Administration Of Aldosterone Research Articles

735 Role of P38 MAPK in aldosterone-induced glomerular injury in natriuretic peptide receptor-A deficient mice

Background: Aldosterone plays an important role in pathophysiology of renal injury. We already reported that natriuretic peptide receptor guanylyl cyclase-A (GC-A) signaling, with potential anti-aldosterone actions, exerts renoprotective effects in various mouse nephropathies including subtotal nephrectomy, immune-mediated renal injury and diabetic nephropathy. Furthermore, we demonstrated that GC-A knockout (KO) mice with aldosterone and sodium overload exhibited accelerated hypertension with massive proteinuria (∼300-fold urinary albumin excretion from baseline). Aldosterone infusion also increased phosphorylation of ERK and p38 MAP kinase (MAPK) mainly in podocytes of GC-A KO mice, but their role remains elusive. Methods: To explore the role of p38 MAPK in this model, we investigated effects of p38 MAPK inhibition. Two weeks after uninephrectomy, mice were administered with aldosterone (0.2 μg/kg/min) subcutaneously using an osmotic minipump with 6% sodium diet, and with or without hydralazine or a p38 MAPK inhibitor (FR167653, 33 mg/kg/day) in drinking water. We examined BP, albumin excretion, and histological findings. Results: Aldosterone administration increased systolic BP by ∼50 mmHg in GC-A KO mice compared with wild-type mice, and FR treatment modestly but significantly decreased BP. Although hydralazine treatment normalized BP in GC-A KO, urinary albumin excretion did not change. Interestingly, urinary albumin excretion was dramatically decreased by 90% in aldosterone-infused GC-A KO with FR treatment. Glomerular hypertrophy, mesangial expansion, and podocyte injury were markedly ameliorated in these mice with FR treatment. Conclusions: These results suggest that p38 MAPK plays a crucial role connecting aldosterone and GC-A pathways, and could be a potential target against aldosterone-induced glomerular injury.

Expression of osmotic stress protein 94 in murine endolymphatic hydrops model

Conclusion: The up-regulation of osmotic stress protein 94 (OSP94) in the murine endolymphatic hydrops (EH) models suggests that OSP94 might be involved in cellular adaptation in response to ionic and osmotic stress in the murine inner ear. Objectives: The purpose of the present study was to investigate the expression of OSP94 in cochlear tissues of the murine EH models and control animals. Methods: Nine adult BALB/c mice were treated with both intratympanic injection of lipopolysaccharide and intraperitoneal administration of aldosterone to induce EH. Nine mice were used as control animals. The expression level of OSP94 in the EH and control groups was compared using immunohistochemistry and real-time RT-PCR. Results: Immunohistochemical staining of tissues in the EH group showed an up-regulation of OSP94 expression in the cochlea, especially in the stria vascularis and Reissner's membrane. Quantitative real-time PCR analysis also showed that transcription of the OSP94 gene in the cochlea was significantly up-regulated in the EH group.

Aldosterone Blunts Tubuloglomerular Feedback by Activating Macula Densa Mineralocorticoid Receptors

Chronic aldosterone administration increases glomerular filtration rate, whereas inhibition of mineralocorticoid receptors (MRs) markedly attenuates glomerular hyperfiltration and hypertension associated with primary aldosteronism or obesity. However, the mechanisms by which aldosterone alters glomerular filtration rate regulation are poorly understood. In the present study, we hypothesized that aldosterone suppresses tubuloglomerular feedback (TGF) via activation of macula densa MR. First, we observed the expression of MR in macula densa cells isolated by laser capture microdissection and by immunofluorescence in rat kidneys. Second, to investigate the effects of aldosterone on TGF in vitro, we microdissected the juxtaglomerular apparatus from rabbit kidneys and perfused the afferent arteriole and distal tubule simultaneously. Under control conditions, TGF was 2.8±0.2 μm. In the presence of aldosterone (10(-8) mol/L), TGF was reduced by 50%. The effect of aldosterone to attenuate TGF was blocked by the MR antagonist eplerenone (10(-5) mol/L). Third, to investigate the effect of aldosterone on TGF in vivo, we performed micropuncture, and TGF was determined by maximal changes in stop-flow pressure P(sf) when tubular perfusion rate was increased from 0 to 40 nL/min. Aldosterone (10(-7) mol/L) decreased ΔP(sf) from 10.1±1.4 to 7.7±1.2 mm Hg. In the presence of l-NG-monomethyl arginine citrate (10(-3) mol/L), this effect was blocked. We conclude that MRs are expressed in macula densa cells and can be activated by aldosterone, which increases nitric oxide production in the macula densa and blunts the TGF response.

