Deoxycorticosterone Acetate Hypertension Research Articles

O98 NEW APPROACHES TARGETING THE RENIN-ANGIOTENSIN SYSTEM: BRAIN ACE2 ACTIVATION FOLLOWING BRAIN AMINOPEPTIDASE A BLOCKADE BY FIRIBASTAT IN SALT-DEPENDENT HYPERTENSION

Background and Objective: In the brain, aminopeptidase A (APA), a membrane-bound zinc metalloprotease, generates angiotensin III from angiotensin II. Brain angiotensin III exerts a tonic stimulatory effect on the control of blood pressure (BP) in hypertensive rats and increases vasopressin release. Blocking brain angiotensin III formation by the APA inhibitor prodrug RB150/firibastat normalizes arterial BP in hypertensive deoxycorticosterone acetate (DOCA)-salt rats. Ang II and Ang III have similar affinities for AT1 receptors and blocking brain AngIII formation by RB150 decreases arterial BP without inducing Ang II accumulation. Methods: We therefore hypothesized that another metabolic pathway of brain angiotensin II, such as the conversion of angiotensin II into angiotensin 1-7 (Ang 1-7) by angiotensin-converting enzyme 2 (ACE2) might be activated following brain APA inhibition. To answer this question, we use surgical animal models that could allow us to monitor awake blood pressure responses to intracerebroventricular (icv) injections and measure enzymatic activity. Results: We found that the intracerebroventricular administration of RB150/firibastat in conscious DOCA-salt rats both inhibited brain APA activity and induced an increase in brain ACE2 activity. Then, we showed that the decreases in BP and vasopressin release resulting from brain APA inhibition with RB150/firibastat were reduced if ACE2 was concomitantly inhibited by MLN4760, a potent ACE2 inhibitor, or if the Mas receptor (MasR) was blocked by A779, a MasR antagonist. Conclusions: Our findings suggest that in the brain, the increase in ACE2 activity resulting from APA inhibition by RB150/firibastat treatment, subsequently increasing Ang 1-7 and activating the MasR while blocking angiotensin III formation, contributes to the antihypertensive effect and the decrease in vasopressin release induced by RB150/firibastat. RB150/firibastat treatment constitutes an interesting therapeutic approach to improve BP control in hypertensive patients by inducing in the brain renin-angiotensin system, hyperactivity of the beneficial ACE2/Ang 1-7/MasR axis while decreasing that of the deleterious APA/Ang II/Ang III/ATI receptor axis.

Bioactive Peptides from Ruditapes philippinarum Attenuate Hypertension and Cardiorenal Damage in Deoxycorticosterone Acetate-Salt Hypertensive Rats.

Hypertension is a common disease that affects human health and can lead to damage to the heart, kidneys, and other important organs. In this study, we investigated the regulatory effects of bioactive peptides derived from Ruditapes philippinarum (RPP) on hypertension and organ protection in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. We found that RPPs exhibited significant blood pressure-lowering properties. Furthermore, the results showed that RPPs positively influenced vascular remodeling and effectively maintained a balanced water-sodium equilibrium. Meanwhile, RPPs demonstrated anti-inflammatory potential by reducing the serum levels of inflammatory cytokines (TNF-α, IL-2, and IL-6). Moreover, we observed the strong antioxidant activity of RPPs, which played a critical role in reducing oxidative stress and alleviating hypertension-induced damage to the aorta, heart, and kidneys. Additionally, our study explored the regulatory effects of RPPs on the gut microbiota, suggesting a possible correlation between their antihypertensive effects and the modulation of gut microbiota. Our previous studies have demonstrated that RPPs can significantly reduce blood pressure in SHR rats. This suggests that RPPs can significantly improve both essential hypertension and DOAC-salt-induced secondary hypertension and can ameliorate cardiorenal damage caused by hypertension. These findings further support the possibility of RPPs as an active ingredient in functional anti-hypertensive foods.

EDITORIAL: Mitochondrial Function and Blood Pressure Regulation: From Bioenergetics to Pathophysiology

