BP or other parameters, we performed Pearson correlation coefficient and least-squares linear regression tests on our study subjects. As shown in Fig. 4, PRR immunoreactivity in the SFO was significantly correlated with SBP (r = 0.559, P = 0.013) but not with DBP (r = 0.233, P = 0.337), age (r = 0.132, P = 0.589), or body mass incex (r = 0.181, P = 0.457). Least-squares linear regression analyses indicated that PRR immunoreactivity in the SFO was significantly correlated with SBP, suggesting the potential importance of SFO PRR levels in human hypertension development. Distinct sex difference in the incidence and severity of human hypertension have been well established (68a, 70), and age and body mass index are factors known to affect BP. To determine whether these variables affect the relationship between PRR and SBP, we performed a multiple regression between PRR and SBP controlling for age, body mass index, and sex. As shown in Table 2, PRR levels remained positively correlated with SBP (P = 0.032) after controlling for these parameters, none of which significantly contributed to PRR levels in our subjects (Table 3).

Fig. 4.

(Pro)renin receptor (PRR) immunoreactivity is positively correlated with systolic blood pressure (SBP). A−D: correlation of the relative intensity of PRR immunoreactivity with SBP (A), diastolic blood pressure (DBP; B), age (C), and body mass index (BMI; D).

Table 2.

Multiple regressions between the (pro)renin receptor and systolic blood pressure controlling for age, body mass index, and sex

Variable	Degrees of Freedom	Parameter Estimate	SE	t Value	Pr > |t|
Intercept	1	121.68391	10.07623	12.08	<0.0001
Systolic blood pressure	1	0.14464	0.06067	2.38	0.0318*
Age	1	−0.02282	0.10260	−0.22	0.8272
Body mass index	1	0.00233	0.16813	0.01	0.9891
Sex	1	3.67177	2.74916	1.34	0.2030

Parameter estimates are shown (n = 19).

Table 3.

Multiple regressions between the (pro)renin receptor and heart rate controlling for age, body mass index, and sex

Variable	Degrees of Freedom	Parameter Estimate	SE	t Value	Pr > |t|
Intercept	1	131.87894	9.01423	14.63	<0.0001
Heart rate	1	0.06144	0.03908	1.57	0.1382
Age	1	0.04434	0.11070	0.40	0.6948
Body mass index	1	−0.04678	0.18663	−0.25	0.8057
Sex	1	5.88905	3.13371	1.88	0.0812

Parameter estimates are shown (n = 19).

DISCUSSION

Since the first description of the PRR by Nguyen et al. (45, 46), accumulating evidence has supported its functional importance in animal models of hypertension, hypertension-related chronic kidney diseases, and diabetic end-organ complications (5, 6, 14, 43, 68, 69). PRR was recently discovered to be the major contributor to endogenous brain ANG II formation and to play an important role in the development of neurogenic hypertension in animal models (30). However, information on the role of PRR in humans is limited (25, 50), and no data on expression levels of PRR in the hypertensive human brain are available, likely reflecting difficulties in obtaining human brain tissue from BP regulatory regions. In the present study, we demonstrate, for the first time, that in the human SFO, PRR is expressed in neurons and microglia but not in astrocytes. More importantly, PRR immunoreactivity was significantly higher in hypertensive human subjects, regardless of antihypertensive medication status or type of drug taken. In addition, there was a significant correlation between SFO PRR levels and BP, indicating the potential clinical significance of SFO PRR in human hypertension.

In the brains of both normotensive and hypertensive adult rodents, PRR is expressed in most neurons but not in astrocytes (32, 33, 57, 69). By comparison, studies using primary cultured cells and cell lines have reported that PRR is expressed in neurons and microglia cells but also to a lesser extent in astrocytes (52, 57, 58). PRR is expressed in the pituitary and hypothalamus of the adult human brain and is colocalized with vasopressin and oxytocin neurons in the supraoptic nucleus (65). However, whether PRR is expressed in microglial cells in humans has not been previously investigated. In the present study, we found that in the SFO, PRR is expressed in neurons and microglia but not in astrocytes. These data are in agreement with previous animal studies and establish a similar cellular distribution of PRR in human brains; they also provide new information on expression of SFO PRR in microglia in humans. The expression level of PRR is regulated by a variety of hypertensive stimuli, both in cultured cells in vitro and in vivo rodent models of hypertension and diabetes, suggesting that expression of PRR is not dependent on BP per se since there are no pressure effects in an in vitro setting. The SFO tissues used in our study were collected postmortem, confirming this concept, at least in the timeframe of our study.

Localization of PRR to neurons and microglia in the SFO establishes a foundation for the formation of ANG II locally in this brain region. Because of the permeability of its BBB, the SFO is in a unique position to sense circulating factors (e.g., prorenin) as well as to interact with prorenin generated locally in the brain. A number of elegant studies have provided evidence for the endogenous expression of prorenin in the brain (29, 32, 60). There is also a significant amount of prorenin in the circulation, although it is predominantly inactive in the absence of association with PRR (6). In fact, the level of prorenin is ~10-fold higher than that of active renin in healthy individuals (8, 47) but can be as much as 100-fold higher when active renin is suppressed (7). Evidence for the local production of ANG II in the brain, including the SFO, has been well established in animal models (34, 55, 61). We propose that PRR within the SFO acts in a regionally restricted manner to activate prorenin either from the circulation or produced locally and thereby contributes to ANG II formation in the SFO. Identification of the key source(s) of the prorenin that interacts with PRR in the SFO awaits future investigations.

