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.

No evidence for brain renin-angiotensin system activation during DOCA-salt hypertension.

Brain renin-angiotensin system (RAS) activation is thought to mediate deoxycorticosterone acetate (DOCA)-salt hypertension, an animal model for human primary hyperaldosteronism. Here, we determined whether brainstem angiotensin II is generated from locally synthesized angiotensinogen and mediates DOCA-salt hypertension. To this end, chronic DOCA-salt-hypertensive rats were treated with liver-directed siRNA targeted to angiotensinogen, the angiotensin II type 1 receptor antagonist valsartan, or the mineralocorticoid receptor antagonist spironolactone (n = 6-8/group). We quantified circulating angiotensinogen and renin by enzyme-kinetic assay, tissue angiotensinogen by Western blotting, and angiotensin metabolites by LC-MS/MS. In rats without DOCA-salt, circulating angiotensin II was detected in all rats, whereas brainstem angiotensin II was detected in 5 out of 7 rats. DOCA-salt increased mean arterial pressure by 19 ± 1 mmHg and suppressed circulating renin and angiotensin II by >90%, while brainstem angiotensin II became undetectable in 5 out of 7 rats (<6 fmol/g). Gene silencing of liver angiotensinogen using siRNA lowered circulating angiotensinogen by 97 ± 0.3%, and made brainstem angiotensin II undetectable in all rats (P<0.05 vs. non-DOCA-salt), although brainstem angiotensinogen remained intact. As expected for this model, neither siRNA nor valsartan attenuated the hypertensive response to DOCA-salt, whereas spironolactone normalized blood pressure and restored brain angiotensin II together with circulating renin and angiotensin II. In conclusion, despite local synthesis of angiotensinogen in the brain, brain angiotensin II depended on circulating angiotensinogen. That DOCA-salt suppressed circulating and brain angiotensin II in parallel, while spironolactone simultaneously increased brain angiotensin II and lowered blood pressure, indicates that DOCA-salt hypertension is not mediated by brain RAS activation.

Hypotensive and antihypertensive effects of an aqueous extract from Guinep fruit (Melicoccus bijugatus Jacq) in rats.

Melicoccus bijugatus Jacq (Mb) has been reported to have cardiovascular modulatory effects. In this study, we evaluated the antihypertensive effects and mechanism of action of Mb on NG-Nitro-L-arginine Methyl Ester (L-NAME) and Deoxycorticosterone Acetate (DOCA) rat models. Aqueous extract of Mb fruit (100 mg/kg) was administered for 6 weeks to rats by gavage and blood pressure was recorded. Effects of the extract on vascular reactivity was evaluated using isolated organ baths, and tissues were collected for biochemical and histological analysis. The systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) were significantly (P < 0.05) reduced with extract (100 mg/kg) administration and treatment compared to the hypertensive models. Mb (100 µg/mL) reduced the vascular contractility induced by phenylephrine (PE), and caused a dose-dependent relaxation of PE-induced contraction of aortic vascular rings. The vasorelaxation properties seemed to be endothelium dependent, as well as nitric oxide (NO) and guanylyl cyclase, but not prostaglandin dependent. Histomicrograph of transverse sections of the ventricles from the Mb group did not show abnormalities. The extract significantly (P < 0.05) reduced an L-NAME induced elevation of cardiac output and Creatine Kinase Muscle-Brain (CKMB), but had no significant impact on the activities of arylamine N-acetyltransferase. In conclusion, Mb significantly decreased blood pressure in hypertensive models. The extract possesses the ability to induce endothelium dependent vasodilation, which is dependent on guanylyl cyclase but not prostaglandins.

Neuronal (pro)renin receptor regulates deoxycorticosterone-induced sodium intake.

Increased sodium appetite is a physiological response to sodium deficiency; however, it has also been implicated in disease conditions such as congestive heart failure, kidney failure, and salt-sensitive hypertension. The central nervous system is the major regulator of sodium appetite and intake behavior; however, the neural mechanisms underlying this behavior remain incompletely understood. Here, we investigated the involvement of the (pro)renin receptor (PRR), a component of the brain renin-angiotensin system, in the regulation of sodium intake in a neuron-specific PRR knockout (PRRKO) mouse model generated previously in our laboratory. Sodium intake following deoxycorticosterone (DOCA) stimulation was tested by assessing the preference of mice for 0.9% saline or regular water in single-animal metabolic cages. Blood pressure was monitored in conscious, freely moving mice by a telemetry system. We found that saline intake and total fluid intake were significantly reduced in PRRKO mice following DOCA treatment compared with that in wild-type (WT) mice, whereas regular water intake was similar between the genotypes. Sodium preference and total sodium intake were significantly reduced in PRRKO mice compared with WT mice. PRRKO mice also excreted less urine and urinary sodium compared with WT mice following DOCA treatment, whereas potassium excretion was similar between the two groups. Finally, we found that the sodium balance, calculated by subtracting urinary sodium excretion from sodium intake, was greater in WT mice than in PRRKO mice. Collectively, these findings suggest that the neuronal PRR plays a regulatory role in DOCA-induced sodium intake.