The mitochondrion is the powerhouse of all living cells as it provides the energy needed to maintain obligatory regulatory functions.1 The generation of adenosine triphosphate (ATP) via oxidative phosphorylation underlies the principal role of the mitochondrion in cell survival. Aside this basic contribution to energy generation, the mitochondria has been established to regulate cell death (apoptosis), redox and ion signaling.2 The crosstalk between redox signaling and a myriad of pathological disorders created a nexus between the mitochondrion and the cardiorenal system.3,4 Similarly, the high distribution of mitochondria in organs of the cardiorenal system, meant that these organs such as the kidney, are subject to the effect of mitochondria-induced alterations in redox signaling.5 For instance, mitochondrial dysfunction has been linked to the pathophysiology of kidney disorders.6 Considering the intricate link between the kidneys and blood pressure regulation, mitochondrial dysfunction was suggested to contribute significantly to distortions in renal control of blood pressure. Recently, it was reported that the tricarboxylic acid (TCA) cycle plays a role in the etiology of genetic hypertension.7 This novel iscovery linked the activity of the TCA cycle enzyme, fumarase to a reduction in nitric oxide production and an upregulation in redox signaling in the renal medulla of salt-sensitive rats.7,8 In these animals, an innate mutation in the fumarase enzyme, reduced its activity and increased cellular levels of its substrate, fumarate. Hence, the role of these TCA cycle intermediaries was shifted from being ‘mere’ participants in the generation of energy to endogenous ligands with biochemical targets that alter renal function and by extension, blood pressure. Furthermore, fumarate was shown to reduce blood pressure and modulate the expression of genes that ameliorated hypertension induced renal damage in deoxycorticosterone acetate (DOCA) hypertension, a non-genetic form of hypertension.9 Subsequently, succinate, the upstream product of fumarate was reported to directly stimulate GPR91 receptors to increase blood pressure.10 These actions of fumarate and its intermediaries, exceed the renal system as reports have shown a cardioprotective role via upregulation of nuclear erythroid factor-2 (Nrf2).11 Fumarate is now known to regulate the expression of genes such as hypoxia inducible factor (HIF-1), transforming growth factor (TGF-β), kidney injury molecule (KIM-1) amongst others. What is evident from the foregoing is that the mitochondrion is no longer just an idle energy-generating center. It is now listed as a probable etiology in hypertension, and this has opened new vistas of possibilities as it relates to the pathophysiology of hypertension.8 Is it possible that these intermediaries are involved in the physiological control of blood pressure? Could they also be exerting direct vasoactive effects? Is it likely that they may be modulating the expression of genes that underlie vascular/organ remodeling? And finally, is it possible that mitochondrial dysfunction could partly explain the etiology of idiopathic hypertension? As, far reaching as these insights may be, it is not completely out of place to be optimistic as the foray into these previously uncharted areas of mitochondrial metabolism progress. What is very clear is that there is now a paradigm shift in the function of the mitochondria in blood pressure regulation from that of a bioenergetic center to pathophysiological axis which contributes significantly to the etiology of hypertension.

Medullary overexpression of sphingosine‐1‐phosphate receptor 1 transgene attenuates sodium retention and hypertension in deoxycorticosterone acetate (DOCA) salt‐treated mice

Our previous studies have showed that sphingosine‐1‐phosphate (S1P) agonist stimulates urinary sodium excretion and the S1P type 1 receptor (S1PR1) knockout in the collecting duct led to increased sodium retention and enhanced salt‐sensitive hypertension under the treatment of deoxycorticosterone (DOCA) and high salt. We hypothesized that the increase in medullary S1PR1 would attenuate sodium retention by promoting sodium excretion in such model. Male C57BL/6J mice around 25g bodyweight received intramedullary transfection of S1PR1 plasmids to achieve renal medullary S1PR1 overexpression (OE). The mice were then implanted with the silastic DOCA sheet subcuntaneously and 1% NaCl drinking water to induce salt‐sensitive hypertension (SS‐HTN). Thereafter, the mice underwent either acute or chronic assessments on blood pressure and urinary sodium excretion. Under DOCA‐salt challenge, mice with S1PR1‐OE showed increased urine volume (UV) and urinary sodium excretion (UNAV) in responses to the acute sodium loading (IV of normal saline at 5% body weight in 30 min); the UV and UNAV were 12.51±2.31 vs. 28.50±4.23mL/min/gKW and 0.93±0.27 vs. 4.72±1.12mmole/min/gKW (p<0.05) in control and S1PR1‐OE mice, respectively. The mice with S1PR1‐OE also showed enhanced pressure natriuretic responses (UV, 18.73±7.57 vs. 40.61±6.43mL/min/gKW, p<0.05; UNAV, 3.58±1.77 vs. 9.52±1.38nmole/min/gKW, p<0.05). In the long‐term DOCA‐salt treatment, S1PR1‐OE mice showed less daily sodium balance with the greater sodium excretion (2.72±0.56 vs. 5.60±0.72mmole/day, p<0.05) and ameliorated sodium retention (3.05±0.39 vs. 1.65±0.39mmole/72hr, p<0.05), which was accompanied with the attenuation of SS‐HTN (SBP 134.2±6.79 vs. 109.8±3.54mmHg, p<0.05). The level of alpha subunit of epithelial sodium channel (αENaC) was not changed. However, the beta subunit (βENaC) was significantly decreased (55%, p<0.01). Although ENaCγ was significantly increased (350%) in the S1PR1‐OE group, immunostaining showed that the DOCA‐stimulated translocation of γENaC onto the apical membrane was inhibited. These results suggested that the overexpression of S1PR1 in the renal medulla attenuates sodium retention by the inhibition of ENaC function in the DOCA‐induced SS‐HTN.

Changes in Gut Microbiota Induced by Doxycycline Influence in Vascular Function and Development of Hypertension in DOCA-Salt Rats.

