DOCA-salt Hypertensive Rats Research Articles

Abstract P383: Increased renal afferent nerve activity and peak response to pro-inflammatory cytokine interleukin 6 in isolated dorsal root ganglion neurons from hypertensive rats

Renal afferent nerves are reported to mediate the progression of hypertension in preclinical models, putatively via an aberrant increase in activation. The underlying source of this nerve activation is unknown but we hypothesized renal inflammatory cytokines are a primary driver. We previously reported interleukin 6 (IL-6) is increased in the kidneys of hypertensive rats, and intrarenal administration of IL-6 can activate renal afferent nerves. Though these data are promising, the direct effects of IL-6 on afferent renal nerves remain unclear. We hypothesized that isolated renal afferent neurons from hypertensive rats will have increased activity and responsiveness to inflammatory cytokine IL-6 compared to normotensive controls. To test our hypothesis, we tested the direct effects of interleukin-6 (IL-6) on renal afferent nerve action potentials (AP) generated in freshly isolated dorsal root ganglion (DRG) neurons. To model salt-sensitive hypertension, uninephrectomized rats (n=6) were administered deoxycorticosterone acetate (DOCA; 100mg, sc) and 0.9% saline for drinking water for 21 days. Control rats (n=6) were uninephrectomized. To label renal afferent nerves, neuronal tracer DiI was introduced by intrarenal injection 6 days prior to DRG collection. DRGs from T10-L1 were isolated, enzymatically digested and dissociated, and cultured overnight for whole-cell patch-clamp the following day. DiI-positive neurons from both control and DOCA rats generated AP during the delivery of 100, 200, and 300 pA current steps. Data were analyzed by two-way, repeat-measures ANOVA (α=.05). At baseline, DOCA neurons had a higher (p<0.05) mean AP frequency compared to controls (no difference in AP mean peak amplitude or AP duration). In both groups, increasing concentrations of IL-6 (0. 4, 4, and 40 ng/ml) elicited a dose-dependent increase in AP peak amplitude compared to baseline. No difference was detected in IL-6 responses between DOCA and controls. Overall, these data supported our original hypothesis that pro-inflammatory cytokine IL-6 would increase renal afferent nerve activity, and that said activity is increased in hypertensive DOCA-salt rats. IL-6 accumulation triggered by end organ damage due to hypertension might lead to increased afferent renal nerve activity, which exacerbates the hypertensive phenotype through a sympatho-excitatory response. Additional measurements of isolated DRG responses to pro-inflammatory mediators are underway for direct comparison.

Egg white hydrolysate modulates the renin-angiotensin system in mesenteric perivascular adipose tissue in doca-salt hypertensive rats and restores its anticontractile ability

Perivascular adipose tissue (PVAT) regulates vascular tonus with an anticontractile effect, which may be dysfunctional in hypertension. An Egg White Hydrolysate (EWH), a bioactive food, restores endothelium dysfunction and reduces blood pressure levels in malignant hypertension. We aimed to evaluate whether dietary supplementation with EWH interferes with the PVAT function in the mesenteric resistance arteries (MRA) of DOCA-salt hypertensive animals and the mechanisms involved. Male rats were divided into 4 groups and treated for 8 weeks. For 4 weeks, DOCA-salt or vehicle-treated rats (SHAM) were induced, and after that, some rats were co-treated with DOCA-salt or vehicle plus EWH (1 g/kg/day) for 4 weeks more. MRA and/or their PVAT (mPVAT) were used for functional, molecular, and biochemical analysis. The anticontractile effect of mPVAT in response to norepinephrine (NE) was observed only in MRA rings with PVAT of the SHAM group, while this effect was abolished in DOCA-salt rings. The EWH treatment did not change the anticontractile effect of mPVAT in SHAM but partially restored it in DOCA-salt rats. Acute aliskiren incubation reduced NE-induced contraction only in MRA with PVAT of DOCA-salt rats. EWH prevented the overactivation of the renin levels and ACE activity in mPVAT of DOCA-salt rats; while did not change AT1R expression, but increased AT2R expression. In mPVAT, EWH blocked the increase of MDA, ROS and pro-inflammatory cytokines observed in DOCA-salt rats. EWH improved PVAT dysfunction in MRA of severe hypertension. This beneficial effect could be mediated by an ameliorated redox balance, inflammatory state, and RAS axis.

