Mineralocorticoid receptor activation: a major contributor to salt-induced renal injury and hypertension in young rats

Excessive salt intake is known to preferentially increase blood pressure (BP) and promote kidney damage in young, salt-sensitive hypertensive human and animal models. We have suggested that mineralocorticoid receptor (MR) activation plays a major role in kidney injury in young rats. BP and urinary protein were compared in young (3-wk-old) and adult (10-wk-old) uninephrectomized (UNx) Sprague-Dawley rats fed a high (8.0%)-salt diet for 4 wk. The effects of the MR blocker eplerenone on BP and renal injury were examined in the high-salt diet-fed young UNx rats. Renal expression of renin-angiotensin-aldosterone (RAA) system components and of inflammatory and oxidative stress markers was also measured. The effects of the angiotensin receptor blocker olmesartan with or without low-dose aldosterone infusion, the aldosterone synthase inhibitor FAD286, and the antioxidant tempol were also studied. Excessive salt intake induced greater hypertension and proteinuria in young rats than in adult rats. The kidneys of young salt-loaded rats showed marked histological injury, overexpression of RAA system components, and an increase in inflammatory and oxidative stress markers. These changes were markedly ameliorated by eplerenone treatment. Olmesartan also ameliorated salt-induced renal injury but failed to do so when combined with low-dose aldosterone infusion. FAD286 and tempol also markedly reduced urinary protein. UNx rats exposed to excessive salt at a young age showed severe hypertension and renal injury, likely primarily due to MR activation and secondarily due to angiotensin receptor activation, which may be mediated by inflammation and oxidative stress.     

Aldosterone and the kidney

The mineralocorticoid aldosterone is a key regulator of blood pressure, fluid and electrolyte homeostasis, and acts via the mineralocorticoid receptor (MR). In recent years, an increasing number of studies revealed deleterious effects of aldosterone via its receptor. Especially in patients with primary hyperaldosteronism (PHA) a significant higher risk of developing cardiovascular comorbidities and comortalities was reported. Also renal insufficiency is clearly increased in patients with PHA indicating a role of aldosterone and the MR in the pathogenesis of renal injury. It has been shown that aldosterone in combination with an elevated salt intake, leads to renal inflammation, fibrosis, podocyte injury, and mesangial cell proliferation. This review focuses on the current knowledge of aldosterone effects in the kidney and highlights this topic from 2 perspectives: from clinical medicine and from experimental studies.     

Mineralocorticoid receptor antagonism protects the aorta from vascular smooth muscle cell proliferation and collagen deposition in a rat model of adrenal aldosterone-producing adenoma

The number of patients with adrenal aldosterone-producing adenomas (APAs) has gradually increased. However, even after adenoma resection, some patients still suffer from high systolic blood pressure (SBP), which is possibly due to great arterial remodeling. Moreover, mineralocorticoid receptors (MRs) were found to be expressed in vascular smooth muscle cells (VSMCs). This study aims to determine whether MR antagonism protects the aorta from aldosterone-induced aortic remolding. Male rats were subcutaneously implanted with an osmotic minipumps and randomly divided into four groups: control; aldosterone (1 μg/h); aldosterone plus a specific MR antagonist, eplerenone (100 mg/kg/day); and aldosterone plus a vasodilator, hydralazine (25 mg/kg/day). After 8 weeks of infusion, aortic smooth muscle cell proliferation and collagen deposition, as well as the MDM2 and TGF-β1 expression levels in the aorta, were examined. Model rats with APAs were successfully constructed. Compared with the control rats, the model rats exhibited (1) marked SBP elevation, (2) no significant alteration in aortic morphology, (3) increased VSMC proliferation and MDM2 expression in the aorta, and (4) enhanced total collagen and collagen III depositions in the aorta, accompanied with up-regulated expression of TGF-β1. These effects were significantly inhibited by co-administration with eplerenone but not with hydralazine. These findings suggested that specific MR antagonism protects the aorta from aldosterone-induced VSMC proliferation and collagen deposition.     