Previous experiments in animals and humans show that shifts in microbiota and its metabolites are linked to hypertension. The present study investigates whether doxycycline (DOX, a broad-spectrum tetracycline antibiotic) improves dysbiosis, prevent cardiovascular pathology and attenuate hypertension in deoxycorticosterone acetate (DOCA)-salt rats, a renin-independent model of hypertension. Male Wistar rats were randomly assigned to three groups: control, DOCA-salt hypertensive rats, DOCA-salt treated with DOX for 4 weeks. DOX decreased systolic blood pressure, improving endothelial dysfunction and reducing aortic oxidative stress and inflammation. DOX decreased lactate-producing bacterial population and plasma lactate levels, improved gut barrier integrity, normalized endotoxemia, plasma noradrenaline levels and restored the Treg content in aorta. These data demonstrate that DOX through direct effects on gut microbiota and its non-microbial effects (anti-inflammatory and immunomodulatory) reduces endothelial dysfunction and the increase in blood pressure in this low-renin form of hypertension.

LIT01-196, a Metabolically Stable Apelin-17 Analog, Normalizes Blood Pressure in Hypertensive DOCA-Salt Rats via a NO Synthase-dependent Mechanism.

Apelin is a neuro-vasoactive peptide that plays a major role in the control of cardiovascular functions and water balance, but has an in-vivo half-life in the minute range, limiting its therapeutic use. We previously developed LIT01-196, a systemically active metabolically stable apelin-17 analog, produced by chemical addition of a fluorocarbon chain to the N-terminal part of apelin-17. LIT01-196 behaves as a potent full agonist for the apelin receptor and has an in vivo half-life in the bloodstream of 28 min after intravenous (i.v.) and 156 min after subcutaneous (s.c.) administrations in conscious normotensive rats. We aimed to investigate the effects of LIT01-196 following systemic administrations on arterial blood pressure, heart rate, fluid balance and electrolytes in conscious normotensive and hypertensive deoxycorticosterone acetate (DOCA)-salt rats. Acute i.v. LIT01-196 administration, in increasing doses, dose-dependently decreases arterial blood pressure with ED50 values of 9.8 and 3.1 nmol/kg in normotensive and hypertensive rats, respectively. This effect occurs for both via a nitric oxide-dependent mechanism. Moreover, acute s.c. LIT01-196 administration (90 nmol/kg) normalizes arterial blood pressure in conscious hypertensive DOCA-salt rats for more than 7 h. The LIT01-196-induced blood pressure decrease remains unchanged after 4 consecutive daily s.c. administrations of 90 nmol/kg, and does not induce any alteration of plasma sodium and potassium levels and kidney function as shown by the lack of change in plasma creatinine and urea nitrogen levels. Activating the apelin receptor with LIT01-196 may constitute a novel approach for the treatment of hypertension.

Effects of firibastat in combination with enalapril and hydrochlorothiazide on blood pressure and vasopressin release in hypertensive DOCA-salt rats

In the brain, aminopeptidase A (APA) generates angiotensin III, one of the effector peptides of the brain renin-angiotensin system (RAS), exerting tonic stimulatory control over blood pressure (BP) in hypertensive rats. Oral administration of firibastat, an APA inhibitor prodrug, in hypertensive rats, inhibits brain APA activity, blocks brain angiotensin III formation and decreases BP. In this study, we evaluated the efficacy of firibastat in combination with enalapril, an angiotensin I-converting enzyme inhibitor, and hydrochlorothiazide (HCTZ), in conscious hypertensive deoxycorticosterone acetate (DOCA)-salt rats, which display high plasma arginine-vasopressin levels, low circulating renin levels and resistance to treatment by systemic RAS blockers. We determined mean arterial BP, heart rate, plasma arginine-vasopressin levels and renin activity in DOCA-salt rats orally treated with firibastat, enalapril or HCTZ administered alone or in combination. Acute oral firibastat administration (30 mg/kg) induced a significant decrease in BP, whereas enalapril (10 mg/kg) or HCTZ (10 mg/kg) administered alone induced no significant change in BP in conscious DOCA-salt rats. The BP decrease induced by acute and nine-day chronic tritherapy [Firibastat+Enalapril+HCTZ] was significantly greater than that observed after bitherapy [Enalapril+HCTZ]. Interestingly, the chronic administration of a combination of firibastat with [Enalapril+HCTZ] reduced plasma arginine-vasopressin levels by 62% relative to those measured in DOCA-salt rats receiving bitherapy. Our data show that tritherapy with firibastat, enalapril and HCTZ improves BP control and arginine-vasopressin release in an experimental salt-dependent model of hypertension, paving the way for the development of new treatments for patients with currently difficult-to-treat or resistant hypertension.

Brain ACE2 activation following brain aminopeptidase A blockade by firibastat in salt-dependent hypertension.

