Colon-specific deletion of epithelial sodium channel causes sodium loss and aldosterone resistance.

Aldosterone promotes electrogenic sodium reabsorption through the amiloride-sensitive epithelial sodium channel (ENaC). Here, we investigated the importance of ENaC and its positive regulator channel-activating protease 1 (CAP1/Prss8) in colon. Mice lacking the αENaC subunit in colonic superficial cells (Scnn1a(KO)) were viable, without fetal or perinatal lethality. Control mice fed a regular or low-salt diet had a significantly higher amiloride-sensitive rectal potential difference (∆PDamil) than control mice fed a high-salt diet. In Scnn1a(KO) mice, however, this salt restriction-induced increase in ∆PDamil did not occur, and the circadian rhythm of ∆PDamil was blunted. Plasma and urinary sodium and potassium did not change with regular or high-salt diets or potassium loading in control or Scnn1a(KO) mice. However, Scnn1a(KO) mice fed a low-salt diet lost significant amounts of sodium in their feces and exhibited high plasma aldosterone and increased urinary sodium retention. Mice lacking the CAP1/Prss8 in colonic superficial cells (Prss8(KO)) were viable, without fetal or perinatal lethality. Compared with controls, Prss8(KO) mice fed regular or low-salt diets exhibited significantly reduced ∆PDamil in the afternoon, but the circadian rhythm was maintained. Prss8(KO) mice fed a low-salt diet also exhibited sodium loss through feces and higher plasma aldosterone levels. Thus, we identified CAP1/Prss8 as an in vivo regulator of ENaC in colon. We conclude that, under salt restriction, activation of the renin-angiotensin-aldosterone system in the kidney compensated for the absence of ENaC in colonic surface epithelium, leading to colon-specific pseudohypoaldosteronism type 1 with mineralocorticoid resistance without evidence of impaired potassium balance.

Effects of thyromimetic drugs on aldosterone-dependent sodium transport in the toad bladder.

Aldosterone increases transepithelial Na+ transport in the urinary bladder of Bufo marinus. The response is characterized by 3 distinct phases: 1) a lag period of about 60 min, ii) an initial phase (early response) of about 2 hr during which Na+ transport increases rapidly and transepithelial electrical resistance falls, and iii) a late phase (late response) of about 4 to 6 hr during which Na+ transport still increases significantly but with very little change in resistance. Triiodothyronine (T3, 6 nM) added either 2 or 18 hr before aldosterone selectively antagonizes the late response. T3 per se (up to 6 nM) has no effect on base-line Na+ transport. The antagonist activity of T3 is only apparent after a latent period of about 6 to 8 hr. It is not rapidly reversible after a 4-hr washout of the hormone. The effects appear to be selective for thyromimetic drugs since reverse T3 (rT3) is inactive and isopropyldiiodothyronine (isoT2) is more active than T3. The relative activity of these analogs corresponds to their relative affinity for T3 nuclear binding sites which we have previously described. Our data suggest that T3 might control the expression of aldosterone by regulating gene expression, e.g. by the induction of specific proteins, which in turn will inhibit the late mineralocorticoid response, without interaction with the early response.

Testosterone: a specific competitive antagonist of aldosterone in the toad bladder.

Testosterone (100 nM to 40 microM) antagonized the effect of aldosterone (10 nM) on Na+ transport in the toad bladder measured in vitro as short-circuit current (SCC). Half-maximal inhibition occurred at an antagonist-agonist molar ratio of 150:1. The antagonist action of testosterone was reversed by addition of more aldosterone. The antagonism was specific in the sense that testosterone (20 microM) did not inhibit the response of the SCC to oxytocin (50 mU/ml). By itself, testosterone (up to 20 microM) had no agonist activity on base-line SCC. Finally, testosterone (500 nM to 20 microM) specifically displaced [3H]aldosterone (5 nm) from its cytoplasmic and nuclear binding sites in bladders incubated in vitro at 25 or 0 degrees C and labeled at steady state. There was a significant linear correlation between the effect of testosterone on the aldosterone-dependent SCC and its effect on [3H]aldosterone binding sites in the cytoplasm and in the nucleus. We conclude that 1) testosterone is a specific competitive antagonist of aldosterone, and 2) [3H]aldosterone nuclear and cytoplasmic binding sites could be mineralocorticoid receptors, mediating the action of aldosterone on Na+ transport.

The aldosterone-mineralocorticoid receptor pathway exerts anti-inflammatory effects in endotoxin-induced uveitis.