Da-Chuan-Xiong Decoction Ameliorates Sodium Sensitivity and Plasma Norepinephrine via Attenuation of Brain Oxidative Stress in the DOCA-Salt Hypertensive Rats.

Da-Chuan-Xiong Decoction (DCXD) is an aqueous extract from a classic Chinese herbal formula composed of dried rhizomes of Ligusticum chuanxiong Hort and Gastrodia elata Bl. in the mass ratio of 4 : 1. It has been long used to treat chronic cardiovascular disease caused by blood stasis and wind pathogen in the clinic. This experimental study aimed to investigate the blood pressure (BP)-lowering effect of DCXD treatment on hypertension and underlying mechanisms. Male Sprague-Dawley rats were used in the experiment, and the hypertensive models were created by administering deoxycorticosterone acetate (DOCA) in conjunction with a high salt intake in uninephrectomized rats. DCXD was administered to hypertensive rats by oral gavage daily at a dose of 5 g/kg or 2.5 g/kg bodyweight for 28 days. The brain sodium sensitivity, ENaC function, superoxide anion level, NADPH oxidase activity, and expression of ENaC, p67phox, p47phox, and Rac1 in the paraventricular nucleus were assessed by using the appropriate methods. The 28 days of DCXD (5 g/kg) treatment significantly reduced the increased BP effectively, inhibited the enhanced heart index, kidney index, and 24 h urinary protein, and improved the progressive pathological changes of heart and kidney, which was comparable to that of the positive control amlodipine. DCXD treatment also caused a marked reduction in plasma norepinephrine and induced a significant improvement in brain sodium sensitivity and ENaC function in DOCA-salt hypertensive rats. Rats in DCXD-treated groups also exhibited decreased superoxide anion levels and NADPH oxidase activity in the paraventricular nucleus. The level of ENaC, p67phox, and Rac1 protein expression in the paraventricular nucleus was significantly downregulated by DCXD treatment in DOCA-salt hypertensive rats. These findings indicate that the depressor action and sympathetic inhibition of DCXD on salt-sensitive hypertension may be by ameliorating brain sodium sensitivity, modulating ENaC function, and inhibiting the expression of ENaC and NADPH oxidase in the hypothalamic paraventricular nucleus.

Tonic NMDAR Currents of NR2A-Containing NMDARs Represent Altered Ambient Glutamate Concentration in the Supraoptic Nucleus.

NMDA receptors (NMDARs) modulate glutamatergic excitatory tone in the brain via two complementary modalities: a phasic excitatory postsynaptic current and a tonic extrasynaptic modality. Here, we demonstrated that the tonic NMDAR-current (I NMDA) mediated by NR2A-containing NMDARs is an efficient biosensor detecting the altered ambient glutamate level in the supraoptic nucleus (SON). I NMDA of magnocellular neurosecretory cells (MNCs) measured by nonselective NMDARs antagonist, AP5, at holding potential (V holding) -70 mV in low concentration of ECF Mg2+ ([Mg2+]o) was transiently but significantly increased 1-week post induction of a DOCA salt hypertensive model rat which was compatible with that induced by a NR2A-selective antagonist, PEAQX (I PEAQX) in both DOCA-H2O and DOCA-salt groups. In agreement, NR2B antagonist, ifenprodil, or NR2C/D antagonist, PPDA, did not affect the holding current (I holding) at V holding -70 mV. Increased ambient glutamate by exogenous glutamate (10 mM) or excitatory amino acid transporters (EAATs) antagonist (TBOA, 50 mM) abolished the I PEAQX difference between two groups, suggesting that attenuated EAATs activity increased ambient glutamate concentration, leading to the larger I PEAQX in DOCA-salt rats. In contrast, only ifenprodil but not PEAQX and PPDA uncovered I NMDA at V holding +40 mV under 1.2 mM [Mg2+]o condition. I ifenprodil was not different in DOCA-H2O and DOCA-salt groups. Finally, NR2A, NR2B, and NR2D protein expression were not different in the SON of the two groups. Taken together, NR2A-containing NMDARs efficiently detected the increased ambient glutamate concentration in the SON of DOCA-salt hypertensive rats due to attenuated EAATs activity.