(-)-Epicatechin Reduces Blood Pressure and Improves Left Ventricular Function and Compliance in Deoxycorticosterone Acetate-Salt Hypertensive Rats.

(−)-Epicatechin (E) is a flavanol found in green tea and cocoa and has been shown to attenuate tumour necrosis factor alpha (TNF-α)-mediated inflammation, improve nitric oxide levels, promote endothelial nitric oxide synthase (eNOS) activation and inhibit NADPH oxidase. This study investigated the effect of 28 days of low epicatechin dosing (1 mg/kg/day) on the cardiovascular function of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Wistar rats (n = 120, 8 weeks of age) underwent uninephrectomy and were randomised into four groups (uninephrectomy (UNX), UNX + E, DOCA, DOCA + E). DOCA and DOCA + E rats received 1% NaCl drinking water along with subcutaneous injections of 25 mg deoxycorticosterone-acetate (in 0.4 mL of dimethylformamide) every fourth day. UNX + E and DOCA + E rats received 1 mg/kg/day of epicatechin by oral gavage. Single-cell micro-electrode electrophysiology, Langendorff isolated-heart assessment and isolated aorta and mesenteric organ baths were used to assess cardiovascular parameters. Serum malondialdehyde concentration was used as a marker of oxidative stress. Myocardial stiffness was increased and left ventricular compliance significantly diminished in the DOCA control group, and these changes were attenuated by epicatechin treatment (p < 0.05). Additionally, the DOCA + E rats showed significantly reduced blood pressure and malondialdehyde concentrations; however, there was no improvement in left ventricular hypertrophy, electrophysiology or vascular function. This study demonstrates the ability of epicatechin to reduce blood pressure, prevent myocardial stiffening and preserve cardiac compliance in hypertrophied DOCA-salt rat hearts.

Salt supplementation ameliorates developmental kidney defects in COX-2-/- mice.

Deficiency of cyclooxygenase-2 (COX-2) activity in the early postnatal period causes impairment of kidney development leading to kidney insufficiency. We hypothesize that impaired NaCl reabsorption during the first days of life is a substantial cause for nephrogenic defects observed in COX-2-/- mice and that salt supplementation corrects these defects. Daily injections of NaCl (0.8 mg·g-1·day-1) for the first 10 days after birth ameliorated impaired kidney development in COX-2-/- pups resulting in an increase in glomerular size and fewer immature superficial glomeruli. However, impaired renal subcortical growth was not corrected. Increasing renal tubular flow by volume load or injections of KCl did not relieve the renal histomorphological damage. Administration of torsemide and spironolactone also affected nephrogenesis resulting in diminished glomeruli and cortical thinning. Treatment of COX-2-/- pups with NaCl/DOCA caused a stronger mitigation of glomerular size and induced a slight but significant growth of cortical tissue mass. After birth, renal mRNA expression of NHE3, NKCC2, ROMK, NCCT, ENaC, and Na+/K+-ATPase increased relative to postnatal day 2 in wild-type mice. However, in COX-2-/- mice, a significantly lower expression was observed for NCCT, whereas NaCl/DOCA treatment significantly increased NHE3 and ROMK expression. Long-term effects of postnatal NaCl/DOCA injections indicate improved kidney function with normalization of pathologically enhanced creatinine and urea plasma levels; also, albumin excretion was observed. In summary, we present evidence that salt supplementation during the COX-2-dependent time frame of nephrogenesis partly reverses renal morphological defects in COX-2-/- mice and improves kidney function.

Flax lignan concentrate reverses alterations in blood pressure, left ventricular functions, lipid profile and antioxidant status in DOCA-salt induced renal hypertension in rats.