In the brain, aminopeptidase A (APA), a membrane-bound zinc metalloprotease, generates angiotensin III from angiotensin II. Brain angiotensin III exerts a tonic stimulatory effect on the control of blood pressure (BP) in hypertensive rats and increases vasopressin release. Blocking brain angiotensin III formation by the APA inhibitor prodrug RB150/firibastat normalizes arterial BP in hypertensive deoxycorticosterone acetate (DOCA)-salt rats without inducing angiotensin II accumulation. We therefore hypothesized that another metabolic pathway of brain angiotensin II, such as the conversion of angiotensin II into angiotensin 1-7 (Ang 1-7) by angiotensin-converting enzyme 2 (ACE2) might be activated following brain APA inhibition. We found that the intracerebroventricular (icv) administration of RB150/firibastat in conscious DOCA-salt rats both inhibited brain APA activity and induced an increase in brain ACE2 activity. Then, we showed that the decreases in BP and vasopressin release resulting from brain APA inhibition with RB150/firibastat were reduced if ACE2 was concomitantly inhibited by MLN4760, a potent ACE2 inhibitor, or if the Mas receptor (MasR) was blocked by A779, a MasR antagonist. Our findings suggest that in the brain, the increase in ACE2 activity resulting from APA inhibition by RB150/firibastat treatment, subsequently increasing Ang 1-7 and activating the MasR while blocking angiotensin III formation, contributes to the antihypertensive effect and the decrease in vasopressin release induced by RB150/firibastat. RB150/firibastat treatment constitutes an interesting therapeutic approach to improve BP control in hypertensive patients by inducing in the brain renin-angiotensin system, hyperactivity of the beneficial ACE2/Ang 1-7/MasR axis while decreasing that of the deleterious APA/Ang II/Ang III/ATI receptor axis.

Sodium butyrate ameliorates deoxycorticosterone acetate/salt-induced hypertension and renal damage by inhibiting the MR/SGK1 pathway.

Our recent work demonstrates that infusion of sodium butyrate (NaBu) into the renal medulla blunts angiotensin II-induced hypertension and improves renal injury. The present study aimed to test whether oral administration of NaBu attenuates salt-sensitive hypertension in deoxycorticosterone acetate (DOCA)/salt-treated rats. Uninephrectomized male Sprague-Dawley (SD) rats were treated with DOCA pellets (150 mg/rat) plus 1% NaCl drinking water for 2 weeks. Animals received oral administration of NaBu (1 g/kg) or vehicle once per day. Our results showed that NaBu administration significantly attenuated DOCA/salt-increased mean arterial pressure from 156 ± 4 mmHg to 136 ± 1 mmHg. DOCA/salt treatment markedly enhanced renal damage as indicated by an increased ratio of kidney weight/body weight, elevated urinary albumin, extensive fibrosis, and inflammation, whereas kidneys from NaBu-treated rats exhibited a significant reduction in these renal damage responses. Compared to the DOCA/salt group, the DOCA/salt-NaBu group had ~30% less salt water intake and decreased Na+ and Cl- excretion in urine but no alteration in 24-h urine excretion. Mechanistically, NaBu inhibited the protein levels of several sodium transporters stimulated by DOCA/salt in vivo, such as βENaC, γENaC, NCC, and NKCC-2. Further examination showed that NaBu downregulated the expression of mineralocorticoid receptor (MR) and serum and glucocorticoid-dependent protein kinase 1 (SGK1) in DOCA/salt-treated rats or aldosterone-treated human renal tubular duct epithelial cells. These results provide evidence that NaBu may attenuate DOCA/salt-induced hypertension and renal damage by inhibiting the MR/SGK1 pathway.

Probiotic Bifidobacterium breve prevents DOCA-salt hypertension.

Many probiotics that affect gut microbial ecology have been shown to produce beneficial effects on renin-angiotensin-dependent rodent models and human hypertension. We hypothesized that Bifidobacterium breve CECT7263 (BFM) would attenuate hypertension in deoxycorticosterone acetate (DOCA)-salt rats, a renin-independent model of hypertension. Rats were randomly divided into five groups: control, DOCA-salt, treated DOCA-salt-BFM, treated DOCA-salt-butyrate, and treated DOCA-salt-acetate, for 5weeks. BFM prevented the increase in systolic blood pressure, cardiac weight, and renal damage induced by DOCA-salt. BFM increased acetate-producing bacterial population and gut acetate levels, improved colonic integrity, normalized endotoxemia, plasma trimethylamine (TMA) levels, and restored the Th17 and Treg content in mesenteric lymph nodes and aorta. Furthermore, BFM improved nitric oxide-dependent vasorelaxation induced by acetylcholine in aortic rings and reduced NADPH oxidase activity in DOCA-salt animals. These protective effects were mimicked by acetate, but not by butyrate supplementation. These data demonstrate that BFM induces changes in gut microbiota linked with attenuation of endothelial dysfunction and increase in blood pressure in this low-renin form of hypertension. These beneficial effects seem to be mediated by increased acetate and reduced TMA production by gut microbiota, thus, improving gut integrity and restoring Th17/Tregs polarization and endotoxemia.

Discovery of a Potent and Selective TRPC5 Inhibitor, Efficacious in a Focal Segmental Glomerulosclerosis Model.

