It is well accepted that high dietary salt intake accelerates both hypertension and target organ damage. We have previously shown that eplerenone attenuates sustained elevated systolic blood pressure in Dahl salt-sensitive (SS) rats. In the present study, we investigated the role of eplerenone on vascular endothelial growth factor (VEGF) expression because we suspected that eplerenone treatment may trigger a unique mechanism that relies on the downregulation of VEGF.
Dahl SS rats were fed a high salt (8% NaCl) diet for 3 weeks and then switched to normal salt (0.3% NaCl) diet with or without treatment with eplerenone (100 mg/kg/day), enalapril (30 mg/kg/day) and their combination for an additional 3 weeks.
In addition to reducing blood pressure, eplerenone inhibited glomeruli sclerosis and suppressed the expression of VEGF and endothelial nitric oxide synthase mRNA as well as protein levels.
Based on these findings, we suggest that in part, VEGF stimulation of endothelial nitric oxide synthase plays a significant role in the eplerenone-induced reversal of the renal and vascular damage caused by high dietary salt intake.
Mineralcorticoid receptor antagonist; Hypertension; Nitric oxide synthase; Dahl rat
Salt-induced hypertension in the Dahl rat is associated with increases in angiotensin II, aldosterone, free radical generation and endothelial dysfunction. However, little is known about the specific mechanism(s) associated with the end-organ damage effects of aldosterone. We hypothesised that eplerenone reduces kidney damage by blocking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity.
Dahl salt-sensitive rats fed either a low-salt (LS) or high-salt (HS) diet were treated with aldosterone in the presence of eplerenone or apocynin. Indirect blood pressure was measured prior to start of diet and weekly thereafter. Levels of plasma nitric oxide (NO) and urinary 8-isoprostane were measured following treatment. Protein levels of selected subunits of NADPH were assessed by western blot.
Eplerenone and apocynin inhibited the rise in blood pressure induced by HS and/or aldosterone. This observation was accompanied with a parallel change in kidney protein levels of NADPH oxidase 4 (NOX-4) and p22phox. Aldosterone and high salt were associated with lower NO levels and greater renal oxidative stress.
NADPH oxidase is associated with the vascular and renal remodelling observed in high dietary salt intake. Aldosterone-induced expression of NOX-4 plays a pivotal role in the end-organ damage effect of aldosterone, as eplerenone tended to reduce kidney damage and inhibit NOX expression.
Aldosterone; eplerenone; hypertension; NADPH oxidase inhibitor
Hypertension is a leading contributor to cardiovascular mortality worldwide. Despite this, its underlying mechanism(s) and the role of excess salt in cardiorenal dysfunction are unclear. Previously, we have identified cross-talk between mineralocorticoid receptor (MR), a nuclear transcription factor regulated by the steroid aldosterone, and the small GTPase Rac1, which is implicated in proteinuric kidney disease. We here show that high-salt loading activates Rac1 in the kidneys in rodent models of salt-sensitive hypertension, leading to blood pressure elevation and renal injury via an MR-dependent pathway. We found that a high-salt diet caused renal Rac1 upregulation in salt-sensitive Dahl (Dahl-S) rats and downregulation in salt-insensitive Dahl (Dahl-R) rats. Despite a reduction of serum aldosterone levels, salt-loaded Dahl-S rats showed increased MR signaling in the kidneys, and Rac1 inhibition prevented hypertension and renal damage with MR repression. We further demonstrated in aldosterone-infused rats as well as adrenalectomized Dahl-S rats with aldosterone supplementation that salt-induced Rac1 and aldosterone acted interdependently to cause MR overactivity and hypertension. Finally, we confirmed the key role of Rac1 in modulating salt susceptibility in mice lacking Rho GDP–dissociation inhibitor α. Therefore, our data identify Rac1 as a determinant of salt sensitivity and provide insights into the mechanism of salt-induced hypertension and kidney injury.
Mineralocorticoid receptor (MR) antagonists attenuate renal injury in salt-sensitive hypertensive rats with low plasma aldosterone levels. We hypothesized that oxidative stress causes MR activation in high-salt-fed Dahl salt-sensitive rats. Furthermore, we determined if MR activation persisted and induced renal injury, even after switching from a high- to a normal-salt diet.
Methods and Findings
High-salt feeding for 4 weeks increased dihydroethidium fluorescence (DHE, an oxidant production marker), p22phox (a NADPH oxidase subunit) and serum and glucocorticoid-regulated kinase-1 (SGK1, an MR transcript) in glomeruli, compared with normal-salt feeding, and these changes persisted 4 weeks after salt withdrawal. Tempol treatment (0.5 mmol/L) during high-salt feeding abolished the changes in DHE fluorescence, p22phox and SGK1. Dietary salt reduction after a 4-week high-salt diet decreased both blood pressure and proteinuria, but was associated with significantly higher proteinuria than in normal control rats at 4 weeks after salt reduction. Administration of tempol during high-salt feeding, or eplerenone, an MR antagonist (100 mg/kg/day), started after salt reduction, recovered proteinuria to normal levels at 4 weeks after salt reduction. Paraquat, a reactive oxygen species generator, enhanced MR transcriptional activity in cultured rat mesangial cells and mouse podocytes.