Activation of the aldosterone/mineralocorticoid receptor system and protective effects of mineralocorticoid receptor antagonism in retinal ischemia-reperfusion injury

The purpose of this project was to investigate the effects of the mineralocorticoid receptor antagonist against retinal ischemia-reperfusion injury and identify the aldosterone/mineralocorticoid receptor (MR) system in the rat retina. Retinal ischemia was induced by increasing intraocular pressure to 130 mmHg. Rats were treated with the angiotensin II type 1 receptor (AT1-R) antagonist (candesartan), MR antagonist (spironolactone), or aldosterone. Retinal damage was evaluated at 7 days after the ischemia by measuring the retinal thickness and the number of retinal ganglion cells. Pretreatment with candesartan, spironolactone, or candesartan and spironolactone significantly inhibited retinal ischemic injury. However, there was no protective effect against retinal ischemia-reperfusion injury provided by the combined aldosterone with candesartan treatment. Additionally, pretreatment with aldosterone alone also did not provide any neuroprotective effects against retinal ischemia-reperfusion injury. When rats were treated via local administration of aldosterone in the absence of ischemia, the number of retinal ganglion cells decreased while the retinal thickness remained unchanged. The present findings demonstrated the existence of a local aldosterone/MR system in the retina. Our results also demonstrated that an MR antagonist can attenuate subsequent ischemic damage in the rat retina.

Activation of the Mineralocorticoid Receptor Increases Striatin Levels

Aldosterone (ALDO), a critical regulator of sodium homeostasis, mediates its effects via activation of the mineralocorticoid receptor (MR) through mechanisms that are not entirely clear. Striatin, a membrane associated protein, interacts with estrogen receptors in endothelial cells. We studied the effects of MR activation in vitro and in vivo on striatin levels in vascular tissue. We observed that dietary sodium restriction was associated with increased striatin levels in mouse heart and aorta and that striatin and MR are present in the human endothelial cell line, (EA.hy926), and in mouse aortic endothelial cells (MAEC). Further, we show that MR co-precipitates with striatin in vascular tissue. Incubation of EA.hy926 cells with ALDO (10(-8) mol/l for 5-24 h) increases striatin protein and mRNA expression, an effect that was inhibited by canrenoic acid, an MR antagonist. Consistent with these observations, incubation of MAEC with ALDO increased striatin levels that were likewise blocked by canrenoic acid. To test the in vivo relevance of these findings, we studied two previously described mouse models of increased ALDO levels. Intraperitoneal ALDO administration augmented the abundance of striatin protein in mouse heart. We also observed that in a murine model of chronic ALDO-mediated cardiovascular damage following treatment with N(G)-nitro-L-arginine methyl ester plus angiotensin II an increased abundance of striatin protein in heart and kidney tissue. Our results provide evidence that increased striatin levels is a component of MR activation in the vasculature and suggest that regulation of striatin by ALDO may modulate estrogen's nongenomic effects.

The effect of acute bilateral adrenalectomy on vasopressor responses to catecholamines in dogs

The effect of acute bilateral adrenalectomy on the pressor responses to adrenaline, noradrenaline and isoprenaline was studied in anaesthetized dogs. The responses to all the three catecholamines were reduced by adrenalectomy. Treatment with cortisone, cyclic AMP partially restored the responsiveness. Desocycorticosterone, aldosterone, hydrocortisone, phenylbutazone or infusion of either saline and noradrenaline failed to improve the impaired pressor responses seen in adrenalectomized dogs. Treatment with corticosterone alone. combined administration of aldosterone and hydrocortisone or cortisone followed by cyclic 3',5'-AMP also restored catecholamine responses amost to normal. The pressor responses to catecholmaines in dogs were also reduced by metyrapone-induced cortical insufficiency. Administration of corticosterone, cortisone or cyclic AMP slightly improved these responses; the recovery was not, however, as effective as that noted in the adrenalectomized condition.