CONTEXT: Earlier we reported cardioprotective, antihyperlipidemic, and in vitro antioxidant activity of flax lignan concentrate (FLC) obtained from the seeds of Linum usitatissimum L. (Linaceae). OBJECTIVE: To investigate the effect of FLC in deoxycorticosterone acetate (DOCA)-salt induced experimental renal hypertension in rats. MATERIALS AND METHODS: Hypertension was induced in uninephrectomized (UNTZD) male Wistar rats (230-280 g) by injecting DOCA (25 mg/kg, subcutaneously, twice weekly) and supplementing 1% NaCl in drinking water for 5 weeks. The rats were divided in six groups. Captopril (30 mg/kg, p.o.) and FLC (200, 400 and 800 mg/kg, p.o.) were administered daily to the rats of groups III-VI, respectively, for 5 weeks. Various hemodynamic and biochemical parameters were investigated as well as histology of kidney and heart were carried out. RESULTS: In this study, the FLC (400 and 800 mg/kg) significantly (p < 0.01, p < 0.001) decreased the systolic blood pressure, diastolic blood pressure, and mean arterial blood pressure. It also significantly (p < 0.01, p < 0.001) decreased elevated end diastolic pressure (EDP), dP/dt max and dP/dt min, organs weights (kidney and heart) and activities of hepatic, renal and cardiac marker enzymes in the serum. Furthermore, FLC (400 and 800 mg/kg) significantly (p < 0.01, p < 0.001) restored altered antioxidant status, serum electrolyte level, lipid profile values, and histological abnormalities. Captopril (30 mg/kg) showed maximum antihypertensive effect but low dose of FLC (200 mg/kg) was not enough to show the antihypertensive activity. CONCLUSION: FLC possessed antihypertensive effect via modulation of endogenous enzymes in DOCA-salt induced renal hypertension in rats.

Development of a Model of Chronic Kidney Disease in the C57BL/6 Mouse with Properties of Progressive Human CKD.

Chronic kidney disease (CKD) is a major healthcare problem with increasing prevalence in the population. CKD leads to end stage renal disease and increases the risk of cardiovascular disease. As such, it is important to study the mechanisms underlying CKD progression. To this end, an animal model was developed to allow the testing of new treatment strategies or molecular targets for CKD prevention. Many underlying risk factors result in CKD but the disease itself has common features, including renal interstitial fibrosis, tubular epithelial cell loss through apoptosis, glomerular damage, and renal inflammation. Further, CKD shows differences in prevalence between the genders with premenopausal women being relatively resistant to CKD. We sought to develop and characterize an animal model with these common features of human CKD in the C57BL/6 mouse. Mice of this genetic background have been used to produce transgenic strains that are commercially available. Thus, a CKD model in this strain would allow the testing of the effects of numerous genes on the severity or progression of CKD with minimal cost. This paper describes such a mouse model of CKD utilizing angiotensin II and deoxycorticosterone acetate as inducers.

Protective effects of grape seed proanthocyanidins on cardiovascular remodeling in DOCA-salt hypertension rats.

Cardiovascular remodeling, as a hallmark of hypertension-induced pathophysiology, causes substantial cardiovascular morbidity and mortality. There is increasing evidence that has demonstrated a broad spectrum of pharmacological and therapeutic benefits of grape seed proanthocyanidins (GSP) against oxidative stress and cardiovascular diseases. In this study, 180- to 200-g SD rats treated with DOCA (120 mg/week sc with 1% NaCl and 0.2% KCl in drinking water) and GSP (150, 240, 384 mg/kg) or amlodipine (ALM) (5 mg/kg) for 4 weeks were recruited. The protective effects of GSP on blood pressure and cardiovascular remodeling in rats with DOCA-salt-induced hypertension were investigated. Our results indicated that DOCA-salt could induce hypertension, cardiovascular remodeling and dysfunction, oxidative stress and the release of endothelin-1 (ET-1) and could increase JNK1/2 and p38MAPK phosphorylation. GSP or ALM treatments significantly improved hypertension, cardiovascular remodeling and dysfunction and oxidative stress, restrained the release of ET-1 and down-regulated the JNK1/2 and p38MAPK phosphorylation. These findings demonstrate that GSP has protective effects against increase of blood pressure induced by DOCA-salt hypertension and cardiovascular remodeling by inhibiting the reactive oxygen species/mitogen-activated protein kinase pathway via restraining the release of ET-1.

Role of the monocyte chemoattractant protein-1/C-C chemokine receptor 2 signaling pathway in transient receptor potential vanilloid type 1 ablation-induced renal injury in salt-sensitive hypertension.