These results suggest that oxidative stress plays an important role in glomerular MR activation in Dahl salt-sensitive rats. Persistent MR activation even after reducing salt intake could limit the beneficial effects of salt restriction.
1. The mechanisms by which excessive salt causes hypertension involve more than retention of sodium and water by the kidneys and are far from clear. Mineralocorticoids act centrally to increase salt appetite, sympathetic drive and vasopressin release, resulting in hypertension that is prevented by the central infusion of mineralocorticoid receptor (MR) antagonists. The MR has similar affinity for aldo and the glucocorticoids corticosterone or cortisol. Specificity is conferred in transport epithelia by the co-localization of the MR with 11β-hydroxysteroid dehydrogenase type 2. Co-expression also occurs in some neurons, notably those of the nucleus tractus solitarius that are activated by sodium depletion and aldo and mediate salt seeking behavior.
2. The salt-induced hypertension of the Dahl Salt Sensitive rat is mitigated by the central infusion of a mineralocorticoid antagonist even though circulating aldo is normal or reduced in SS. Contrary to reports that salt appetite in the SS rat is depressed, we found that it is increased compared to the Spraque-Dawley (SD).
3. Extra-adrenal aldo synthesis in the brain occurs in minute amounts that could only be relevant locally. mRNA for aldo synthase enzyme and aldo concentration in the brain of the Dahl SS are increased compared to SD rats. The central infusion of inhibitors of aldo synthesis lowers salt-induced hypertension in the SS, suggesting a role for excessive aldo synthesis in the brain. Brain MR, particularly those in the PVN, regulate inflammatory processes that are exacerbated by sodium and lead to cardiovascular dysfunction.
Elevated C-reactive protein (CRP) may contribute to elevated arterial pressure in Ang II-dependent hypertension. However, the in vivo effects of Ang II and of mineralocorticoid receptor (MR) antagonism on CRP during Ang II-dependent hypertension have not been examined. In addition, urinary CRP excretion as a method to monitor the progression of Ang II-induced inflammation has not been evaluated.
Urine samples were collected from three groups (n = 10/group) of rats: 1) normotensive control, 2) angiotensin II infused (Ang II; 60 ng/min), and 3) Ang II + eplerenone (epl; 25 mg/d). A diet containing epl (0.1 %) was provided after 1 week of Ang II infusion.
After 28 d, Ang II increased SBP from 136 ± 5 to 207 ± 8 mmHg; this response in SBP was not altered following MR antagonism (215 ± 6 mmHg). Ang II-infusion increased plasma CRP from 14 ± 2 to 26 ± 3 μg/mL and increased urinary CRP excretion nearly 8-fold (143 ± 26 vs 1102 ± 115 ng/d). Treatment with eplerenone reduced plasma CRP by 25 % and urinary immunoreactive CRP (irCRP) by 34 % in Ang II-infused rats suggesting that aldosterone contributes to the CRP-associated inflammatory response in Ang II-dependent hypertension.
The increase in SBP preceded the increase in irCRP excretion by at least 4 days suggesting that CRP does not significantly contribute to increased arterial blood pressure in Ang II-dependent hypertension. The blockade of MR reduced plasma CRP and urinary irCRP excretion demonstrating the contribution of aldosterone to the Ang II-induced generation of CRP. Furthermore, urinary CRP may serve as a non-invasive index for monitoring cardiovascular inflammation during hypertension.
aldosterone; eplerenone; inflammation; mineralocorticoids; spironolactone
The enzymes required for aldosterone synthesis from cholesterol are expressed in rat and human brains. The hypertension of Dahl salt-sensitive (SS) rats is mitigated by the intracerebroventricular (i.c.v.) infusion of antagonists of the mineralocorticoid receptor (MR) and downstream effectors of mineralocorticoid action, as well as ablations of brain areas that also abrogate mineralocorticoid–salt excess hypertension in normotensive rats. We used real time RT-PCR to measure mRNA of aldosterone synthase and 11β-hydroxylase, the requisite enzymes for the last step in the synthesis of aldosterone and corticosterone, respectively, MR and the determinants of MR ligand specificity, 11β-hydroxysteroid dehydrogenase types 1 and 2 (11β-HSD1&2) and hexose-6-phosphate dehydrogenase (H6PDH). A combination of extraction and ELISA was used to measure aldosterone concentrations in tissue and urine of SS and Sprague–Dawley (SD) rats. Aldosterone synthase mRNA expression was higher in the brains and lower in the adrenal glands of SS compared with SD rats. The amounts of mRNA for MR, 11β-hydroxylase, 11β-HSD1&2 and H6PD were similar. Aldosterone concentrations were greater in brains of SS than SD rats, yet, in keeping with the literature, the circulating and total aldosterone production of aldosterone in SS rats were not. The selective inhibitor of aldosterone synthase, FAD286, was infused i.c.v. or subcutaneously in a cross-over blood pressure study in hypertensive SS rats further challenged by a high-salt diet. The i.c.v. infusion of FAD286, at a dose that had no effect systemically, significantly and reversibly lowered blood pressure in SS rats. Aldosterone synthesis in brains of SS rats is greater than in SD rats and is important in the genesis of their salt-sensitive hypertension.
