A colonic mineralocorticoid receptor cell model expressing epithelial Na channels

In the distal colon, the epithelial sodium channel (ENaC) is rate limiting for sodium absorption. Progress in the molecular characterization of ENaC expression and trafficking in response to the mineralocorticoid aldosterone has been hampered, since no epithelial colonic cell line existed expressing functional ENaC stimulated by nanomolar aldosterone via mineralocorticoid receptor (MR). Here, we present a human colonic epithelial cell line inducibly expressing the MR (HT-29/B6-Tet-On-MR) which exhibits aldosterone-dependent ENaC-mediated sodium transport in the presence of the short-chain fatty acid butyrate. Butyrate was necessary for high-level expression of MR which allowed for aldosterone-dependent upregulation of β- and γ-ENaC expression. As butyrate alone was not capable of promoting ENaC-mediated sodium transport, aldosterone-induced GILZ (glucocorticoid-induced leucine zipper protein) was identified as a candidate factor increasing apical ENaC levels.     

Juvenile hypertension, the role of genetically altered steroid metabolism.

The importance of hypertension in the pediatric population is not as well appreciated as in adults. This might be related in part to the lower prevalence of high blood pressure in this age group. As with height and weight, blood pressure increases with age during childhood. The underlying causes of significant hypertension in children differ considerably from those in adults: while the prevalence of hypertension in pediatrics is lower than in adults, clinically identifiable causes of hypertension are common. Abnormalities in steroid biosynthesis have been known for years to cause hypertension in some cases of congenital adrenal hyperplasia. In these patients, hypertension usually accompanies a characteristic phenotype with abnormal sexual differentiation. Recently, the molecular basis of four forms of severe hypertension transmitted on an autosomal basis has been elucidated: (a) the glucocorticoid-remediable aldosteronism (GRA), (b) the syndrome of apparent mineralocorticoid excess (AME), (c) activating mutation of the mineralocorticoid receptor and (d) Liddle's syndrome. All these conditions are characterized primarily by low or low-normal plasma renin, normal or low serum potassium and salt-sensitive hypertension, indicating an increased mineralocorticoid effect. These forms of juvenile hypertension are a consequence of abnormal biosynthesis, metabolism or action of steroid hormones: (a) GRA is due to expression of a chimeric gene produced by fusion of 11beta-hydroxylase aldosterone-synthase genes. Expression of the chimeric enzyme occurs in the zona fasciculata of the adrenal cortex under the control of ACTH and can be suppressed by administration of glucocorticoids. (b) AME is caused by mutations of the 11beta-hydroxysteroid dehydrogenase type 2 enzyme, an enzyme that metabolizes cortisol into its receptor inactive keto-form cortisone, thus protecting the mineralocorticoid receptor (MR) from occupation by glucocorticoids. (c) The activating mutation of the MR results in constitutive MR activity and alters receptor specificity, with progesterone and other steroids lacking 21-hydroxyl groups becoming potent agonists. (d) Liddle's syndrome is due to mutations in the beta or gamma chain of the epithelial sodium channel in distal renal tubule cells. The hyperactivity of this channel caused by the mutations results in increased sodium reabsorption. With the advent of molecular biology in clinical practice it has become evident that some genetic defect may present with a more discrete phenotype, with only moderate hypertension with or without hypokalemia as presenting feature. Considering that hypertension in children and adolescents is often 'nonessential', a search for disorders should be integral part of the diagnostic work-up in young patients with hypertension.     

Mineralocorticoid effector mechanism in human mononuclear leukocytes

Mineralocorticoid receptors and mineralocorticoid effector mechanism were determined in mononuclear leukocytes (MNL) from normal subjects. The hierarchy of affinities of competitors for the receptor was similar to that described in other non-classical target tissues for aldosterone. In spite of the relative high affinity of cortisol for the receptor, these binding sites are occupied in vivo by aldosterone and play a mineralocorticoid effect in terms of electrolyte content of the cells. The effect of aldosterone is to prevent the loss of electrolytes due to incubation in medium alone and this action is reversed by addition of actinomycin D. In addition, the incubation of the MNL with aldosterone plus human alpha ANP leads to complete block of the action of aldosterone alone. This effect is not mediated by binding of alpha ANP to mineralocorticoid receptors but is probably related to a some postereceptorial effect of aldosterone at the level of plasma membrane. We conclude that the model of MNL is a good tool for studying mineralocorticoid receptors regulation and consequent effector mechanism in humans.     

Arterial smooth muscle effects of aldosterone and vasopressin: action on ionic fluxes

We have shown previously that aldosterone injected s.c. to adrenalectomized rats has a mineralocorticoid specific action on the transmembrane movements of sodium and potassium from the rat tail artery. These effects appeared to be partly due to an unknown humoral factor. Indeed, the late in vivo effects of aldosterone on 22Na and 86Rb effluxes are suppressed or reduced after in vitro exposure to the hormone. In rats perfused with a specific antagonist of the pressor effect of vasopressin, the in vitro administration of aldosterone induced a kinetic action similar to that observed after in vitro exposure to the mineralocorticoid. Vasopressin exerts a direct action on 22Na and 86Rb effluxes. These effects were correlated in the time with the late in vivo effects of aldosterone. Moreover, vasopressin appears to potentiate the in vitro effects of aldosterone on 22Na and 86Rb effluxes. It is not yet possible to ascertain if this effect is additive or permissive.     