The nonselective Ca2+-permeable transient receptor potential (TRP) channels play important roles in diverse cellular processes, including actin remodeling and cell migration. TRP channel subfamily C, member 5 (TRPC5) helps regulate a tight balance of cytoskeletal dynamics in podocytes and is suggested to be involved in the pathogenesis of proteinuric kidney diseases, such as focal segmental glomerulosclerosis (FSGS). As such, protection of podocytes by inhibition of TRPC5 mediated Ca2+ signaling may provide a novel therapeutic approach for the treatment of proteinuric kidney diseases. Herein, we describe the identification of a novel TRPC5 inhibitor,GFB-8438, by systematic optimization of a high-throughput screening hit, pyridazinone 1. GFB-8438 protects mouse podocytes from injury induced by protamine sulfate (PS) in vitro. It is also efficacious in a hypertensive deoxycorticosterone acetate (DOCA)-salt rat model of FSGS, significantly reducing both total protein and albumin concentrations in urine.

Selective inhibition of histone deacetylase 8 improves vascular hypertrophy, relaxation, and inflammation in angiotensin II hypertensive mice

BackgroundThe dysregulation of histone deacetylase (HDAC) protein expression or its enzyme activity is implicated in a variety of diseases. Cardiac HDAC6 and HDAC8 enzyme activity induced by deoxycorticosterone acetate (DOCA) hypertension was attenuated by sodium valproate, a pan-HDAC inhibitor. However, the HDAC6-selective inhibitor, tubastatin A, did not attenuate angiotensin II-induced hypertension. The purpose of this study was to investigate whether PCI34051, an HDAC8-selective inhibitor, can modulate angiotensin II-induced hypertension and its regulatory mechanism.MethodsAn angiotensin II-regulated mouse model was used in this study. Animals received vehicle or PCI34051 (3 mg·kg − 1·day− 1) via intraperitoneal injection. Systolic blood pressure was measured by the tail-cuff method. Blood vessel thickness was measured following hematoxylin and eosin staining, VCAM-1 immunohistochemistry was performed in the aortas, and mRNA expression of renin-angiotensin system components, inflammation markers, and NADPH oxidase (Nox) was determined by RT-PCR. The effect of PCI34051 on vasorelaxation was studied in rat aortic rings, and its effect on nitric oxide (NO) production was determined using DAF-FM DA, a fluorescent dye, in human umbilical vascular endothelial cells (HUVECs).ResultsPCI34051 administration reduced systolic blood pressure via downregulation of angiotensin II receptor type 1 (AT1) mRNA expression. PCI34051 treatment attenuated vascular hypertrophy by decreasing E2F3 and GATA6 mRNA expression. Vascular relaxation after PCI34051 treatment was more dependent on vascular endothelial cells and it was blocked by an NO synthase (NOS) inhibitor. In addition, NO production increased in HUVECs after PCI34051 treatment; this was decreased by the NOS inhibitor. The expression of inflammatory molecules and adhesion molecules VCAM-1 and ICAM-1 decreased in the aortas of angiotensin II-infused mice after PCI34051 administration. However, PCI34051 did not affect Nox or its regulatory subunits.ConclusionsPCI34051 lowered high blood pressure through modulation of arterial remodeling, vasoconstriction, and inflammation in an angiotensin II-induced hypertension model. We suggest that HDAC8 could be a potential therapeutic target for hypertension.

DOCA‐salt hypertension and the role of the OVLT‐sympathetic‐gut microbiome axis

Limited studies suggest an association of gut microbiome changes with certain forms of hypertension, but a cause/effect relationship has yet to be fully defined. The deoxycorticosterone acetate (DOCA)‐salt model of hypertension in rats is known to have a neurogenic component linked to increased sympathetic nervous system activity. Indeed, we have recently shown the hypertensive response in DOCA‐treated rats requires the organum vasculosum of the lamina terminalis (OVLT), a circumventricular organ that has direct connections to the PVN to increase sympathetic activity. Furthermore, celiac ganglionectomy also supports a role of splanchnic sympathetic innervation to the gut in the hypertensive response to DOCA. Taken together, we hypothesize an OVLT‐sympathetic‐gut (OSG) microbiome axis is involved in the development of hypertension. Having previously reported that the OVLT plays a significant role in the development of DOCA‐salt hypertension, the present study was designed to test the hypothesis that the OVLT mediates changes in the gut microbiome associated with concomitant hypertension. In uninephrectomized OVLT lesioned (OVLTx; n = 5) and SHAM (n = 4) Sprague‐Dawley rats consuming a 2% NaCl diet and 0.9% NaCl drinking solution, 24‐hour mean arterial pressure (MAP) was recorded telemetrically 5 days before and 21 days after DOCA implantation (100 mg/rat; SQ). Microbiome analyses were performed on regional gut samples (jejunum, ileum, cecum, and colon) at the end of the study and compared to normal control rats (n=8). The chronic pressor response to DOCA was attenuated in OVLTx rats such that MAP increased to 157±7 mmHg in SHAM rats by Day 21 of DOCA compared to only 127±5 mmHg in OVLTx rats. Moreover, DOCA hypertension in SHAM rats was associated with decreased levels of Lactobacillus species in the ileum. However, these changes were mitigated and offset by lesion of the OVLT and these bacterial populations in OVLT lesioned rats with attenuated hypertension more closely resembled those in normal control rats. Using Spearman correlation, increasing blood pressure on day 21 correlated with increased Clostridia levels in all tissues, as well as increasing Prevotella and decreasing Bacteroides. We conclude that DOCA‐salt hypertension is associated with specific microbiome changes in the gut, and the attenuated hypertensive effects of DOCA‐salt in OVLT lesioned rats is mediated in part through counteracting changes in these bacterial populations.Support or Funding InformationSupported by Merial Veterinary Scholars Research Grant and University of Minnesota Grant In AidThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Decrease in Tregs Exacerbates DOCA‐Salt Hypertension and Renal Injury in Female Rats