Salt sensitivity of blood pressure (BP) is speculated to be a characteristic in obesity-induced hypertension. To elucidate the influence of obesity on salt-sensitive hypertension, we examined the effect of fat loading on BP, renal damage, and their progression induced by salt excess in Dahl salt-sensitive (S) rats. High fat (HF: 45% fat diet: 8 weeks) diet increased BP with greater weight gain and visceral fat accumulation than low fat (10% fat) diet. In HF-fed rats, plasma glucose, plasma insulin, and urinary catecholamine increased, and urinary protein tended to be elevated. Moreover, excessive salt (8% salt diet: 8 weeks)-induced hypertension and proteinuria was accelerated in HF-fed rats. Therefore, fat loading increased BP in Dahl S rats possibly through insulin-resistance and sympathetic excitation. Moreover, fat loading accelerated salt-induced BP elevation and renal damage, suggesting excessive intake of both fat and salt, such as a civilized diet, exert the synergic harmful effects.
obesity; salt sensitivity of blood pressure; urinary protein; insulin resistance; sympathetic nervous system
Studies suggest that the inflammatory cytokine, TNF-α plays a role in the prognosis of end-stage renal diseases. We have previously shown that TNF-α inhibition slowed the progression of hypertension and renal damage in angiotensin II salt-sensitive hypertension. Thus, we hypothesize that TNF-α contributes to renal inflammation in a model of mineralocorticoid-induced hypertension. Four groups of rats (n=5-6) were studied for 3 weeks with the following treatments 1) placebo, 2) placebo + TNF-α inhibitor, etanercept (1.25 mg/kg/day, sc), 3) deoxycorticosterone acetate plus 0.9 % NaCl to drink (DOCA-salt), or 4) DOCA-salt + etanercept. Mean arterial blood pressure (MAP) measured by telemetry increased in DOCA-salt rats compared to baseline (177±4 vs. 107±3 mmHg, P<0.05) and TNF-α inhibition had no effect in the elevation of MAP in these rats (177±8 mmHg). Urinary protein excretion significantly increased in DOCA-salt rats compared to placebo (703±76 vs. 198±5 mg/day, respectively); etanercept lowered the proteinuria (514±64 mg/day, P < 0.05 vs. DOCA-salt alone). Urinary albumin excretion followed a similar pattern in each group. Urinary MCP-1 and ET-1 excretion were also increased in DOCA-salt rats compared to placebo (MCP-1: 939±104 vs. 43±7 ng/day, and ET-1: 3.30±0.29 vs. 1.07±0.03 fmol/day, respectively, both P<0.05); TNF-α inhibition significantly decreased both MCP-1 and ET-1 excretion (409±138 ng/day and 2.42±0.22 fmol/day, respectively, both P < 0.05 vs. DOCA-salt alone). Renal cortical NFκB activity also increased in DOCA-salt hypertensive rats and etanercept treatment significantly reduced this effect. These data support the hypothesis that TNF-α contributes to the increase in renal inflammation in DOCA-salt rats.
salt; DOCA; renal inflammation; blood pressure; TNF-α; etanercept; NFκB
Salt-sensitive hypertension is common in the aged population. Increased fruit and vegetable intake reduces hypertension, but its effect on eventual diastolic dysfunction is unknown. This relationship is tested in the Dahl Salt-Sensitive (Dahl-SS) rat model of salt-sensitive hypertension and diastolic dysfunction. Table grape powder contains phytochemicals that are relevant to human diets. For 18 weeks, male Dahl-SS rats were fed one of five diets: low salt (LS), a low salt + grape powder (LSG), high salt (HS), a high salt grape powder (HSG), or high salt vasodilator hydralazine (HSH). Compared to the HS diet,the HSG diet lowered blood pressure and improved cardiac function; reduced systemic inflammation; reduced cardiac hypertrophy, fibrosis, and oxidative damage; and increased cardiac glutathione. The HSH diet similarly reduced blood pressure but did not reduce cardiac pathogenesis. The LSG diet reduced cardiac oxidative damage and increased cardiac glutathione. In conclusion, physiologically relevant phytochemical intake reduced salt-sensitive hypertension and diastolic dysfunction.
Heart failure; Diet; Fruits; Vegetables
The mineralocorticoid receptor has been implicated in the pathogenesis of chronic cardiorenal disease. Statins improve renal remodeling and dysfunction in patients with proteinuric kidney diseases. We aimed to clarify the beneficial effects and mechanisms of action of statins in renal insufficiency.