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.

Abstract 019: Perimenopause Exacerbates Hypertension In Doca-salt Rats

Blood pressure (BP) is lower in premenopausal women than in age-matched men, but after menopause BP rates and the incidence of hypertension (HTN) show opposite tendencies. The mechanisms by which fluctuating ovarian hormone environment during that period impacts the onset and development of HTN are poorly understood, and strategies to reduce HTN risk in peri- and postmenopausal females are lacking. An ovary-intact rat model of perimenopause using the chemical, 4-vinylcyclohexene diepoxide (VCD), has been developed to accelerate the natural process of slow follicular loss. Daily dosing with VCD for 15 days selectively destroys primordial and primary follicles in ovaries of mice and rats by accelerating atresia processes, thus inducing precocious perimenopause/menopause. Hypothesis: The induction of perimenopause by VCD will increase susceptibility to the development of HTN in DOCA-salt rats. Female Sprague-Dawley rats at post-natal day 28 received subcutaneous injection of VCD (160mg/kg) or corn oil (control) for 15 days. 57 days after the first injections, nephrectomy and telemetric pressure transducer implantation were performed. After 2 weeks of recovery, a 100 mg/kg DOCA silicone or placebo pellet was implanted subcutaneously. The rats that received DOCA were provided 0.9% saline during the 21 days of DOCA protocol and mean arterial pressure (MAP) was continuously evaluated. Rats were housed weekly in metabolic cages for 24 hours to measure saline intake and urine volume. After protocol completion the animals were decapitated, and blood was collected for the measurement of anti-Mullerian hormone (AMH) and estradiol (E2) by ELISA. The AMH plasma levels were reduced in the periestropause rats (VCD + DOCA, n=9) compared to oil (Oil + DOCA, n=6) confirming the VCD-induced follicular depletion. The steady state increase in MAP (day 21 of DOCA-salt) was higher in periestropause (±45.7mmHg) compared to oil (±28.6 mmHg). Urine volume was higher in periestropause rats compared to oil. No differences were observed in the saline intake between the groups. Our data demonstrate that periestropause rats showed greater susceptibility to the development of HTN in a DOCA-salt rat model.

Small artery remodeling and stiffening in deoxycorticosterone acetate-salt hypertensive rats involves the interaction between endogenous ouabain/Na + K + -ATPase/cSrc signaling.