BackgroundT regulatory cells (Tregs) have previously been shown to protect against increases in blood pressure (BP) in male experimental models of hypertension, however less is known regarding the role of Tregs in BP control in females. Our lab has previously reported that there is a sex difference in the renal T cell profile in DOCA, where DOCA results in a decrease in Tregs in males while females maintain their Tregs. The current study tested the hypothesis that Treg maintenance attenuates the increase in blood pressure (BP) to DOCA‐salt in females.MethodsFemale (n=4–7) 13‐week‐old Sprague Dawley rats underwent an uninephrectomy followed by the implantation of a time release 200mg Deoxycorticosterone Acetate (DOCA) pellet and 0.9% saline to drink ad libitum. Rats were then randomized to receive a vehicle or anti‐CD25 injection (IP 250 μg/week) weekly for 3 weeks and BP was measured by telemetry. 24‐hour urinary protein excretion was measured via Bradford Assay at baseline and weekly thereafter for the duration of the study. At the end of the study, kidneys were isolated for flow cytometric analysis of T cells and a terminal plasma sample was collected to measure the kidney injury marker Tim1/Kim1.ResultsAnti‐CD25 injections in DOCA hypertensive females significantly decreased renal (4.2 ± 0.6 vs. 6 ± 0.7; p=0.03) and circulating Tregs compared to DOCA alone (3.6 ± 0.5 vs. 5.5 ± .6 p=0.05). 3 weeks of DOCA‐salt treatment significantly increased BP (101 ± 3 to 160 ± 5; p=<.0001), and decreasing Tregs exacerbated the DOCA‐salt induced increase in BP (109 ± 1 to 188 ± 4; p=0.002). We calculated area under the curve for each BP tracing, and the overall BP load in the rats treated with anti‐CD25 was significantly greater than in rats with DOCA‐salt alone (3327 ± 37 vs. 3662 ± 39; p=0.033). DOCA‐salt also increased urinary protein excretion from baseline to week 3 (3 mg/day ± 0.4 to 34 mg/day ± 9; p=0.01) and this increase was exacerbated with anti‐CD25 treatment (2 mg/day ± 0.5 to 86 mg/day ± 17; p=0.003). Tim1/Kim1 was also increased in DOCA hypertensive females compared to uninephrectomized controls, and this increase was greater in rats treated with anti‐CD25 (control: 292 ± 25 pg/ml; DOCA: 415 ± 57pg/ml; DOCA+anti‐CD25: 589 ± 105; p=0.04).DiscussionOur data suggests that the maintenance of Tregs in DOCA‐salt hypertensive females attenuates increases in BP and renal injury. Future studies will determine if this effect if mediated by the anti‐inflammatory and anti‐hypertensive effects of IL‐10.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TRPV4 activation in rat carotid artery in DOCA hypertension involves eNOS and endothelium-derived contractile factor (EDCF)

ABSTRACTAim: Role of TRPV4 channel in regulation of endothelial function in the carotid artery in deoxycorticosterone acetate (DOCA) model of hypertension in rat was studied.Methods: 8–10 weeks old albino Wistar rats divided into three groups namely Control, UNX and hypertensive animals. Vascular smooth muscle response was studied in isolated carotid artery of rat with acetylcholine, sodium nitroprusside, GSK1016790A (GSK) in presence and absence of L-NAME and indomethacin.Results: At the end of the 6th week, the mean systolic blood pressure was increased in DOCA-treated hypertensive rats (166 ± 8 mm Hg) compared to Control and UNX (125 ± 5 mm Hg). ACh (10–9 to 10–5 M) produced almost 100% relaxation in Control (Emax = 97.48 ± 1.06 %) and UNX animals (Emax = 93.16 ± 2.33 %) which was attenuated in DOCA-treated hypertensive animals (Emax = 70.85 ± 1.65 %). No significant changes seen in SNP (10–12 to 10–5 M) induced relaxation. GSK1016790A (10–12 to 10–7 M)-mediated relaxation was significantly attenuated in DOCA-treated hypertensive animals (Emax = 25.58 ± 13.60%) compared to the control (Emax = 80.59 ± 6.86%) and UNX (Emax = 87.32 ± 2.01%) animals. L-NAME (10–4 M) potently blocked GSK-induced relaxation, and a contractile response to GSK was observed in presence of L-NAME in all the three groups of animals which was sensitive to indomethacin (10–5 M).Conclusion: TRPV4 may regulate the vascular tone of rat carotid artery through an attenuated NO pathway and stimulation of the release of contractile prostanoids in the DOCA hypertensive rats.