Methods and results
Dahl salt-sensitive rats fed a high-salt diet were treated from 12 to 20 weeks of age with vehicle, the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin, the synthetic cathepsin inhibitor E64d, or a low or high dosage of pitavastatin (1 or 3 mg/kg daily). Rats fed a low-salt diet served as controls. Rats on the high-salt diet developed massive proteinuria and glomerulosclerosis; these changes were attenuated by both doses of pitavastatin. The amounts of mRNAs or proteins for mineralocorticoid receptor, angiotensin-converting enzyme, angiotensin II type 1 receptor (AT1R), monocyte chemoattractant protein-1, osteopontin, macrophage infiltration, and NADPH subunits (gp91phox, p22phox, and Rac1) were significantly higher in the failing kidneys of vehicle-treated rats than in the kidneys of control rats. Either dose of pitavastatin significantly attenuated these changes. These effects of pitavastatin were mimicked by those of apocynin and E64d. Pretreatment with pitavastatin and apocynin inhibited mRNA and protein of mineralocorticoid receptor induced by angiotensin II in cultured podocytes.
The beneficial effects of pitavastatin are likely attributable, at least in part, to attenuation of the mineralocorticoid receptor-dependent inflammatory mediator, matrix protein, and cathepsin expressions induced by AT1R-mediated NADPH oxidase activation in the kidneys of a salt-induced hypertensive Dahl salt-sensitive rat model.
hypertension; mineralocorticoid receptor; oxidative stress; renal insufficiency; salt; statin
Insulin resistance has been extensively investigated during the past decade because of its proposed role in initiating a cluster of cardiovascular risk factors including hypertension. Insulin resistance is an inherited genetic trait that precedes hypertension in Dahl salt-sensitive (S) rats, and is not present in Dahl salt-resistant (R) rats. Owing to the co-existence of insulin resistance and salt sensitivity of blood pressure in Dahl S, but not R rats, Dahl S rats are used to elucidate the role of dietary salt as a potential link in exacerbating both phenotypes (insulin resistance and salt sensitivity). In light of available data, examining the impact of dietary salt on insulin resistance in Dahl S rats in terms of salt concentration and duration of exposure helps answer the following question: What percentage of dietary salt and for what duration of exposure would we expect an enhanced insulin resistance in Dahl S rats? This commentary gathers all available research done on insulin resistance in Dahl S rats in an attempt to unravel dietary salt contribution to insulin resistance in Dahl S rats.
One major precursor of carbonyl stress, methylglyoxal (MG), is elevated in the plasma of chronic kidney disease (CKD) patients, and this precursor contributes to the progression of vascular injury, hypertension and renal injury in diabetic nephropathy patients. This molecule induces salt-sensitive hypertension via a reactive oxygen species-mediated pathway. We examined the role of MG in the pathogenesis of hypertension and cardio–renal injury in Dahl salt-sensitive (Dahl S) rats, which is a rat model of CKD. Nine-week-old Dahl S rats were fed a 1% NaCl diet, and 1% MG was added to their drinking water for up to 12 weeks. Blood pressure and cardio–renal injuries were compared with rats treated with tap water alone. The angiotensin II receptor blocker (ARB), candesartan (10 mg kg−1 day−1), was administered to MG Dahl S rats to determine the impact of this drug on the pathogenesis of MG-induced CKD. A progressive increase in systolic blood pressure was observed (123±1–148±5 mm Hg) after 12 weeks of MG administration. MG administration significantly increased urinary albumin excretion, glomerular sclerosis, tubular injury, myocardial collagen content and cardiac perivascular fibrosis. MG also enhanced the renal expression of Nɛ-carboxyethyl-lysine (an advanced glycation end product), 8-hydroxydeoxyguanosine (a marker of oxidative stress), macrophage (ED-1) positive cells (a marker of inflammation) and nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase activity. Candesartan treatment for 4 weeks significantly reduced these parameters. These results suggest that MG-induced hypertension and cardio–renal injury and increased inflammation and carbonyl and oxidative stress, which were partially preventable by an ARB.
carbonyl stress; chronic kidney disease; methylglyoxal; salt sensitivity
In humans, salt intake has been suggested to influence blood pressure (BP) on a wide range of time scales ranging from several hours or days to many months or years. Detailed time course data collected in the Dahl salt-sensitive rat strain suggest that the development of salt-induced hypertension may consist of several distinct phases or components that differ in their timing and reversibility. To better understand these components, the present study sought to model the dynamics of salt-induced hypertension in the Dahl salt sensitive (Dahl-S) rat using 3 sets of time course data.