Our recent studies indicate that the transient receptor potential vanilloid type 1 (TRPV1) channel may act as a potential regulator of monocyte/macrophage recruitment to reduce renal injury in salt-sensitive hypertension. This study tests the hypothesis that deletion of TRPV1 exaggerates salt-sensitive hypertension-induced renal injury due to enhanced inflammatory responses via monocyte chemoattractant protein-1 (MCP-1)/C-C chemokine receptor 2 (CCR2)-dependent pathways. Wild type (WT) and TRPV1-null mutant (TRPV1(-/-)) mice were subjected to uninephrectomy and deoxycorticosterone acetate (DOCA)-salt treatment for four weeks with or without the selective CCR2 antagonist, RS504393. DOCA-salt treatment increased systolic blood pressure (SBP) to the same degree in both strains, but increased urinary excretion of albumin and 8-isoprostane and decreased creatinine clearance with greater magnitude in TRPV1(-/-) mice compared to WT mice. DOCA-salt treatment also caused renal glomerulosclerosis, tubulointerstitial injury, collagen deposition, monocyte/macrophage infiltration, proinflammatory cytokine and chemokine production, and NF-κB activation in greater degree in TRPV1(-/-) mice compared to WT mice. Blockade of the CCR2 with RS504393 (4 mg/kg/day) had no effect on SBP in DOCA-salt-treated WT or TRPV1(-/-) mice compared to their respective controls. However, treatment with RS504393 ameliorated renal dysfunction and morphological damage, and prevented the increase in monocyte/macrophage infiltration, cytokine/chemokine production, and NF-κB activity in both DOCA-salt hypertensive strains with a greater effect in DOCA-salt-treated TRPV1(-/-) mice compared to DOCA-salt-treated WT mice. No differences in CCR2 protein expression in kidney were found between DOCA-salt-treated WT and TRPV1(-/-) mice with or without RS504393 treatment. Our studies for the first time indicate that deletion of TRPV1 aggravated renal injury in salt-sensitive hypertension via enhancing MCP-1/CCR2 signaling-dependent inflammatory responses.

Consumption of a high n-3 polyunsaturated fatty acid diet during gradual mild physiological stress in rats.

n-3 Polyunsaturated fatty acids (n-3PUFAs) may be beneficial for anxiety and depression under stressful conditions. Studies however, typically utilise physical or sudden physiological stress, while gradual physiological stress is also relevant to human conditions. Using deoxycorticosterone acetate (DOCA) administration to induce gradual physiological stress, this study investigated the impact of n-3PUFAs under gradual physiological stress in rats. Animals (aged 2 months) (N=8-12/group) received daily injections of DOCA or vehicle and were concurrently fed a high n-3PUFA or control diet for eight weeks. Behavioural measures were taken throughout. Behavioural tests and physiological measures were conducted after six and eight weeks respectively. DOCA administration decreased plasma renin, plasma proteins and relative adrenal weight, and increased water intake, relative kidney weight, and anxiety in the open field. These findings demonstrate disruptions to the renin-angiotensin-aldosterone system, a result of mild physiological stress, that also impact on anxiety behaviours. No effects of n-3PUFAs were found.

HDAC inhibition suppresses cardiac hypertrophy and fibrosis in DOCA-salt hypertensive rats via regulation of HDAC6/HDAC8 enzyme activity.

Background : Inhibition of histone deacetylase (HDAC) was reported to suppress cardiac hypertrophy and fibrosis in various hypertrophic animal models. However, the HDAC expression profile and HDAC enzyme activity have not yet been investigated in DOCA-salt hypertensive rats. Methods : Unilaterally nephrectomized rats were implanted with DOCA strips. DOCA-salt rats then received a control diet with vehicle or valproate. We measured the expression of cardiac hypertrophic markers, class I HDACs, class II HDACs, fibrosis, and HDAC enzyme activity. Results : Here we report that sodium valproate inhibits the cardiac hypertrophy accompanied by fibrosis in the heart of chronic hypertensive rats. We show that expression of GATA6 and HDAC6 is upregulated in DOCA-salt hypertension. In addition, HDAC6 and HDAC8 enzyme activity is attenuated by sodium valproate. Conclusion : These results suggest that a novel HDAC6- and HDAC8-selective inhibitor is needed to treat or prevent pathological cardiac hypertrophy. © 2013 S. Karger AG, Basel.