We have previously shown that the eye is a mineralocorticoid-sensitive organ and we now question the role of mineralocorticoid receptor (MR) in ocular inflammation. The endotoxin-induced uveitis (EIU), a rat model of human intraocular inflammation, was induced by systemic administration of lipopolysaccharide (LPS). Evaluations were made 6 and 24 hours after intraocular injection of aldosterone (simultaneous to LPS injection). Three hours after onset of EIU, the MR and the glucocorticoid metabolizing enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11β-HSD2) expression were down-regulated in iris/ciliary body and the corticosterone concentration was increased in aqueous humor, altering the normal MR/glucocorticoid receptor (GR) balance. At 24 hours, the GR expression was also decreased. In EIU, aldosterone reduced the intensity of clinical inflammation in a dose-dependent manner. The clinical benefit of aldosterone was abrogated in the presence of the MR antagonist (RU26752) and only partially with the GR antagonist (RU38486). Aldosterone reduced the release of inflammatory mediators (6 and 24 hours: TNF-α, IFN-γ, MIP-1α) in aqueous humor and the number of activated microglia/macrophages. Aldosterone partly prevented the uveitis-induced MR down-regulation. These results suggest that MR expression and activation in iris/ciliary body could protect the ocular structures against damages induced by EIU.

Effects of thyroid hormones and aldosterone on mineralocorticoid binding sites in the toad bladder.

In the urinary bladder of the toad Bufo marinus triiodothyronine selectively inhibits the late effect of aldosterone on Na+ transport. We have investigated whether T3 might mediate its antimineralocorticoid action by controlling: i) the level of aldosterone binding sites in the soluble (cytosolic) pool isolated from tissues treated with T3 (60 nM) for up to 20 hr of incubation; ii) the kinetics of uptake of 3H-aldosterone into cytoplasmic and nuclear fractions after 2 or 20 hr of exposure to T3. The number and the affinity of Type I (high affinity, low capacity) and Type II (low affinity, high capacity) cytosolic binding sites (measured at 0 degrees C) did not vary significantly after 18 hr of exposure to T3, while aldosterone-dependent Na+ transport was significantly inhibited. In addition, T3 did not modify the kinetics of uptake (90 min) of 3H-aldosterone into cytoplasmic and nuclear fractions of toad bladder incubated in vitro at 25 degrees C. By contrast, aldosterone itself was able to down-regulate its cytosolic and nuclear binding sites after an 18-hr exposure to the steroid hormone (10 or 80 nM). T3 slightly (20%) but significantly potentiated the down regulation of nuclear binding sites. In conclusion, T3 does not appear to have major effects on the regulation of the aldosterone receptor, which could explain in a simple manner its antimineralocorticoid action.

Nedd4-2 modulates renal Na+-Cl- cotransporter via the aldosterone-SGK1-Nedd4-2 pathway.

Regulation of renal Na(+) transport is essential for controlling blood pressure, as well as Na(+) and K(+) homeostasis. Aldosterone stimulates Na(+) reabsorption by the Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) and by the epithelial Na(+) channel (ENaC) in the late DCT, connecting tubule, and collecting duct. Aldosterone increases ENaC expression by inhibiting the channel's ubiquitylation and degradation; aldosterone promotes serum-glucocorticoid-regulated kinase SGK1-mediated phosphorylation of the ubiquitin-protein ligase Nedd4-2 on serine 328, which prevents the Nedd4-2/ENaC interaction. It is important to note that aldosterone increases NCC protein expression by an unknown post-translational mechanism. Here, we present evidence that Nedd4-2 coimmunoprecipitated with NCC and stimulated NCC ubiquitylation at the surface of transfected HEK293 cells. In Xenopus laevis oocytes, coexpression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreased NCC activity and surface expression. SGK1 prevented this inhibition in a kinase-dependent manner. Furthermore, deficiency of Nedd4-2 in the renal tubules of mice and in cultured mDCT(15) cells upregulated NCC. In contrast to ENaC, Nedd4-2-mediated inhibition of NCC did not require the PY-like motif of NCC. Moreover, the mutation of Nedd4-2 at either serine 328 or 222 did not affect SGK1 action, and mutation at both sites enhanced Nedd4-2 activity and abolished SGK1-dependent inhibition. Taken together, these results suggest that aldosterone modulates NCC protein expression via a pathway involving SGK1 and Nedd4-2 and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.

Control of transepithelial Na+ transport and Na-K-ATPase by oxytocin and aldosterone.