Endogenous ouabain (EO) increases in some patients with hypertension and in rats with volume-dependent hypertension. When ouabain binds to Na + K + -ATPase, cSrc is activated, which leads to multieffector signaling activation and high blood pressure (BP). In mesenteric resistance arteries (MRA) from deoxycorticosterone acetate (DOCA)-salt rats, we have demonstrated that the EO antagonist rostafuroxin blocks downstream cSrc activation, enhancing endothelial function and lowering oxidative stress and BP. Here, we examined the possibility that EO is involved in the structural and mechanical alterations that occur in MRA from DOCA-salt rats. MRA were taken from control, vehicle-treated DOCA-salt or rostafuroxin (1 mg/kg per day, for 3 weeks)-treated DOCA-salt rats. Pressure myography and histology were used to evaluate the mechanics and structure of the MRA, and western blotting to assess protein expression. DOCA-salt MRA exhibited signs of inward hypertrophic remodeling and increased stiffness, with a higher wall:lumen ratio, which were reduced by rostafuroxin treatment. The enhanced type I collagen, TGFβ1, pSmad2/3 Ser465/457 /Smad2/3 ratio, CTGF, p-Src Tyr418 , EGFR, c-Raf, ERK1/2 and p38MAPK protein expression in DOCA-salt MRA were all recovered by rostafuroxin. A process combining Na + K + -ATPase/cSrc/EGFR/Raf/ERK1/2/p38MAPK activation and a Na + K + -ATPase/cSrc/TGF-1/Smad2/3/CTGF-dependent mechanism explains how EO contributes to small artery inward hypertrophic remodeling and stiffening in DOCA-salt rats. This result supports the significance of EO as a key mediator for end-organ damage in volume-dependent hypertension and the efficacy of rostafuroxin in avoiding remodeling and stiffening of small arteries.

Role of Brain Endothelin Receptor Type B (ETB) in the Regulation of Tyrosine Hydroxylase in the Olfactory Bulb of DOCA-Salt Hypertensive Rats.

We previously reported that endothelins (ETs) regulate tyrosine hydroxylase (TH) activity and expression in the olfactory bulb (OB) of normotensive and hypertensive animals. Applying an ET receptor type A (ETA) antagonist to the brain suggested that endogenous ETs bind to ET receptor type B (ETB) to elicit effects. The aim of the present work was to evaluate the role of central ETB stimulation on the regulation of blood pressure (BP) and the catecholaminergic system in the OB of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. DOCA-salt hypertensive rats were infused for 7 days with cerebrospinal fluid or IRL-1620 (ETB receptor agonist) through a cannula placed in the lateral brain ventricle. Systolic BP (SBP) and heart rate were recorded by plethysmography. The expression of TH and its phosphorylated forms in the OB were determined by immunoblotting, TH activity by a radioenzymatic assay, and TH mRNA by quantitative real-time polymerase chain reaction. Chronic administration of IRL-1620 decreased SBP in hypertensive rats but not in normotensive animals. Furthermore, the blockade of ETB receptors also decreased TH-mRNA in DOCA-salt rats, but it did not modify TH activity or protein expression. These findings suggest that brain ETs through the activation of ETB receptors contribute to SBP regulation in DOCA-salt hypertension. However, the catecholaminergic system in the OB does not appear to be conclusively involved although mRNA TH was reduced. Present and previous findings suggest that in this salt-sensitive animal model of hypertension, the OB contributes to chronic BP elevation.

Dietary Magnesium Supplementation Modifies Blood Pressure and Cardiovascular Function in Mineralocorticoid-Salt Hypertensive Rats but Not in Normotensive Rats

The purpose of this study was to determine the effect of dietary magnesium supplementation on blood pressure and cardiovascular function of Sprague-Dawley normotensive and mineralocorticoid-salt (DOCA-salt) hypertensive rats. The rats were pairfed for 5 wk a purified diet containing either a normal or magnesium-supplemented diet (1.5 or 10 g/kg diet). Magnesium supplementation significantly lowered blood pressure levels in hypertensive rats, but not in normotensive rats. Heart rate was not affected in either group. The blood pressure-lowering effect of magnesium supplementation in DOCA-salt hypertensive rats was associated with a lower in vivo cardiovascular reactivity to norepinephrine and angiotensin II. Norepinephrine reactivity in isolated aortae from DOCA-salt hypertensive rats was not modified by magnesium supplementation. However, endothelium-dependent relaxation to acetylcholine was improved and could be related to the release of endothelial relaxant factors. Magnesium supplementation did not affect cardiac hemodynamics in isolated heart from either normotensive or DOCA-salt hypertensive rats. Furthermore, no protective effects upon myocardial ischemia and ventricular arrhythmias were demonstrated. These findings suggest that the lowering effect of magnesium supplementation on blood pressure in hypertensive rats may be related to a vascular effect of magnesium that reduces vascular tone. Mechanisms related to the pathophysiological development of mineralocorticoid-salt hypertension may be involved.