The role of 11β-hydroxysteroid dehydrogenase type 2 in human hypertension

Cortisol and aldosterone have the same in vitro affinity for the mineralocorticoid receptor (MR), although in vivo only aldosterone acts as a physiologic agonist of the MR, despite circulating levels of cortisol in humans and corticosterone in rodents being three orders of magnitude higher than aldosterone levels. In mineralocorticoid target organs the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) inactivates 11-hydroxy steroids, to their inactive keto-forms, thus protecting the nonselective MR from activation by glucocorticoids. The gene is highly expressed in all sodium-transporting epithelia, particularly in the kidney and colon, but also in human placenta and vascular wall. Mutations in the HSD11B2 gene cause a rare monogenic juvenile hypertensive syndrome called apparent mineralocorticoid excess (AME). In AME, compromised 11βHSD2 enzyme activity results in activation of the MR by cortisol, causing sodium retention, hypokalaemia, and salt-dependent hypertension. Whereas mutations or inhibition of 11βHSD2 by licorice have been clearly shown to produce a congenital or acquired syndrome of mineralocorticoid excess, the questions remaining are the extent to which subtle abnormalities in MR/11βHSD2 mechanisms may contribute to essential hypertension. Studies in patients with essential hypertension showed a prolonged half-life of cortisol and an increased ratio of urinary cortisol to cortisone metabolites, suggesting a deficient 11βHSD2 activity. These abnormalities may be genetically determined, as suggested by the association of a microsatellite flanking the HSD11B2 gene with hypertension in black patients with end-stage kidney disease and with salt sensitivity of blood pressure in healthy subjects. These findings indicate that variants of the HSD11B2 gene may contribute to the enhanced blood pressure response to salt and possibly to hypertension in humans.     

Mineralocorticoid receptor blockade improves diastolic function independent of blood pressure reduction in a transgenic model of RAAS overexpression

There is emerging evidence that aldosterone can promote diastolic dysfunction and cardiac fibrosis independent of blood pressure effects, perhaps through increased oxidative stress and inflammation. Accordingly, this investigation was designed to ascertain if mineralocorticoid receptor blockade improves diastolic dysfunction independently of changes in blood pressure through actions on myocardial oxidative stress and fibrosis. We used young transgenic (mRen2)27 [TG(mRen2)27] rats with increases in both tissue ANG II and circulating aldosterone, which manifests age-related increases in hypertension and cardiac dysfunction. Male TG(mRen2)27 and age-matched Sprague-Dawley rats were treated with either a low dose (～1 mg·kg(-1)·day(-1)) or a vasodilatory, conventional dose (～30 mg·kg(-1)·day(-1)) of spironolactone or placebo for 3 wk. TG(mRen2)27 rats displayed increases in systolic blood pressure and plasma aldosterone levels as well as impairments in left ventricular diastolic relaxation without changes in systolic function on cine MRI. TG(mRen2)27 hearts also displayed hypertrophy (left ventricular weight, cardiomyoctye hypertrophy, and septal wall thickness) as well as fibrosis (interstitial and perivascular). There were increases in oxidative stress in TG(mRen2)27 hearts, as evidenced by increases in NADPH oxidase activity and subunits as well as ROS formation. Low-dose spironolactone had no effect on systolic blood pressure but improved diastolic dysfunction comparable to a conventional dose. Both doses of spironolactone caused comparable reductions in ROS/3-nitrotryosine immunostaining and perivascular and interstitial fibrosis. These data support the notion mineralocorticoid receptor blockade improves diastolic dysfunction through improvements in oxidative stress and fibrosis independent of changes in systolic blood pressure.     

Tempol prevents genotoxicity induced by vorinostat: role of oxidative DNA damage

Vorinostat is a member of histone deacetylase inhibitors, which represents a new class of anticancer agents for the treatment of solid and hematological malignancies. Studies have shown that these drugs induce DNA damage in blood lymphocytes, which is proposed to be due to the generation of oxidative lesions. The increase in DNA damage is sometimes associated with risk of developing secondary cancer. Thus, finding a treatment that limits DNA damage caused by anticancer drugs would be beneficial. Tempol is a potent antioxidant that was shown to prevent DNA damage induced by radiation. In this study, we aimed to investigate the harmful effects of vorinostat on DNA damage, and the possible protective effects of tempol against this damage. For that, the spontaneous frequency of sister chromatid exchanges (SCEs), chromosomal aberrations (CAs), and 8-hydroxy-2-deoxy guanosine (8-OHdG) levels were measured in cultured human lymphocytes treated with vorinostat and/or tempol. The results showed that vorinostat significantly increases the frequency of SCEs, CAs and 8-OHdG levels in human lymphocytes as compared to control. These increases were normalized by the treatment of cells with tempol. In conclusion, vorinostat is genotoxic to lymphocytes, and this toxicity is reduced by tempol. Such results could set the stage for future studies investigating the possible usefulness of antioxidants co-treatment in preventing the genotoxicity of vorinostat when used as anticancer in human.     

The epithelial sodium channel (ENaC): Mediator of the aldosterone response in the vascular endothelium?