The first component of the model ("Acute-Reversible") consisted of a linear transfer function to account for the rapid and reversible effects of salt on BP (ie. acute salt sensitivity, corresponding with a depressed slope of the chronic pressure natriuresis relationship). For the second component ("Progressive-Irreversible"), an integrator function was used to represent the relatively slow, progressive, and irreversible effect of high salt intake on BP (corresponding with a progressive salt-induced shift of the chronic pressure natriuresis relationship to higher BP levels). A third component ("Progressive-Reversible") consisted of an effect of high salt intake to progressively increase the acute salt-sensitivity of BP (ie. reduce the slope of the chronic pressure natriuresis relationship), amounting to a slow and progressive, yet reversible, component of salt-induced hypertension. While the 3 component model was limited in its ability to follow the BP response to rapid and/or brief transitions in salt intake, it was able to accurately follow the slower steady state components of salt-induced BP changes. This model exhibited low values of mean absolute error (1.92 ± 0.23, 2.13 ± 0.37, 2.03 ± 0.3 mmHg for data sets 1 - 3), and its overall performance was significantly improved over that of an initial model having only 2 components. The 3 component model performed well when applied to data from hybrids of Dahl salt sensitive and Dahl salt resistant rats in which salt sensitivity varied greatly in its extent and character (mean absolute error = 1.11 ± 0.08 mmHg).
Our results suggest that the slow process of development of salt-induced hypertension in Dahl-S rats over a period of many weeks can be well represented by a combination of three components that differ in their timing, reversibility, and their associated effect on the chronic pressure natriuresis relationship. These components are important to distinguish since each may represent a unique set of underlying mechanisms of salt-induced hypertension.
The upregulation of cyclooxygenase (COX) expression by aldosterone (ALDO) or high salt diet intake is very interesting and complex in the light of what is known about the role of COX in renal function. Thus, in this study, we hypothesize that apocynin (APC) and/or eplerenone (EPL) inhibit ALDO/salt-induced kidney damage by preventing the production of prostaglandin E2 (PGE2).
Dahl salt-sensitive rats on either a low-salt or high-salt diet were treated with ALDO (0.2 mg pellet) in the presence of EPL (100 mg/kg/day) or APC (1.5 mM). Indirect blood pressure, prostaglandins and ALDO levels and histological changes were measured.
Cyclooxygenase-2 (COX-2) levels were upregulated in the renal tubules and peritubular vessels after high-salt intake, and APC attenuated renal tubular COX-2 protein expression induced by ALDO. Plasma PGE2 levels were significantly reduced by ALDO in the rats fed a low-salt diet when compared to rats fed a high-salt diet. PGE2 was blocked by EPL but increased in the presence of APC.
The beneficial effects of EPL may be associated with an inhibition of PGE2. The mechanism underlying the protective effects of EPL is clearly distinct from that of APC and suggests that these agents can have differential roles in cardiovascular disease.
Aldosterone; mineralcorticoid receptor blocker; NADPH oxidase inhibitor; hypertension; cyclooxgenase
Postmenopausal women (PMW) are at greater risk for salt-sensitive hypertension and insulin resistance than premenopausal women. Peroxisome proliferator activated receptor-gamma (PPARγ) agonists reduce blood pressure (BP) and insulin resistance in humans. As in PMW, ovariectomy (OVX) increases salt sensitivity of BP and body weight in Dahl salt sensitive (DS) rats. This study addressed whether rosiglitazone (ROSI), a PPARγ agonist, attenuates salt-sensitive hypertension in intact (INT) and OVX DS rats, and if so, whether insulin resistance, nitric oxide (NO), oxidative stress, and/or renal inflammation were contributing mediators. Telemetric BP was similar in OVX and INT on low salt diet (0.3% NaCl), but was higher in OVX than INT on high salt (8% NaCl). ROSI reduced BP in OVX and INT on both low and high salt diet, but only attenuated salt sensitivity of BP in OVX. Nitrate/nitrite excretion (NOx; index of NO) was similar in INT and OVX on low salt diet, and ROSI increased NOx in both groups. High salt diet increased NOx in all groups but ROSI only increased NOx in OVX rats. OVX females exhibited insulin resistance, increases in body weight, plasma leptin, cholesterol, numbers of renal cortical macrophages, and renal MCP-1 and osteopontin mRNA expression compared to INT. ROSI reduced cholesterol and macrophage infiltration in OVX, but not INT. In summary, PPAR-gamma activation reduces BP in INT and OVX females, but attenuates the salt sensitivity of BP in OVX only likely due to increases in NO and in part to reductions in renal resident macrophages and inflammation.
ovariectomy; menopause; hypertension; inflammation; oxidative stress
Accumulating evidence supports the role of the mineralocorticoid receptor (MR) in the pathogenesis of diabetic nephropathy. These findings have generated renewed interest in novel MR antagonists with improved selectivity against other nuclear hormone receptors and a potentially reduced risk of hyperkalemia. Characterization of novel MR antagonists warrants establishing translatable biomarkers of activity at the MR receptor. We assessed the translatability of urinary sodium to potassium ratio (Na+/K+) and plasma aldosterone as biomarkers of MR antagonism using eplerenone (Inspra®), a commercially available MR antagonist. Further we utilized these biomarkers to demonstrate antagonism of MR by PF-03882845, a novel compound.
The effect of eplerenone and PF-03882845 on urinary Na+/K+ and plasma aldosterone were characterized in Sprague-Dawley rats and spontaneously hypertensive rats (SHR). Additionally, the effect of eplerenone on these biomarkers was determined in healthy volunteers. Drug exposure-response data were modeled to evaluate the translatability of these biomarkers from rats to humans.