JS-HR-12: BASIC RESEARCH: ELUCIDATION OF “MOSAIC” PATHOGENESIS OF HYPERTENSION

Basic research plays critical roles in elucidating the “mosaic” pathogenesis of hypertension and developing its treatment. In the field of brain and autonomic nervous system, Chen et al. demonstrated that mild cold exposure elicits autonomic dysregulation, such as increased sympathetic activity and decreased baroreflex sensitivity, and poor sleep quality, causing blood pressure (BP) elevation in normotensive rats. This finding may have critical implications for the cardiovascular event occurrence with low ambient temperature. Domingos-Souza et al. showed that the ability of baroreflex activation to modulate hemodynamics and induce lasting vascular adaptation is critically dependent on the electrical parameters and duration of carotid sinus stimulation in spontaneously hypertensive rats (SHRs), proposing a rationale for improving baroreflex activation therapy in humans. In the kidney, Kasacka et al. showed that activity of Wnt/β-catenin pathway is increased in SHRs and 2K1C hypertensive rats, while it is inhibited in DOCA-salt rats. Intrarenal renin-angiotensin system (RAS) is involved in BP regulation. In renal damage with an impaired glomerular filtration barrier, liver-derived angiotensinogen filtered through damaged glomeruli regulates intrarenal RAS activity. Matsuyama et al. further showed that the glomerular filtration of liver-derived angiotensinogen depending on glomerular capillary pressure causes circadian rhythm of the intrarenal RAS. Otsuki et al. revealed that TWIST1, a transcription factor regulating mesodermal embryogenesis, transcriptionally upregulates complement 3, which has been demonstrated to activate intrarenal RAS, in the glomerular mesangial cells from SHRs. The RAS in the vascular system also contributes to BP regulation. Soring nexins (SNXs) are cellular sorting proteins and can regulate the expression and function of G protein-coupled receptors (GPCRs). Liu C et al. demonstrated that SNX1 knockout mice exhibit hypertension through vasoconstriction mediated by increased expression of GPCR AT1R within the arteries. Liu X et al. found that sirtuin 6 (SIRT6) expression is downregulated in the aortae of aged rats and showed that SIRT6 knockdown enhances angiotensin II-induced vascular adventitial aging by activating NF-κB pathway in vitro studies. The studies investigating extracellular vesicles (EVs) and gut microbiota have been increased. Ochiai-Homma et al. showed that pendrin in urinary EVs can be a useful biomarker for diagnosis and treatment of primary aldosteronism. Wu et al. demonstrated that captopril has the potential to rebalance the dysbiotic gut microbiota of DOCA-salt hypertensive rats. Several reports indicate the potential treatment for hypertension and the associated organ damages: rivaroxaban in cardiac hypertrophy; nebivolol, maximakinin, and Pinggan-Qianyang in hypertension; and melatonin and crocin in gestational hypertension.

Investigation of the relationship between serum adropin levels, oxidative stress biomarkers, and blood pressure in DOCA-salt hypertensive rats