In the kidney the epithelial sodium channel (ENaC) is regulated by the mineralocorticoid hormone aldosterone, which is essential for long-term blood pressure control. Evidence has accumulated showing that ENaC is expressed in endothelial cells. Moreover, its activity modifies the biomechanical properties of the endothelium. Therefore, the vascular system is also an important target for aldosterone and responds to the hormone with an increase in cell volume, surface area, and mechanical stiffness. These changes occur in a concerted fashion from minutes to hours and can be prevented by the specific sodium channel blocker amiloride and the mineralocorticoid receptor (MR) blocker spironolactone. Aldosterone acts on cells of the vascular system via genomic and non-genomic pathways. There is evidence that the classical cytosolic MR could mediate both types of response. Using a nanosensor covalently linked to aldosterone, binding sites at the plasma membrane were identified by atomic force microscopy. The interaction of aldosterone and this newly identified surface receptor could precede the slow classic genomic aldosterone response resulting in fast activation of endothelial ENaC. Recent data suggest that aldosterone-induced ENaC activation initiates a sequence of cellular events leading to a reduced release of vasodilating nitric oxide. We propose a model in which ENaC is the key mediator of aldosterone-dependent blood pressure control in the vascular endothelium.     

The role of progesterone metabolism and androgen synthesis in renal blood pressure regulation.

11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) plays a crucial role in converting hormonally active cortisol into inactive cortisone, conferring specificity onto the human mineralocorticoid receptor (MR). Progesterone binds with even higher affinity to the MR, but acts as an MR antagonist. How aldosterone is able to keep its function as predominant MR ligand in clinical situations with high progesterone concentrations, such as pregnancy, is not clear. We have shown in vitro that the human kidney possesses an effective enzyme system that metabolizes progesterone to inactive metabolites in a process similar to the inactivation of cortisol by 11beta-HSD2. In studies on patients with adrenal insufficiency, we have shown that the in vivo anti-mineralocorticoid activity of progesterone is diminished by inactivating metabolism of progesterone, local formation of the deoxycorticosterone mineralocorticoid from progesterone, and inhibition of 11beta-HSD2 by progesterone and its metabolites resulting in decreased inactivation of cortisol and hence increased MR binding by cortisol. The enzymes involved in progesterone metabolism are also responsible for the capability of the human kidney to convert pregnenolone to DHEA and androstenedione leading to the formation of active androgens, testosterone and 5alpha-DH-testosterone. Locally produced androgens might be responsible for the observed difference in blood pressure between men and women and higher susceptibility to hypertension in men.     

Role of aldosterone in the initial stage of 2 forms of experimental vasorenal hypertension]

The role of rat adrenal mineralocorticoid function in the development of experimental reno-vascular hypertension was studied. Na-retention action of aldosterone was blocked by means of verospiron. Daily excretion of aldosterone proved to be significantly increased in rats with unilateral stenosis of the renal artery (the contralateral kidney was intact). In ischemia of the only kidney there was seen no change in the activation of adrenal mineralocorticoid function. Administration of verospiron strongly suppressed the development of the first form of reno-vascular hypertension and had no effect on the blood pressure in rats with stenosis of the only kidney.     

Aldosterone increases kidney tubule cell oxidants through calcium-mediated activation of NADPH oxidase and nitric oxide synthase

Chronic hyperaldosteronism has been associated with an increased cancer risk. We recently showed that aldosterone causes an increase in cell oxidants, DNA damage, and NF-κB activation. This study investigated the mechanisms underlying aldosterone-induced increase in cell oxidants in kidney tubule cells. Aldosterone caused an increase in both reactive oxygen and reactive nitrogen (RNS) species. The involvement of the activation of NADPH oxidase in the increase in cellular oxidants was demonstrated by the inhibitory action of the NADPH oxidase inhibitors DPI, apocynin, and VAS2870 and by the migration of the p47 subunit to the membrane. NADPH oxidase activation occurred as a consequence of an increase in cellular calcium levels and was mediated by protein kinase C. The prevention of RNS increase by BAPTA-AM, W-7, and L-NAME indicates a calcium-calmodulin activation of NOS. A similar pattern of effects of the NADPH oxidase and NOS inhibitors was observed for aldosterone-induced DNA damage and NF-κB activation, both central to the pathogenesis of chronic aldosteronism. In summary, this paper demonstrates that aldosterone, via the mineralocorticoid receptor, causes an increase in kidney cell oxidants, DNA damage, and NF-κB activation through a calcium-mediated activation of NADPH oxidase and NOS. Therapies targeting calcium, NOS, and NADPH oxidase could prevent the adverse effects of hyperaldosteronism on kidney function as well as its potential oncogenic action.     