Pathophysiological Roles of Aldosterone and Mineralocorticoid Receptor in the Kidney

Aldosterone, a steroid hormone, has traditionally been viewed as a key regulator of fluid and electrolyte homeostasis, as well as blood pressure, through the activation of mineralocorticoid receptor (MR). However, a number of studies performed in the last decade have revealed an important role of aldosterone/MR in the pathogenesis of renal injury. Aldosterone/MR-induced renal tissue injury is associated with increased renal inflammation and oxidative stress, fibrosis, mesangial cell proliferation, and podocyte injury, probably through genomic and non-genomic pathways. However, our preliminary data have indicated that acute administration of aldosterone or a selective MR antagonist, eplerenone, does not change blood pressure, heart rate, or renal blood flow. These data suggest that aldosterone/MR induces renal injury through mechanisms that are independent of acute changes in systemic and renal hemodynamics. In this review, we will briefly summarize the roles of aldosterone/MR in the pathogenesis of renal injury, focusing on the underlying mechanisms that are independent of systemic and renal hemodynamic changes.

Aldosterone and the cardiovascular system: a dangerous association.

Initial studies have focussed on the actions of aldosterone in renal electrolyte handling and, as a consequence, blood pressure control. More recently, attention has primarily been focussed on its actions on the heart and vascular system, where it is locally produced. Aldosterone by binding mineralocorticoid receptors causes oxidative stress, fibrosis and triggers an inflammatory response in the cardiovascular system. All these effects could be underlying the role of aldo-sterone on cardiac and vascular remodelling associated with different pathological situations. At the vascular level, aldo-sterone affects endothelial function because administration of aldosterone to rats impaired endothelium-dependent relaxations. In addition, the administration of mineralocorticoid receptor antagonists ameliorates endothelium-dependent relaxation in models of both hypertension and atherosclerosis, and in patients with heart failure. Several mechanisms can participate in this effect, including production of vasoconstrictor factors and a reduction in nitric oxide levels. This reduction can involve both a decrease in its production as well as an increase in its degradation by reactive oxygen species. Aldosterone can produce oxidative stress by the activation of transcription factors such as the NF-κB system, which can also trigger an inflammatory process through the production of different cytokines. At cardiac level, high levels of aldosterone can also adversely impact heart function by producing cardiac hypertrophy, diastolic dysfunction and electrical remodelling through changes in ionic channels. All these effects can explain the beneficial effect of mineralocorticoid blockade in the cardiovascular system.

Nitric Oxide-mediated Dilation of Arterioles to Intraluminal Administration of Aldosterone

The nature of the rapid action of aldosterone on blood vessels, whether endothelium-dependent dilation or smooth muscle-dependent constriction is predominant, is still in dispute. In this study, we administered aldosterone intraluminally or extraluminally to isolated mesenteric and cerebral arterioles of male Wistar rats. Extraluminal administration of aldosterone (10(-11) or 10(-7) M) elicited a transient vasodilatation. The peak response appeared at approximately 5 minutes. In contrast, intraluminal administration of aldosterone elicited a greater and sustained dilation. When aldosterone (10(-12)-10(-7) M) was administered extraluminally in a cumulative manner, dose-dependent vasodilator responses were elicited, except a reduced dilation was observed to 10(-7) M aldosterone. The dilations were significantly inhibited by spironolactone (10(-7) M), a mineralocorticoid receptor antagonist or Nomega-nitro-l-arginine methyl ester (3 x 10(-4) M), a NO synthesis inhibitor. In endothelium-denuded vessels, extraluminal aldosterone induced a dose-dependent vasoconstrictor response. Scavenging superoxide with Tempol (10(-4) M) sustained the extraluminal aldosterone (10(-11) or 10(-7) M)-induced dilation, whereas inhibition of NO synthesis or removal of the endothelium abolished intraluminal aldosterone-induced dilation. Dilation to 10(-7) M aldosterone was significantly enhanced after inhibition of NAD(P)H-oxidase with apocynin (10(-5) M). Furthermore, in the presence of endothelial dysfunction, induced by chronic inhibition of NO synthesis, intraluminal administration of aldosterone failed to dilate the arterioles. We conclude that in physiological conditions, acute elevation of aldosterone will evoke mainly an endothelium-dependent NO-mediated dilation.