Background/Aim: Adropin is involved in the pathophysiology and development of cardiovascular diseases, such as hypertension. The aim of this study was to investigate the effects of adropin in serum, potential use as a biochemical biomarker of oxidative stress, and effects on blood pressure in deoxycorticosterone acetate (DOCA) salt hypertensive rats.
 Methods: Eighteen male Sprague-Dawley rats were divided into two groups: (1) Control (C) and (2) Hypertensive (H). Systolic and diastolic blood pressures (SBP and DBP, respectively), and mean blood pressure (MBP) were measured using the tail-cuff method. At the end of the study, serum endothelin-1 (ET-1), adropin, nitric oxide (NO), total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) were also analyzed.
 Results: Significant increases in SBP, DBP, MBP, cardiac hypertrophy index (CHI), and left ventricular hypertrophy index (LVCI) in the H group compared with the C group were found. Serum levels of ET-1, TOS, and OSI were significantly higher in the H group and serum levels of NO, adropin, and TAS were lower than in the C group. A negative correlation between serum adropin levels and the variables SBP, DBP, MBP, TOS, OSI, CHI, and LVHI was found. Adropin levels were positively correlated positively with serum NO levels in both groups.
 Conclusion: Serum adropin levels decreased in hypertensive DOCA-salt rats. Lower serum adropin levels were found to be significantly associated with hypertension and may play a role in this disease. However, further comprehensive and diverse studies are needed.

Captopril Alleviates Chondrocyte Senescence in DOCA-Salt Hypertensive Rats Associated with Gut Microbiome Alteration.

Gut microbiota is the key controller of healthy aging. Hypertension and osteoarthritis (OA) are two frequently co-existing age-related pathologies in older adults. Both are associated with gut microbiota dysbiosis. Hereby, we explore gut microbiome alteration in the Deoxycorticosterone acetate (DOCA)-induced hypertensive rat model. Captopril, an anti-hypertensive medicine, was chosen to attenuate joint damage. Knee joints were harvested for radiological and histological examination; meanwhile, fecal samples were collected for 16S rRNA and shotgun sequencing. The 16S rRNA data was annotated using Qiime 2 v2019.10, while metagenomic data was functionally profiled with HUMAnN 2.0 database. Differential abundance analyses were adopted to identify the significant bacterial genera and pathways from the gut microbiota. DOCA-induced hypertension induced p16INK4a+ senescent cells (SnCs) accumulation not only in the aorta and kidney (p < 0.05) but also knee joint, which contributed to articular cartilage degradation and subchondral bone disturbance. Captopril removed the p16INK4a + SnCs from different organs, partially lowered blood pressure, and mitigated cartilage damage. Meanwhile, these alterations were found to associate with the reduction of Escherichia-Shigella levels in the gut microbiome. As such, gut microbiota dysbiosis might emerge as a metabolic link in chondrocyte senescence induced by DOCA-triggered hypertension. The underlying molecular mechanism warrants further investigation.

Elucidation of Anti-Hypertensive Mechanism by a Novel Lactobacillus rhamnosus AC1 Fermented Soymilk in the Deoxycorticosterone Acetate-Salt Hypertensive Rats.

Dietary intake of fermented soymilk is associated with hypotensive effects, but the mechanisms involved have not been fully elucidated. We investigated the anti-hypertensive effects of soymilk fermented by L. rhamnosus AC1 on DOCA-salt hypertension from the point of view of oxidative stress, inflammatory response and alteration of the gut microbiome. The antioxidant assays in vitro indicated the ethanol extract (EE) of L. rhamnosus AC1 fermented soymilk showed better antioxidative effects than the water extract (WE). Those extracts displayed a hypotensive effect using a tail-cuff approach to measuring blood pressure and improved nitric oxide (NO), angiotensin II (Ang II), tumor necrosis factor-α (TNF-α) and interleukin factor-6 (IL-6) on DOCA-salt hypertensive rats. Furthermore, cardiac and renal fibrosis were attenuated by those extracts. The gut microbiota analysis revealed that they significantly reduced the abundance of phylum Proteobacteria, its family Enterobacteriaceae and genus Escherichia-Shigella. Moreover, metabolomic profiling revealed several potential gut microbiota-related metabolites which appeared to involve in the development and recovery of hypertension. In conclusion, fermented soymilk is a promising nutritional intervention strategy to improve hypertension via reducing inflammation and reverting dysbiotic microbiota.