In Sprague-Dawley rats, eplerenone elicited a rapid effect on urinary Na+/K+ yielding an EC50 that was within 5-fold of the functional in vitro IC50. More importantly, the effect of eplerenone on urinary Na+/K+ in healthy volunteers yielded an EC50 that was within 2-fold of the EC50 generated in Sprague-Dawley rats. Similarly, the potency of PF-03882845 in elevating urinary Na+/K+ in Sprague-Dawley rats was within 3-fold of its in vitro functional potency. The effect of MR antagonism on urinary Na+/K+ was not sustained chronically; thus we studied the effect of the compounds on plasma aldosterone following chronic dosing in SHR. Modeling of drug exposure-response data for both eplerenone and PF-03882845 yielded EC50 values that were within 2-fold of that estimated from modeling of drug exposure with changes in urinary sodium and potassium excretion. Importantly, similar unbound concentrations of eplerenone in humans and SHR rats yielded the same magnitude of elevations in aldosterone, indicating a good translatability from rat to human.
Urinary Na+/K+ and plasma aldosterone appear to be translatable biomarkers of MR antagonism following administration of single or multiple doses of compound, respectively.
For clinical study reference EE3-96-02-004, this study was completed in 1996 and falls out scope for disclosure requirements.
Clinical study reference A6141115: http://clinicaltrials.gov, http://NIHclinicaltrails.gov; NCTID: NCT00990223
Mechanisms promoting the transition from hypertensive heart disease (HHD) to heart failure with preserved ejection fraction (HFpEF) are poorly understood. When inappropriate for salt status, mineralocorticoid (deoxycorticosterone acetate, DOCA) excess causes hypertrophy, fibrosis and diastolic dysfunction. As cardiac mineralocorticoid receptors (MR) are protected from mineralocorticoid binding by the absence of 11-ß hydroxysteroid dehydrogenase, salt-mineralocorticoid induced inflammation is postulated to cause oxidative stress and mediate cardiac effects. While previous studies have focused on salt/nephrectomy in accelerating mineralocorticoid induced cardiac effects, we hypothesized that HHD is associated with oxidative stress and sensitizes the heart to mineralocorticoid, accelerating hypertrophy, fibrosis and diastolic dysfunction.
Methods and Results
Cardiac structure and function, oxidative stress and MR-dependent gene transcription were measured in SHAM operated and transverse aortic constriction (TAC; studied two weeks later) mice without and with DOCA administration, all in the setting of normal salt diet. Compared to SHAM mice, SHAM+DOCA mice had mild hypertrophy without fibrosis or diastolic dysfunction. TAC mice displayed compensated HHD with hypertrophy, increased oxidative stress (osteopontin and NOX4 gene expression) and normal systolic function, filling pressures and diastolic stiffness. Compared to TAC mice, TAC+DOCA mice had similar LV systolic pressure and fractional shortening but more hypertrophy, fibrosis and diastolic dysfunction with increased lung weights consistent with HFpEF. There was progressive activation of markers of oxidative stress across the groups but no evidence of classic MR-dependent gene transcription.
Pressure overload hypertrophy sensitizes the heart to mineralocorticoid excess which promotes the transition to HFpEF independent of classic MR-dependent gene transcription.
Deoxycorticosterone acetate (DOCA); Pressure overload hypertrophy; Mineralocorticoid; Stress,oxidative; Heart failure with preserved ejection fraction
This study tests the hypothesis that dysfunction of transient receptor potential vanilloid type 1 (TRPV1) channels occurs and contributes to the decrease in the glomerular filtration rate (GFR) and sodium/water excretion in Dahl salt-sensitive hypertensive rats. Recirculating Krebs-Henseleit buffer added with inulin was perfused at a constant flow in the isolated kidneys of Dahl salt-sensitive (DS) or Dahl salt-resistant (DR) rats fed a high salt (HS) or low salt (LS) diet for three weeks. Perfusion pressures (PP) were pre-adjusted to three levels (~100, ~150, ~190 mmHg) with or without phenylephrine. Capsaicin (Cap), a selective TRPV1 agonist, in the presence or absence of capsazepine (Capz), a selective TRPV1 antagonist, was perfused. Basal GFR, urine flow rate (UFR) and Na+ excretion (UNaV) were significantly lower in DS-HS than in DR-HS, DS-LS and DR-LS rats. Cap caused pressure-dependent decreases in PP and increases in GFR, UFR and UNaV in all groups, with less magnitude of decreases in PP and increases in GFR, UFR and UNaV in DS-HS than in DR-HS, DS-LS and DR-LS rats. Capz fully blocked the effect of Cap on PP, GFR, UFR and UNaV in all groups. Thus, these results show that TRPV1 function is impaired in the kidney of DS rats fed a high salt diet, which may contribute to the decrease in GFR and renal excretory function in DS rats in face of salt challenge.