An important finding from this study is that PRR immunoreactivity is significantly higher in the SFO of hypertensive subjects compared with normotensive subjects, indicating a potentially important association of SFO PRR with hypertension in humans. These findings are in accord with previous studies have have reported augmented PRR expression in the SFO in both ANG II-induced and DOCA-salt-induced hypertension in mice (32, 33). SFO-targeted PRR knockdown attenuates ANG II-dependent hypertension in mice (32), further indicating the potential clinical significance of blocking SFO PRR in human hypertension associated with elevated sympathetic activity. These PRR knockdown/deletion studies in animal models of hypertension further suggest that PRR is the cause rather than the consequence of hypertension. ANG II, high salt, glucose, and DOCA-salt have been shown to stimulate PRR expression in vitro and in vivo (26, 31, 33, 38, 64, 67). We speculate that, in hypertensive humans, high-salt and/or high-fat diets may be the primary drivers of PRR elevations. It is also possible that the elevation of aldosterone due to obesity and/or elevation of ANG II owing to chronic kidney diseases could amplify the PRR upregulation and subsequent elevation of BP.

In addition to its involvement in ANG II-dependent signaling, PRR is an accessory protein of vacuolar H⁺-ATPase (V-ATPase) (36), which is critical for the acidification of intracellular compartments and cellular pH homeostasis in many cell types (12, 27, 28). In the central nervous system, V-ATPase has important roles in regulating the exocytosis of neurotransmitters by establishing an electrochemical proton gradient (11, 41). To our knowledge, the role of V-ATPase in BP regulation and hypertension in the central nervous system remains undefined. However, by modulating V-ATPase activity in the SFO, PRR could potentially affect neurotransmitter release and contribute to the regulation of BP.

The hypertensive subjects in our study used antihypertensive agents either singly or in combination (Table 1); however, average SBP remained elevated in these subjects. When SBP was grouped according to the antihypertensive treatment status of hypertensive subjects in our small sample size, we found that subjects that received RASBs tended to have lower SBP compared with subjects that received other antihypertensive drugs. The beneficial effects of RASBs on BP control has been previously reported in Caucasians (4). Nevertheless, Pearson correlation and least-squares linear regression analyses revealed that PRR levels in the SFO were significantly correlated with SBP such that higher levels of PRR were associated with higher SBP. Importantly, we found that PRR levels remained elevated regardless of antihypertensive therapy with or without RASBs. Since the SFO lies outside the BBB, oral antihypertensive agents theoretically should be able to act upon it. The RASBs used in our subjects, including angiotensin-converting enzyme 1 inhibitors and angiotensin receptor blockers, target the RAS pathway, but our data suggest that they do not modify SFO PRR. Moreover, the ineffectiveness of RASBs in reducing SFO PRR expression provides evidence that SFO PRR may act independently of the RAS pathway or may function upstream of it (69).

Perspectives

The critical importance of the brain RAS in regulating sympathetic activity and BP is an emerging field of research. Evidence for the presence of the PRR in the human brain, both in neurons and microglia, and its positive correlation with SBP provides a foundation for future functional studies on brain PRR in human hypertension. Expression of the PRR in neurons and microglia suggests a possible role for the PRR in regulating not only neuronal activity but also inflammation and hypertension, a new research avenue in humans. PRR antagonists have been developed and shown to be effective in animal models of hypertension (35, 56, 67). Elevation of PRR in the brains of hypertensive subjects suggests the potential benefit of PRR antagonism in blocking brain ANG II formation or ANG II-independent pathways as an alternative approach for treating essential hypertension. Further investigations of the role of PRR in human hypertension are warranted.

GRANTS

This work was supported, in part, by National Heart, Lung, and Blood Institute Grant R01-HL-122770 and American Heart Association National Center Grant 17IRG33370128 (to Y. Feng).

DISCLAIMERS

The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the granting agencies.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.G.C., J.T.S., Z.X., and Y.F. conceived and designed research; S.G.C., D.P.T., R.Y., C.-Y.F., J.T.S., and Y.F. performed experiments; S.G.C., R.Y., J.T.S., W.Y., and Y.F. analyzed data; S.G.C., D.P.T., R.Y., C.J.W., C.-Y.F., J.T.S., W.Y., Z.X., and Y.F. interpreted results of experiments; S.G.C., R.Y., C.J.W., W.Y., and Y.F. prepared figures; S.G.C. and Y.F. drafted manuscript; S.G.C., D.P.T., R.Y., C.J.W., C.-Y.F., J.T.S., W.Y., Z.X., and Y.F. edited and revised manuscript; S.G.C., D.P.T., R.Y., C.J.W., C.-Y.F., J.T.S., W.Y., Z.X., and Y.F. approved final version of manuscript.