Secreted protein acidic and rich in cysteine (SPARC) and a disintegrin and metalloproteinase with thrombospondin type 1 motif (ADAMTS1) increments by the renin-angiotensin system induce renal fibrosis in deoxycorticosterone acetate-salt hypertensive rats

Secreted protein acidic and rich in cysteine (SPARC), an extracellular matrix (ECM) protein, was recently shown to induce collagen deposition through the production of a disintegrin and metalloproteinase with thrombospondin type 1 motif (ADAMTS1) in the aging heart. ADAMTS1 regulates ECM turnover by degrading ECM components, and its excessive activation contributes to various pathological states, including fibrosis. The present study investigated the pathophysiological regulation and role of SPARC and ADAMTS1 in renal fibrosis using uninephrectomized rats treated with deoxycorticosterone acetate (DOCA, 40 mg/kg/week, subcutaneously) and salt (1% in drinking water). The administration of DOCA and salt gradually and significantly elevated systolic blood pressure during the 3-week treatment period, induced proteinuria, decreased creatinine clearance, and increased NADPH oxidase-derived superoxide production, malondialdehyde concentrations, and monocyte chemoattractant protein-1 and osteopontin expression in the kidneys. Glomerulosclerosis, fibrillar collagen deposition, and transforming growth factor-β expression increased in a time-dependent manner, and SPARC and ADAMTS1 expression showed a similar pattern to these changes. The angiotensin II type-1 receptor blocker losartan suppressed the overexpression of SPARC and ADAMTS1, and an in vitro exposure to angiotensin II induced the production of both SPARC and ADAMTS1 in renal fibroblast NRK-49F cells. Knockdown of the SPARC gene with small interfering RNA reduced all forms (the 110-kDa latent and 87- and 65-kDa bioactive forms) of ADAMTS1 expression as well as collagen production. These results suggest that SPARC is induced by the renin-angiotensin system and may be a fibrogenic factor, at least in part, by producing ADAMTS1 in hypertensive renal disease.

Pharmacological Inhibition of Mammalian Target of Rapamycin Attenuates Deoxycorticosterone Acetate Salt-Induced Hypertension and Related Pathophysiology: Regulation of Oxidative Stress, Inflammation, and Cardiovascular Hypertrophy in Male Rats.

The present study aimed to explore the contribution of mammalian target of rapamycin (mTOR) in deoxycorticosterone acetate (DOCA) salt-induced hypertension and related pathophysiological changes in cardiovascular and renal tissues. DOCA salt loading resulted in an increase in systolic blood pressure, diastolic blood pressure, and mean blood pressure along with the activity of ribosomal protein S6, the effector protein of mTOR. Treatment with rapamycin, the selective inhibitor of mTOR, initiated at the fourth week of DOCA- salt administration normalized the systolic blood pressure and attenuated ribosomal protein S6 activity in the heart, aorta, and kidney. Cardiac and vascular hypertrophy, oxidative stress, and infiltration of macrophages (CD68+), the marker of inflammation, were also reduced in rapamycin-treated, DOCA-salt, hypertensive rats. In addition, renal hypertrophy and dysfunction were also reduced with rapamycin-treated hypertensive rats. Moreover, these pathophysiological changes in DOCA-salt hypertensive rats were associated with increased NADPH oxidase (NOX) activity, gp91phox (formerly NOX2) expression, ERK1/2, and p38 MAPK activities in the heart, aorta, and kidney were minimized by rapamycin. These data indicate that mTOR plays an important role in regulating blood pressure and the development of cardiovascular and renal pathophysiological changes, most likely due to increased NOX expression/activity, ERK1/2, and p38 MAPK activity with macrophages infiltration in the heart, kidney, and aorta. Pharmacological inhibition of mTOR and related signaling pathways could serve as a novel target for the treatment of hypertension.

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.