transient receptor potential vanilloid type 1 channel; Dahl salt-sensitive rats; glomerular filtration rate
Recently, evidence was presented that uninephrectomy induces salt-sensitive hypertension in rats. The increase in blood pressure was abrogated by a mineralocorticoid receptor antagonist but not by an aldosterone synthase inhibitor. Here, we hypothesize that mineralocorticoid receptor activation occurred by an 11beta-hydroxy-glucocorticosteroid, as a consequence of dysregulated 11beta-hydroxysteroid dehydrogenase enzymes. Therefore, 3-week-old Sprague-Dawley rats were either uninephrectomized or sham operated and given a normal (0.4%) or high (8%)-salt diet. At week 8, a telemetric device was implanted, and during week 13 blood pressure continuously measured and urine was collected. The animals were sacrificed thereafter and liver and kidney were harvested. Steroid metabolites were analyzed by GC-MS and mRNA assessed by PCR. Uninephrectomy caused a distinct salt-sensitive hypertension. The increase in blood pressure correlated significantly with a decline in the apparent activity of 11beta-hydroxysteroid dehydrogenase 2 and an increase of 11beta-hydroxysteroid dehydrogenase 1, when urinary corticosterone metabolites were considered. These results were mirrored by the corresponding metabolite ratios assessed in renal and liver tissue. Changes in enzyme activities were in part explained by changes in the mRNA content. In conclusion, mineralocorticoid receptor-dependent salt sensitivity after UNX in rats appears to be mediated by glucocorticoids.
11β-hydroxysteroid; dehydrogenase; hypertension; kidney tubules; salt sensitivity
The Dahl salt-sensitive rat, but not the Dahl salt-resistant rat, develops hypertension and hypovitaminosis D when fed a high salt diet. Since the salt-sensitive rat and salt-resistant rat were bred from the Sprague Dawley rat, the aim of this research was to test the hypothesis that salt-resistant and Sprague Dawley rats would be similar in their vitamin D endocrine system response to high salt intake.
Sprague Dawley, salt-sensitive, and salt-resistant rats were fed high (80 g/kg, 8%) or low (3 g/kg, 3%) salt diets for three weeks. The blood pressure of Sprague Dawley rats increased from baseline to week 3 during both high and low salt intake and the mean blood pressure at week 3 of high salt intake was higher than that at week 3 of low salt intake (P < 0.05). Mean plasma 25-hydroxyvitamin D concentrations (marker of vitamin D status) of Sprague Dawley, salt-sensitive, and salt-resistant rats were similar at week 3 of low salt intake. Mean plasma 25-hydroxyvitamin D concentrations of Sprague Dawley and salt-resistant rats were unaffected by high salt intake, whereas the mean plasma 25-hydroxyvitamin D concentration of salt-sensitive rats at week 3 of high salt intake was only 20% of that at week 3 of low salt intake.
These data indicate that the effect of high salt intake on the vitamin D endocrine system of Sprague Dawley rats at week 3 was similar to that of salt-resistant rats. The salt-sensitive rat, thus, appears to be a more appropriate model than the Sprague Dawley rat for assessing possible effects of salt-sensitivity on vitamin D status of humans.
Extracellular matrix (ECM) remodeling is the hallmark of hypertensive nephropathy. Uncontrolled proteolytic activity due to an imbalance between matrix metalloproteinases and tissue inhibitors of metalloproteinases (MMPs/TIMPs) has been implicated in renovascular fibrosis. We hypothesized that inhibition of MMPs will reduce excess ECM deposition and modulate autophagy to attenuate hypertension. Dahl salt-sensitive (Dahl/SS) and Lewis rats were fed on high salt diet and treated without or with 1.2 mg/kg b.w. of GM6001 (MMP inhibitor) by intraperitoneal injection on alternate days for 4 weeks. Blood pressure (BP), renal cortical blood flow, vascular density, collagen, elastin, and MMPs/TIMPs were measured. GM6001 treatment significantly reduced mean BP in hypertensive Dahl/SS rats. Renal resistive index (RI) was increased in hypertensive Dahl/SS rats and Doppler flowmetry showed reduced cortical perfusion. Barium angiography demonstrated a reduction in terminal branches of renal vasculature. Inhibition of MMPs by GM6001 resulted in a significant improvement in all the parameters including renal function. In hypertensive Dahl/SS rats, protein levels of MMP-9, -2, and -13 were increased including the activity of MMP-9 and -2; TIMP-1 and -2 levels were increased whereas TIMP-3 levels were similar to Lewis controls. Administration of GM6001 reduced the activity of MMPs and increased the levels of TIMP-1, -2, and -3. MMP inhibition reduced type 1 collagen deposition and increased elastin in the intrarenal vessels indicating reduced fibrosis. Autophagy markers were decreased in hypertensive Dahl/SS rats and GM6001 treatment enhanced their levels. We conclude that MMP inhibition (GM6001) reduces adverse renovascular remodeling in hypertension by modulating ECM turnover and stimulating autophagy.