Hypertension and chronic inhibition of endocannabinoid degradation modify the endocannabinoid system and redox balance in rat heart and plasma

The interaction between the endocannabinoid and ROS signaling systems has been demonstrated in different organs. Inhibitors of fatty acid amide hydrolase (FAAH), the key enzyme responsible for degradation of the endocannabinoid anandamide, are postulated to possess anti-hypertensive potential. Here, we compared the effects of hypertension and chronic FAAH inhibition by URB597 on the endocannabinoid system and redox balance in spontaneously hypertensive rats (SHR) and hypertensive deoxycorticosterone acetate (DOCA)-salt rats. Enhanced oxidative stress and lipid peroxidation were found in both hypertension models. Hypertension affected cardiac and plasma endocannabinoid systems in a model-dependent manner: anandamide and 2-arachidonoylglycerol levels decreased in SHR and increased in DOCA-salt. Cardiac CB1 receptor expression increased in both models while higher CB2 receptor expression was only in DOCA-salt. URB597 increased endocannabinoid levels in both models but produced the partial reduction of oxidative stress in DOCA-salt but not in SHR. Notably, URB597 decreased antioxidant defense and increased lipid peroxidation products in normotension. Therefore, the therapeutic potential of FAAH inhibitors should be interpreted cautiously.

Abstract 104: DOCA Induced Natriorexigenic and Hypertensive Effects are Blunted by Central Infusion of AT1 Receptor Biased Agonist TRV120027

TRV120027 (TRV) is biased agonist at the angiotensin AT1 receptor that activates the β-arrestin pathway, causing receptor internalization. AT1 signaling within the hypothalamus is required for the blood pressure, polydipsia, and sodium appetite phenotypes in the deoxycorticosterone acetate (DOCA)-salt model of salt-sensitive hypertension (HTN). We hypothesized that TRV action within the hypothalamus would antagonize the effects of DOCA-salt. Using a novel luciferase-based HiBit tagging assay, we confirmed that TRV decreased AT1 surface density in immortalized mouse hypothalamic N43/5 cells by 35±4% compared to vehicle (n=4, p<0.05), and this was prevented by pretreatment with losartan. TRV (0.05 μg/h, icv n=5-10) or aCSF (n=4-8) were chronically infused into C57BL/6J mice implanted with DOCA (50 mg, sc) pellets, compared with mice undergoing sham surgeries (CTL, n=5-8), and animals were offered chow, water and NaCl ad libitum . DOCA increased fluid intake at baseline (CTL 4±0 vs 15±1 mL/day DOCA+aCSF, p<0.05), 0.15 mol/L NaCl (3.8±0 vs 20±2, p<0.05), 0.3 mol/L (4±0 vs 26±2, p<0.05), and 0.45 mol/L (4±0 vs 28±3, p<0.05). DOCA-induced polydipsia was not affected by TRV at baseline (DOCA+aCSF 19±1 vs 15±0 mL/day, p>0.05), 0.15 mol/L NaCl (27±4 vs 20±2 mL/day, p>0.05), 0.3 mol/L (25±4 vs 26±2 mL/day, p>0.05), or 0.45 mol/L (25±3 vs 28±3 mL/day, p>0.05). However, TRV prevented DOCA-induced increase of NaCl preference at NaCl 0.15 mol/L (DOCA+aCSF 29±1 vs 14±2% DOCA+TRV, p<0.05), 0.30 mol/L (29±3 vs 5±1%, p<0.05), and 0.45 mol/L (29±3 vs 5±1%, p<0.05). DOCA+aCSF exhibited increased heart weight / tibia length (CTL 8±0 vs 10±0 g/m DOCA+aCSF, p<0.05), which was prevented by TRV (8±0, p<0.05). In a separate cohort, DOCA-implanted mice were provided only 0.9% NaCl drink for one week, and mean arterial pressure (MAP) was recorded after acute injection of TRV (2 μg icv n=5) or aCSF (n=5). MAP was elevated with DOCA-salt (141±5 mmHg), and at 40 minutes post injection, TRV decreased MAP by 17±4 mmHg (p<0.05). These data underscore the potential for AT1 receptor-biased agonists as tools to treat subtypes of HTN characterized by salt sensitivity and low circulating renin activity.