Fetal renal and blood pressure responses to steroid infusion after early prenatal treatment with dexamethasone

Maternal infusion of dexamethasone for 48 h early in gestation results in upregulation of mRNA for mineralocorticoid and glucocorticoid (MR and GR) receptors and angiotensin II receptors in ovine fetal kidneys late in gestation. This study sought to determine whether dexamethasone exposure results in changes in renal function and blood pressure responsiveness to infused cortisol or aldosterone in the late-gestation fetus. Merino ewes carrying single fetuses were infused with isotonic saline (Sal; n = 9) or dexamethasone (Dex, 0.48 mg/h; n = 10) for 48 h between days 26 and 28 of gestation (term = 150 days). At 115-122 days, renal function and blood pressure were measured in fetuses during a 4-h infusion of saline, cortisol (100 microg/h), or aldosterone (5 microg/h). Infusions were given in random order at least 2 days apart. Basal blood pressure and renal function were similar in Sal and Dex groups and did not change over the course of saline infusion. Cortisol infusion caused similar increases in blood pressure, urine flow, and glomerular filtration rate (GFR) in the groups. Aldosterone infusion caused a significantly different GFR response between the groups [P(treatment x time) < 0.05], but increase in K excretion and decrease in Na-to-K ratio were similar in the groups. The similar results obtained with cortisol and aldosterone infusion suggest no increased renal functional maturity to those hormones after early prenatal dexamethasone exposure. This suggests that changes in mRNA for MR and GR in kidneys of dexamethasone-exposed fetuses do not result in functional differences and highlights the renin-angiotensin system, as reported previously, as more important in this model.     

Early superoxide scavenging accelerates renal microvascular rarefaction and damage in the stenotic kidney

Renal artery stenosis (RAS), the main cause of chronic renovascular disease (RVD), is associated with significant oxidative stress. Chronic RVD induces renal injury partly by promoting renal microvascular (MV) damage and blunting MV repair in the stenotic kidney. We tested the hypothesis that superoxide anion plays a pivotal role in MV dysfunction, reduction of MV density, and progression of renal injury in the stenotic kidney. RAS was induced in 14 domestic pigs and observed for 6 wk. Seven RAS pigs were chronically treated with the superoxide dismutase mimetic tempol (RAS+T) to reduce oxidative stress. Single-kidney hemodynamics and function were quantified in vivo using multidetector computer tomography (CT) and renal MV density was quantified ex vivo using micro-CT. Expression of angiogenic, inflammatory, and apoptotic factors was measured in renal tissue, and renal apoptosis and fibrosis were quantified in tissue sections. The degree of RAS and blood pressure were similarly increased in RAS and RAS+T. Renal blood flow (RBF) and glomerular filtration rate (GFR) were reduced in the stenotic kidney (280.1 ± 36.8 and 34.2 ± 3.1 ml/min, P < 0.05 vs. control). RAS+T kidneys showed preserved GFR (58.5 ± 6.3 ml/min, P = not significant vs. control) but a similar decreases in RBF (293.6 ± 85.2 ml/min) and further decreases in MV density compared with RAS. These changes were accompanied by blunted angiogenic signaling and increased apoptosis and fibrosis in the stenotic kidney of RAS+T compared with RAS. The current study shows that tempol administration provided limited protection to the stenotic kidney. Despite preserved GFR, renal perfusion was not improved by tempol, and MV density was further reduced compared with untreated RAS, associated with increased renal apoptosis and fibrosis. These results suggest that a tight balance of the renal redox status is necessary for a normal MV repair response to injury, at least at the early stage of RVD, and raise caution regarding antioxidant strategies in RAS.     

Aldosterone acts centrally to increase brain renin-angiotensin system activity and oxidative stress in normal rats

Aldosterone acts upon mineralocorticoid receptors in the brain to increase blood pressure and sympathetic nerve activity, but the mechanisms are still poorly understood. We hypothesized that aldosterone increases sympathetic nerve activity by upregulating the renin-angiotensin system (RAS) and oxidative stress in the brain, as it does in peripheral tissues. In Sprague-Dawley rats, aldosterone (Aldo) or vehicle (Veh) was infused for 1 wk via an intracerebroventricular (ICV) cannula, while RU-28318 (selective mineralocorticoid receptor antagonist), Tempol (superoxide dismutase mimetic), losartan [angiotensin II type 1 receptor (AT(1)R) antagonist], or Veh was infused simultaneously via a second ICV cannula. After 1 wk of ICV Aldo, plasma norepinephrine was increased and mean arterial pressure was slightly elevated, but heart rate was unchanged. These effects were ameliorated by ICV infusion of RU-28318, Tempol or losartan. Aldo increased expression of AT(1)R and angiotensin-converting enzyme (ACE) mRNA in hypothalamic tissue. RU-28318 minimized and Tempol prevented the increase in AT(1)R mRNA; RU-28318 prevented the increase in ACE mRNA. Losartan had no effect on AT(1)R or ACE mRNA. Immunohistochemistry revealed Aldo-induced increases in dihydroethidium staining (indicating oxidative stress) and Fra-like activity (indicating neuronal excitation) in neurons of the hypothalamic paraventricular nucleus (PVN). RU-28318 prevented the increases in superoxide and Fra-like activity in PVN; Tempol and losartan minimized these effects. Acute ICV infusions of sarthran (AT(1)R antagonist) or Tempol produced greater sympathoinhibition in Aldo-treated than in Veh-treated rats. Thus aldosterone upregulates key elements of brain RAS and induces oxidative stress in the hypothalamus. Aldosterone may increase sympathetic nerve activity by these mechanisms.     