Anti-Hypertensive Activity of Hydro Alcoholic Extract of Cìraka Cūraṇam on High Salt Loaded Wistar Albino Rats

Background: Hypertension is called as Silent killer. The most important risk factor for heart diseases and stroke and it may leads to premature death. Several medicinal plants have high effective in anti-hypertensive and anti-thrombotic activity without any side effects. Aim: To evaluate the anti-hypertensive activity of hydro alcoholic extract of Cìraka cūraṇam on deoxycorticosterone acetate (DOCA) salt induced wistar albino rats. Study design: Observational in-vivo study Place and duration of study: Animal house, Dept. of Pharmacology, Arulmigu Kalasalingam College of Pharmacy, Krishnankoil, Srivilliputtur,Tamilnadu. Materials and methods: Antihypertensive activity was conducted on wistar albino rats by determining serum Sodium and Potassium levels by using semi auto analyzer (RA-50, Bayer Diagnostics), using specific kits (Auto span, India) at 500 and 550 nm respectively and left carotid artery (for recording BP) was cannulated under aseptic conditions with polyethylene cannula filled with 1% heparin in normal saline. Rest procedure, which was stated under the 2K1C-model was followed and BP was observed in terms of mm of Hg. Results: The Cìraka cūraṇam possesses strong antihypertensive effect against DOCA-salt hypertensive rats, which is evidenced by a considerable decrease in blood pressures. Keywords: Anti-hypertensive activity, Cìraka cūraṇam, Wistar albino rats, Deoxycorticosterone acetate.

BRAIN-PENETRATING AMINOPEPTIDASE A INHIBITORS FOR NEW TREATMENT OF HYPERTENSION AND HEART FAILURE

Objective: Brain renin-angiotensin system (RAS) hyperactivity is involved in the pathophysiology of hypertension and heart failure (HF). Angiotensin III/(AngIII), one of the main effector peptides of the brain RAS, exerts tonic stimulatory control over blood pressure/(BP) in hypertensive rats and plays a pivotal role in sympathetic hyperactivity and left ventricular/(LV) dysfunction in rats post-myocardial infarction/(MI). Thus, aminopeptidase A/(APA), the enzyme generating brain AngIII represents a potential therapeutic target for the treatment of these pathologies. Our aim was to evaluate the mode of action of firibastat, a brain-penetrating prodrug of the APA inhibitor, EC33, to reduce BP in hypertensive rats and prevent LV dysfunction in mice post-MI. Design and method: Firibastat and MLN4760, an angiotensin-converting enzyme2/(ACE2) inhibitor were administered by central route in hypertensive DOCA-salt rats and mean arterial BP (MABP) was recorded following insertion of a catheter into the femoral artery. Brain APA and ACE2 activities were measured 30 min post-treatment. Two days post-MI induced by the left anterior descending artery ligation, mice were randomized to receive during 4–8 weeks oral treatment with vehicle, firibastat (150 mg/kg) or the ACE inhibitor, enalapril (1 mg/kg). Cardiac function was evaluated by echocardiography Results: 1-Firibastat, when given centrally in hypertensive rats, is immediately cleaved by brain reductases, generating EC33 which inhibits brain APA activity, blocks brain AngIII formation and decreases BP. At the same time, firibastat treatment increases the activity of brain ACE2, majoring the conversion of AngII to Ang1-7, which by acting on the Mas-receptor, participate to the firibastat-induced BP decrease. 2-Chronic firibastat treatment in mice post-MI normalizes brain APA activity (normalizing brain RAS activity) and prevents sympathetic hyperactivity. It reduced LV end-diastolic pressure, LV end-systolic diameter and volume, and increased LV ejection fraction. It decreased the expression of HF and fibrosis biomarkers. Firibastat treatment prevents cardiac dysfunction and attenuates cardiac hypertrophy and fibrosis. Conclusions: Following these data, firibastat was selected for clinical development. Phase IIa/IIb clinical trials provided pharmacological proof-of-principle of firibastat efficacy for decreasing BP in hypertensive patients. The ongoing phase II study/QUORUM will determine if firibastat treatment may constitute a new therapeutic approach for preventing HF post-MI.