Rapid Aldosterone Signaling and Vascular Reactivity: Relax or Don't Do It

Rapid Aldosterone Signaling and Vascular Reactivity: Relax or Don't Do It

Surface expression of sodium channels and transporters in rat kidney: effects of dietary sodium

The abundance of Na transport proteins in the luminal membrane of the rat kidney was assessed using in situ biotinylation and immunoblotting. When animals were fed an Na-deficient diet for 1 wk, the amounts of epithelial Na channel (ENaC) beta-subunit (beta-ENaC) and gamma-subunit (gamma-ENaC) and Na-Cl cotransporter (NCC) protein in the surface fraction increased relative to controls by 1.9-, 3.5-, and 1.5-fold, respectively. The amounts of the luminal Na/H exchanger (NHE3) and the luminal Na-K-2Cl cotransporter (NKCC2) did not change significantly. The increases in ENaC subunits were mimicked by administration of aldosterone for 1 wk, but the increase in NCC was not. When the animals were fed a high-Na (5% NaCl) diet for 1 wk, the surface expression of beta-ENaC increased by 50%, whereas that of the other membrane proteins did not change, relative to controls. The biochemical parameter most strongly affected by dietary Na was the abundance of the 65-kDa cleaved form of gamma-ENaC at the surface. This increased by 8.5-fold with Na depletion and decreased by 40% with Na loading. The overall 14-fold change reflected regulation of the total abundance of the subunit as well as the fraction of the subunit protein in the cleaved form. We conclude that cleavage of gamma-ENaC and its expression at the apical surface play a major role in the regulation of renal Na reabsorption.

Aldosterone-induced endothelial dysfunction of rat aorta: role of poly(ADP-ribose) activation

The aim of this study was to investigate whether activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) contributes to the development of aldosterone-induced endothelial dysfunction and treatment with the potent PARP inhibitor 1,5-isoquinolinediol (3 mg/kg/day, i.p.) could prevent endothelial dysfunction caused by aldosterone. Infusion of subpressor doses of aldosterone with subcutaneously implanted mini-osmotic pumps (0.05 mg/kg/day) to rats for 21 days induced the development of endothelial dysfunction. In order to evaluate endothelial function, isometric tension studies were performed in response to acetylcholine and sodium nitroprusside.Additionally, PAR (the end product of activated PARP) and PARP-1 expressions in the endothelium of thoracic aortas were evaluated by immunohistochemistry. There was a significant loss of endothelium-dependent vasodilatation in response to acetylcholine in aldosterone-infused rats. In animals treated with 1,5-isoquinolinediol, the effect of aldosterone on vascular responsiveness was less than the untreated groups. Immunohistochemical studies demonstrated that aldosterone administration increased PAR and PARP-1 expressions in the endothelium of thoracic aortas, whereas PARP inhibition decreased their expressions to control levels. Our results indicate that PARP activation in the vascular system may be a contributory factor to the impaired endothelial function associated with aldosterone administration.

Attenuation of peripheral salt taste responses and local immune function contralateral to gustatory nerve injury: effects of aldosterone

Dietary sodium restriction coupled with axotomy of the rat chorda tympani nerve (CTX) results in selectively attenuated taste responses to sodium salts in the contralateral, intact chorda tympani nerve. Converging evidence indicates that sodium deficiency also diminishes the activated macrophage response to injury on both the sectioned and contralateral, intact sides of the tongue. Because a sodium-restricted diet causes a robust increase in circulating aldosterone, we tested the hypothesis that changes in neurophysiological and immune responses contralateral to the CTX could be mimicked by aldosterone administration instead of the low-sodium diet. Taste responses in rats with CTX and supplemental aldosterone for 4-6 days were similar to rats with CTX and dietary sodium restriction. Responses to sodium salts were as much as 50% lower compared with sham-operated and vehicle-supplemented rats. The group-related functional differences were eliminated with lingual application of amiloride, suggesting that a major transduction pathway affected was through epithelial sodium channels. Consistent with the functional results, few macrophages were observed on either side of the tongue in rats with CTX and aldosterone. In contrast, macrophages were elevated on both sides of the tongue in rats with CTX and the vehicle. These results show that sodium deficiency or administration of aldosterone suppresses the immune response to neural injury, resulting in attenuation of peripheral gustatory function. They also show a potential key link among downstream consequences of sodium imbalance, taste function, and immune activity.