Human mineralocorticoid receptor expression renders cells responsive for nongenotropic aldosterone actions

The steroid hormone aldosterone is important for salt and water homeostasis as well as for pathological tissue modifications in the cardiovascular system and the kidney. The mechanisms of action include a classical genomic pathway, but physiological relevant nongenotropic effects have also been described. Unlike for estrogens or progesterone, the mechanisms for these nongenotropic effects are not well understood, although pharmacological studies suggest a role for the mineralocorticoid receptor (MR). Here we investigated whether the MR contributes to nongenotropic effects. After transfection with human MR, aldosterone induced a rapid and dose-dependent phosphorylation of ERK1/2 and c-Jun NH2-terminal kinase (JNK) 1/2 kinases in Chinese hamster ovary or human embryonic kidney cells, which was reduced by the MR-antagonist spironolactone and involved cSrc kinase as well as the epidermal growth factor receptor. In primary human aortic endothelial cells, similar results were obtained for ERK1/2 and JNK1/2. Inhibition of MAPK kinase (MEK) kinase but not of protein kinase C prevented the rapid action of aldosterone and also reduced aldosterone-induced transactivation, most probably due to impaired nuclear-cytoplasmic shuttling of MR. Cytosolic Ca2+ was increased by aldosterone in mock- and in human MR-transfected cells to the same extend due to Ca2+ influx, whereas dexamethasone had virtually no effect. Spironolactone did not prevent the Ca2+ response. We conclude that some nongenotropic effects of aldosterone are MR dependent and others are MR independent (e.g. Ca2+), indicating a higher degree of complexity of rapid aldosterone signaling. According to this model, we have to distinguish three aldosterone signaling pathways: 1) genomic via MR, 2) nongenotropic via MR, and 3) nongenotropic MR independent.     

Central infusion of aldosterone synthase inhibitor prevents sympathetic hyperactivity and hypertension by central Na+ in Wistar rats

In Wistar rats, increasing cerebrospinal fluid (CSF) Na+ concentration ([Na+]) by intracerebroventricular (ICV) infusion of hypertonic saline causes sympathetic hyperactivity and hypertension that can be prevented by blockade of brain mineralocorticoid receptors (MR). To assess the role of aldosterone produced locally in the brain in the activation of MR in the central nervous system (CNS), Wistar rats were infused ICV with artificial CSF (aCSF), Na+ -rich (800 mmol/l) aCSF, aCSF plus the aldosterone synthase inhibitor FAD286 (100 microg x kg(-1) x day(-1)), or Na+ -rich aCSF plus FAD286. After 2 wk of infusion, rats treated with Na+ -rich aCSF exhibited significant increases in aldosterone and corticosterone content in the hypothalamus but not in the hippocampus, as well as increases in resting blood pressure (BP) and sympathoexcitatory responses to air stress, and impairment of arterial baroreflex function. Concomitant ICV infusion of FAD286 prevented the Na+ -induced increase in hypothalamic aldosterone but not corticosterone and prevented most of the increases in resting BP and sympathoexcitatory and pressor responses to air stress and the baroreflex impairment. FAD286 had no effects in rats infused with ICV aCSF. In another set of rats, 24-h BP and heart rate were recorded via telemetry before and during a 14-day ICV infusion of Na+ -rich aCSF with or without FAD286. Na+ -rich aCSF without FAD286 caused sustained increases ( approximately 10 mmHg) in resting mean arterial pressure that were absent in the rats treated with FAD286. These data suggest that in Wistar rats, an increase in CSF [Na+] may increase the biosynthesis of corticosterone and aldosterone in the hypothalamus, and mainly aldosterone activates MR in the CNS leading to sympathetic hyperactivity and hypertension.