ROSTAFUROXIN AMELIORATES THE INWARD REMODELING AND STIFFNESS IN RESISTANCE ARTERIES FROM DEOXYCORTICOSTERONE ACETATE-SALT HYPERTENSIVE RATS

Objective: We have shown that rostafuroxin (ROSTA), an antagonist of endogenous ouabain (OUA), block the biding of OUA to Na+ K+ -ATPase and inhibits the cSRC downstream activation in mesenteric resistance arteries (MRA), which ameliorates endothelial dysfunction and oxidative stress, as reduces hypertension in deoxycorticosterone acetate (DOCA)-salt rats. In addition, MRA of DOCA-salt rats also present the hypertrophic inward remodeling with stiffness. Moreover, the chronic treatment with OUA induces hypotrophic inward remodeling with stiffness in MRA. In line with these results we investigate the role of endogenous OUA on remodeling and stiffness in MRA of DOCA-salt rats. Design and method: A hypertensive model was established in uninephrectomized Wistar rats using DOCA-salt. After 5 weeks DOCA-salt treatment, with high systolic blood pressure (SBP) stabilization, this hypertensive model was divided into two groups: DOCA-salt and DOCA-salt co-treated with ROSTA (1 mg/kg per day gavage, 3 weeks). The SBP was measured using the tail-cuff method, and vascular remodeling was assessed in MRA using a pressure myograph. Histological and western blot analysis were performed in MRA. Results: Our results reinforce that ROSTA treatment reduces SBP in DOCA-salt rats, but did not restore it to control levels. For the first time, the results suggest that ROSTA ameliorates the remodeling and stiffness observed in MRA of DOCA-salt rats, enhancing the luminal diameter and distensibility, reducing the cross-section area and wall/lumen ratio as well as shifting to the right the stress-strain curve in MRA. ROSTA restores collagen type I/III ratio, internal elastic lamina thickness, tyrosine kinase Src phosphorylation (Tyr418), as well EGFR, cRaf, ERK, p38MAPK, TGFβ1, CTGF, and collagen I protein expression in MRA from DOCA-salt rats. Conclusions: The results suggested that ROSTA reduces hypertension, the hypertrophic inward remodeling and the augmented wall/lumen ratio in MRA of DOCA-salt rats. In addition, endogenous OUA emerges as an important mechanism to induce vascular stiffness via Na+ K+ -ATPase/ cSRC/ EGFR/ cRas/ ERK/ p38MAPK/ CTGF/ TGFβ1 pathway in MRA. These results reinforce the idea that endogenous OUA is a putative target for the treatment of vascular adjustments present in hypertension.

Role of the OVLT‐gut microbiome axis on the development of DOCA‐salt hypertension

Limited studies suggest an association of gut microbiome changes with certain forms of hypertension, but a cause/effect relationship has yet to be fully defined. The deoxycorticosterone acetate (DOCA)‐salt model of hypertension in rats is known to have a neurogenic component linked to increased sympathetic nervous system activity. Indeed, we have recently shown the hypertensive response in DOCA‐treated rats requires the organum vasculosum of the lamina terminalis (OVLT), a circumventricular organ that has direct connections to the paraventricular nucleus (PVN) to increase sympathetic activity. Furthermore, celiac ganglionectomy also supports a role of splanchnic sympathetic innervation to the gut in the hypertensive response to DOCA. Taken together, we hypothesize an OVLT‐sympathetic‐gut (OSG) microbiome axis is involved in the development of hypertension. Having previously reported that the OVLT plays a significant role in the development of DOCA‐salt hypertension, the present study was designed to test the hypothesis that the OVLT mediates changes in the gut microbiome associated with concomitant hypertension. In uninephrectomized OVLT lesioned (OVLTx; n = 5) and SHAM (n = 2) Sprague‐Dawley rats consuming a 2% NaCl diet and 0.9% NaCl drinking solution, 24‐hour mean arterial pressure (MAP) was recorded telemetrically 5 days before and 21 days after DOCA implantation (100 mg/rat; SQ). Microbiome analyses were performed on regional gut samples at the end of the study and compared to normal rats (n=2). The chronic pressor response to DOCA was attenuated in OVLTx rats such that MAP increased to 153±4 mmHg in SHAM rats by Day 21 of DOCA compared to 127±5 mmHg in OVLTx rats. Moreover, DOCA hypertension in SHAM rats was associated with decreased levels of Lactobacillus species in the jejunum and ileum, and increased Escherichia levels in the cecum compared with normal rats. However, these changes in bacterial genera were mitigated and offset by lesion of the OVLT and these bacterial populations in OVLT lesioned rats with attenuated hypertension more closely resembled those in normal control rats. We conclude that DOCA‐salt hypertension is associated with specific microbiome changes in the gut, and the attenuated hypertensive effects of DOCA‐salt in OVLT lesioned rats is mediated in part through counteracting changes in these bacterial populations.Support or Funding InformationUniversity of Minnesota Grant‐In‐Aid # 107426This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Renal nerves, renal inflammation and hypertension in deoxycorticosterone acetate (DOCA)‐salt hypertension: Who is in the driver's seat?

Renal nerves, renal inflammation and hypertension in deoxycorticosterone acetate (DOCA)‐salt hypertension: Who is in the driver's seat?