Autophagy; collagen; Dahl salt sensitive; extracellular matrix; fibrosis; vascular density
To examine the role of transient receptor potential vanilloid type 4 (TRPV4) channels in development of salt sensitive hypertension, male Dahl salt-sensitive (DS) and -resistant (DR) rats were fed a low (LS) or high (HS) salt diet for 3 weeks. DS-HS but not DR-HS rats developed hypertension. 4α-phorbol 12, 13-didecanoate (4α-PDD, a selective TRPV4 activator, 2.5 mg/kg iv) decreased mean arterial pressure (MAP) in all groups with the greatest effects in DR-HS and the least in DS-HS rats (p<0.05). Depressor effects of 4α-PDD but not dihydrocapsaicin (DHC, a selective TRPV1 agonist, 30 μg/kg iv) were abolished by ruthenium red (RuR, a TRPV4 antagonist, 3mg/kg iv) in all groups. Blockade of TRPV4 with RuR increased MAP in DR-HS rats only (p<0.05). TRPV4 protein contents were decreased in the renal cortex, medulla, and dorsal root ganglia (DRG) in DS-HS compared to DS-LS rats, but increased in DRG and mesenteric arteries (MA) in DR-HS compared to DR-LS rats (p<0.05). MAP responses to blockade of small- and large-/intermediate-conductance Ca2+-activated K+ channels (Maxiκ channels) with apamin and charybdotoxin, respectively, were examined. Apamin (100 μg/kg) plus charybdotoxin (100 μg/kg) abolished 4α-PDD-induced hypotension in DR-LS, DR-HS, and DS-LS rats only. Thus, HS-induced enhancement of TRPV4 function and expression in sensory neurons and resistant vessels in DR rats may prevent salt-induced hypertension possibly via activation of Maxiκ channels given that blockade of TRPV4 elevates MAP. In contrast, HS-induced suppression of TRPV4 function and expression in sensory neurons and kidneys in DS rats may contribute to increased salt sensitivity.
Dahl salt sensitive hypertension; transient receptor potential (TRP) channels; TRPV4; TRPV1; Ca2+-activated K+ channels
In the setting of high salt intake, aldosterone stimulates fibrosis in the heart, great vessels, and kidney of rats. We used uninephrectomized rats treated with angiotensin II and placed on a high salt diet to exaggerate renal fibrosis. We then tested whether mineralocorticoid receptor blockade by spironolactone or aldosterone synthase inhibition by FAD286 have similar effects on end-organ damage and gene expression. Individually, both drugs prevented the hypertensive response to uninephrectomy and high salt intake but not when angiotensin II was administered. Following 4 weeks of treatment with FAD286, plasma aldosterone was reduced, whereas spironolactone increased aldosterone at 8 weeks of treatment. Angiotensin II and high salt treatment caused albuminuria, azotemia, renovascular hypertrophy, glomerular injury, increased plasminogen activator inhibitor-1 (PAI-1), and osteopontin mRNA expression, as well as tubulointerstitial fibrosis in the kidney. Both drugs prevented these renal effects and attenuated cardiac and aortic medial hypertrophy while reducing osteopontin and transforming growth factor-β mRNA expression in the aorta. The two drugs also reduced cardiac interstitial fibrosis but had no effect on that of the perivascular region. Although spironolactone enhanced angiotensin II and salt-stimulated PAI-1 mRNA expression in aorta and heart, spironolactone and FAD286 prevented renal PAI-1 mRNA protein expression. Our study shows that mineralocorticoid receptor antagonism and aldosterone synthase inhibition similarly decrease hypertrophy and interstitial fibrosis of the kidney and heart caused by angiotensin II and high salt.
aldosterone; mineralocorticoid receptor; plasminogen activator inhibitor-1
The conventional antihypertensive therapies including renin–angiotensin–aldosterone system antagonists (converting enzyme inhibitors, receptor blockers, renin inhibitors, and mineralocorticoid receptor blockers), diuretics, β-blockers, and calcium channel blockers are variably successful in achieving the challenging target blood pressure values in hypertensive patients. Difficult to treat hypertension is still a commonly observed problem world-wide. A number of drugs are considered to be used as novel therapies for hypertension. Renalase supplementation, vasopeptidase inhibitors, endothelin antagonists, and especially aldosterone antagonists (aldosterone synthase inhibitors and novel selective mineralocorticoid receptor blockers) are considered an option in resistant hypertension. In addition, the aldosterone antagonists as well as (pro)renin receptor blockers or AT2 receptor agonists might attenuate end-organ damage. This array of medications has now been complemented by a number of new approaches of non-pharmacological strategies including vaccination, genomic interference, controlled breathing, baroreflex activation, and probably most successfully renal denervation techniques. However, the progress on innovative therapies seems to be slow and the problem of resistant hypertension and proper blood pressure control appears to be still persisting. Therefore the regimens of currently available drugs are being fine-tuned, resulting in the establishment of several novel fixed-dose combinations including triple combinations with the aim to facilitate proper blood pressure control. It remains an exciting question which approach will confer the best blood pressure control and risk reduction in this tricky disease.
Renin–angiotensin–aldosterone system; Endothelin; Controlled breathing; Baroreflex; Renal denervation; Renalase; Fixed combinations