Short- and long-term effect of oxytocin on Na+ transport and Na-K-ATPase biosynthesis in the toad bladder, and the potential interaction of this hormone with aldosterone have been studied, leading to the following observations. An early Na+ transport response (oxytocin, 50 mU/ml) peaked at 10-15 min of hormone addition. At maximal stimulation a three- to fourfold increase in Na+ transport was observed, a sustained Na+ transport response (about two-fold control base line) was observed as long as the hormone was present in the medium and for up to 20 h of incubation. Pretreatment for 30 min with actinomycin D (2 micrograms/ml) did not inhibit the early response, but significantly impaired the sustained response, suggesting that de novo protein synthesis was required. The simultaneous addition of the two hormones led within 60 min to a marked potentiation of the action on Na+ transport. This synergism could be mimicked by exogenous cyclic adenosine monophosphate (cAMP). Oxytocin alone (18 h exposure, 50 mU/ml) increased the relative rate of synthesis of both alpha and beta subunits of Na-K-ATPase (1.9- and 1.6-fold, respectively; P less than 0.05), whereas aldosterone (80 nM) increased the relative rate of synthesis of the same subunits (2.6- and 2.2-fold, respectively; P less than 0.02). Finally, in contrast to what was observed at the physiological level, the interaction of oxytocin and aldosterone did not lead to a similar potentiation at the biochemical level, i.e., induction of Na-K-ATPase biosynthesis (2.7- and 2.9-fold, for alpha and beta subunits, respectively; P less than 0.025).

The past, present and future of renin-angiotensin aldosterone system inhibition.

The renin-angiotensin aldosterone system (RAAS) is central to the pathogenesis of cardiovascular disease. RAAS inhibition can reduce blood pressure, prevent target organ damage in hypertension and diabetes, and improve outcomes in patients with heart failure and/or myocardial infarction. This review presents the history of RAAS inhibition including a summary of key heart failure, myocardial infarction, hypertension and atrial fibrillation trials. Recent developments in RAAS inhibition are discussed including implementation and optimization of current drug therapies. Finally, ongoing clinical trials, opportunities for future trials and issues related to the barriers and approvability of novel RAAS inhibitors are highlighted.

Regulation by aldosterone of Na+,K+-ATPase mRNAs, protein synthesis, and sodium transport in cultured kidney cells.

Transepithelial Na+ reabsorption across tight epithelia is regulated by aldosterone. Mineralocorticoids modulate the expression of a number of proteins. Na+,K+-ATPase has been identified as an aldosterone-induced protein (Geering, K., M. Girardet, C. Bron, J. P. Kraehenbuhl, and B. C. Rossier, 1982, J. Biol. Chem., 257:10338-10343). Using A6 cells (kidney of Xenopus laevis) grown on filters we demonstrated by Northern blot analysis that the induction of Na+,K+-ATPase was mainly mediated by a two- to fourfold accumulation of both alpha- and beta-subunit mRNAs. The specific competitor spironolactone decreased basal Na+ transport, Na+,K+-ATPase mRNA, and the relative rate of protein biosynthesis, and it blocked the response to aldosterone. Cycloheximide inhibited the aldosterone-dependent sodium transport but did not significantly affect the cytoplasmic accumulation of Na+,K+-ATPase mRNA induced by aldosterone.

Nedd4-2 modulates renal Na+-Cl- cotransporter via the aldosterone-SGK1-Nedd4-2 pathway.

Regulation of renal Na(+) transport is essential for controlling blood pressure, as well as Na(+) and K(+) homeostasis. Aldosterone stimulates Na(+) reabsorption by the Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) and by the epithelial Na(+) channel (ENaC) in the late DCT, connecting tubule, and collecting duct. Aldosterone increases ENaC expression by inhibiting the channel's ubiquitylation and degradation; aldosterone promotes serum-glucocorticoid-regulated kinase SGK1-mediated phosphorylation of the ubiquitin-protein ligase Nedd4-2 on serine 328, which prevents the Nedd4-2/ENaC interaction. It is important to note that aldosterone increases NCC protein expression by an unknown post-translational mechanism. Here, we present evidence that Nedd4-2 coimmunoprecipitated with NCC and stimulated NCC ubiquitylation at the surface of transfected HEK293 cells. In Xenopus laevis oocytes, coexpression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreased NCC activity and surface expression. SGK1 prevented this inhibition in a kinase-dependent manner. Furthermore, deficiency of Nedd4-2 in the renal tubules of mice and in cultured mDCT(15) cells upregulated NCC. In contrast to ENaC, Nedd4-2-mediated inhibition of NCC did not require the PY-like motif of NCC. Moreover, the mutation of Nedd4-2 at either serine 328 or 222 did not affect SGK1 action, and mutation at both sites enhanced Nedd4-2 activity and abolished SGK1-dependent inhibition. Taken together, these results suggest that aldosterone modulates NCC protein expression via a pathway involving SGK1 and Nedd4-2 and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.