ENaC at the Cutting Edge: Regulation of Epithelial Sodium Channels by Proteases

Epithelial Na+ channels facilitate the transport of Na+ across high resistance epithelia. Proteolytic cleavage has an important role in regulating the activity of these channels by increasing their open probability. Specific proteases have been shown to activate epithelial Na+ channels by cleaving channel subunits at defined sites within their extracellular domains. This minireview addresses the mechanisms by which proteases activate this channel and the question of why proteolysis has evolved as a mechanism of channel activation.

Cardiac L‐type calcium current is increased in a model of hyperaldosteronism in the rat

Accumulating evidence supports the importance of aldosterone as an independent risk factor in the pathophysiology of cardiovascular disease. It has been postulated that aldosterone could contribute to ventricular arrhythmogeneity by modulation of cardiac ionic channels. The aim of this study was to analyse ex vivo the electrophysiological characteristics of the L-type cardiac calcium current (I(CaL)) in a model of hyperaldosteronism in the rat. Aldosterone was administered for 3 weeks, and cardiac collagen deposition and haemodynamic parameters were analysed. In addition, RT-PCR and patch-clamp techniques were applied to study cardiac L-type Ca(2+) channels in isolated cardiomyocytes. Administration of aldosterone induced maladaptive cardiac remodelling that was related to increased collagen deposition, diastolic dysfunction and cardiac hypertrophy. In addition, ventricular myocytes isolated from the aldosterone-treated group showed increased I(CaL) density and conductance and prolongation of the action potential duration. No changes in kinetics or in voltage dependence of activation and inactivation of I(CaL) were observed, but relative expression of Ca(V)1.2 mRNA levels was higher in cardiomyocytes isolated from the aldosterone-treated group. The present study demonstrates that aldosterone treatment induces myocardial fibrosis, cardiac hypertrophy, increase of I(CaL) density, upregulation of L-type Ca(2+) channels and prolongation of action potential duration. It could be proposed that aldosterone, through these mechanisms, might exert pro-arrhythmic effects in the pathological heart.

Dietary salt regulates the phosphorylation of OSR1/SPAK kinases and the sodium chloride cotransporter through aldosterone

Pseudohypoaldosteronism type II (PHAII) is caused by mutations in the WNK1 and WNK4 genes (WNK with-no-lysine kinase). In a mouse model of this disease where a mutant of Wnk4 D561A was knocked in, increased phosphorylation of the sodium chloride cotransporter (NCC) was found and the transporter was concentrated on the apical membrane of the distal tubules. In addition, we recently found that other kinases, such as the oxidative stress response kinase-1/STE20/SPS1-related proline alanine-rich kinase (OSR1/SPAK), also showed increased phosphorylation in these mice. Here we determined whether this kinase cascade is regulated by dietary salt intake. We found that the phosphorylation states of NCC and OSR1/SPAK were increased by low-salt diets and decreased by high-salt diets; a regulation completely lost in the knock-in mice. Increased phosphorylation was reversed by spironolactone and this decreased phosphorylation was reversed by administration of exogenous aldosterone. These studies suggest that that the WNK-OSR1/SPAK-NCC cascade may be a novel effector system of aldosterone action in the kidney.

Consequences of aldosterone administration on cardiac ion currents in rats

Consequences of aldosterone administration on cardiac ion currents in rats

Epithelial Na+channel activation and processing in mice lacking SGK1

Amiloride-sensitive Na(+) channel activity was examined in the cortical collecting ducts of a mouse line (SGK1(-/-)) deficient in the serum- and glucocorticoid-dependent protein kinase SGK1. This activity was correlated with changes in renal Na handling and in the maturation of epithelial Na(+) channel (ENaC) protein. Neither SGK1(-/-) mice nor paired SGK1(+/+) animals expressed detectable channel activity, measured as amiloride-sensitive whole-cell current (I(Na)), under control conditions with standard chow. Administration of aldosterone (0.5 microg/h via osmotic minipump for 7 days) increased I(Na) to a similar extent in SGK1(+/+) (378 +/- 61 pA/cell at -100 mV) and in SGK1(-/-) (350 +/- 57 pA/cell) animals. However, the maturation of ENaC, assessed as the ratio of cleaved to full-length forms of gamma-ENaC, was more pronounced in SGK(+/+) mice. The SGK1(-/-) animals exhibited a salt-wasting phenotype when kept on a low-Na diet for up to 2 days, losing significantly more Na in the urine than wild-type mice. Under these conditions, I(Na) was enhanced more in SGK1(-/-) (94 +/- 14 pA/cell) than in SGK(+/+) (23 +/- 5 pA/cell) genotypes. Despite the larger currents, the ratio of cleaved to full-length gamma-ENaC was lower in the knockout animals. The mice also expressed a smaller amount of Na(+)-Cl(-) cotransporter protein under Na-depleted conditions. These results indicated that SGK1 is essential for optimal processing of ENaC but is not required for activation of the channel by aldosterone.

Increased oxidative stress diminishes NO‐mediated vasodilatation initiated by aldosterone in arterioles

There is increasing evidence that aldosterone has a significant impact on cardiovascular function. In the present study, we investigated direct vasoreactivity to aldosterone in resistance vessels. Mesenteric arterioles were isolated from normal Wistar rats and pressurized at 80 mmHg intravascular pressure in a no‐flow condition. Aldosterone (10−12 to 10−8M) elicited a dose‐dependent vasodilator response, which was significantly inhibited by administration of spironolactone (10−7 M), mineralocorticoid receptor antagonist, removal of the endothelium or inhibition of NO synthesis by Nω‐nitro‐L‐arginine methyl ester (3×10−4M). At a concentration of 10−7M, aldosterone elicited a reduced dilation, which was converted to a vasoconstriction after removal of the endothelium and a greater dilatation after scavenging superoxide with Tempol (10−4M) or inhibition of NAD(P)H‐oxidase with apocynin (10−4M). Intraluminal administration of aldosterone (10−7M) elicited a significantly greater and prolonged vasodilatation than extraluminal administration. These data suggest that the effects of aldosterone on resistance vessels depend on its concentration. In a physiological concentration, aldosterone increases arteriolar diameter via an endothelial NO‐mediated mechanism; however, in pathological concentrations, aldosterone increases oxidative stress, which may reduce NO bioavailability. (Supported by NIH HL‐43023, HL‐68813 and HL‐070653)

Intracerebroventricular injections of adeno‐associated virus‐siRNA gene to silence angiotensin type 1 receptors (AT1) prevent aldosterone‐induced hypertension

Many studies have implicated aldosterone (ALDO) in the progression of renal injury, cardiac remodeling after myocardial infarction, and the development of hypertension. In several cases, these effects of ALDO appear to involve an interaction with the renin‐angiotensin system (RAS). In the present study, we investigated the effect of intracerebroventricular (ICV) injections of recombinant adeno‐associated virus (AAV) carrying small interference (si) RNA silencers of angiotensin type 1 receptors (AT1‐siRNA) or scrambled siRNA (SCM‐siRNA) on ALDO‐induced hypertension in male rats. AAV (2 μ1) was injected ICV at the same time systemic ALDO infusion (750 ng/h) was started. Arterial blood pressure was continuously measured by telemetry. ICV AAV‐AT1‐siRNA injection attenuated the development of ALDO‐induced hypertension (104.3±1.4 to 120.6±1.8 mmHg in SCM‐siRNA, n=6, and 101.1±1.0 to 109.8±0.5 mmHg in AT1‐siRNA, n=5). An immunohistochemical analysis of AT1‐siRNA‐injected rats showed a marked reduction in the expression of AT1 in the subfornical organ and paraventricular nucleus in comparison to SCM‐siRNA rats. These results suggest that the brain RAS is involved in the hypertensinogenic response produced by systemic ALDO administration. (Support: NIH HL–59676 and HL–62261)