Nongenomic effects of aldosterone: cellular aspects and clinical implications

Nongenomic action of aldosterone has been observed in many cell types which often are different from the classic target tissues for mineralocorticoid action, such as vascular cells. As judged from their time scale and insensitivity toward inhibitors of protein synthesis, effects are not mediated by the classic mineralocorticoid receptor pathway. Here we summarize studies on rapid in vitro aldosterone effects, e.g. ion fluxes, and second messengers involved therein. Furthermore, several clinical studies on in vivo aldosterone action have shown rapid effects on cardiovascular parameters, among them baroreflex and vascular resistance. Taken together with the beneficial effect of aldosterone antagonism in heart failure patients that was demonstrated in the Randomized Aldactone Evaluation Study (RALES), aldosterone may be an equally important factor of the renin-angiotensin-aldosterone system in cardiovascular pathogenesis.     

The mineralocorticoid signaling pathway throughout development: Expression,regulation and pathophysiological implications

The mineralocorticoid signaling pathway has gained interest over the past few years, considering not only its implication in numerous pathologies but also its emerging role in physiological processes during kidney, brain, heart and lung development. This review aims at describing the setting and regulation of aldosterone biosynthesis and the expression of the mineralocorticoid receptor (MR), a nuclear receptor mediating aldosterone action in target tissues, during the perinatal period. Specificities concerning MR expression and regulation during the development of several major organs are highlighted. We provide evidence that MR expression is tightly controlled in a tissue-specific manner during development, which could have major pathophysiological implications in the neonatal period.     

High salt intake down-regulates colonic mineralocorticoid receptors, epithelial sodium channels and 11β-hydroxysteroid dehydrogenase type 2

Besides the kidneys, the gastrointestinal tract is the principal organ responsible for sodium homeostasis. For sodium transport across the cell membranes the epithelial sodium channel (ENaC) is of pivotal relevance. The ENaC is mainly regulated by mineralocorticoid receptor mediated actions. The MR activation by endogenous 11β-hydroxy-glucocorticoids is modulated by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). Here we present evidence for intestinal segment specific 11β-HSD2 expression and hypothesize that a high salt intake and/or uninephrectomy (UNX) affects colonic 11β-HSD2, MR and ENaC expression. The 11β-HSD2 activity was measured by means of 3H-corticosterone conversion into 3H-11-dehydrocorticosterone in Sprague Dawley rats on a normal and high salt diet. The activity increased steadily from the ileum to the distal colon by a factor of about 3, an observation in line with the relevance of the distal colon for sodium handling. High salt intake diminished mRNA and protein of 11β-HSD2 by about 50% (p<0.001) and reduced the expression of the MR (p<0.01). The functionally relevant ENaC-β and ENaC-γ expression, a measure of mineralocorticoid action, diminished by more than 50% by high salt intake (p<0.001). The observed changes were present in rats with and without UNX. Thus, colonic epithelial cells appear to contribute to the protective armamentarium of the mammalian body against salt overload, a mechanism not modulated by UNX.     

Paradoxical differences in the receptor binding of two new antimineralocorticoids

Two synthetic derivatives of spironolactone were used to examine various aspects of the mineralocorticoid receptor structure and function. Introduction of a propyl residue in the 7-position of spironolactone produced a molecule (RU 26752) that saturated the aldosterone specific receptor in the 1-10 nM range, and another, more abundant species in the 10-100 nM range which had little affinity for the natural hormone. The specificity for both sites was increased when the methoxycarbonyl group was introduced in the 7-position (ZK 91587). Neither antagonist exhibited affinity for blood serum transcortin or receptors in non-target organs like the lung and the liver. RU 26752-receptor complex was more unstable than the hormone-receptor complex at 35 degrees C but underwent comparable thermal activation as evidenced by binding to DNA cellulose and the 7 S to 4 S shift on sucrose gradients. In contrast, ZK 91587 did not permit thermal activation and greatly labilized the receptor at 35 degrees C. In ion exchange chromatography, two peaks were observed with unactivated ZK 91587-receptor complex, but RU 26752 was bound exclusively to the component eluted with high salt. Molecular filtration revealed two peaks of bound radioactivity with both antimineralocorticoids. These studies reveal important differences in the mechanism of action of two antagonists differing solely in the residue in position 7 of the spironolactone molecule. Such differences could be exploited to purify the mineralocorticoid receptor and clinically to prescribe the appropriate drug with greater precision.     

Immunofluorescence of mineralocorticoid receptors in peripheral lymphocytes: Presence of receptor-like activity in patients with the autosomal dominant form of pseudohypoaldosteronism, and its absence in the recessive form

Pseudohypoaldosteronism is a syndrome characterized by salt wasting and a failure to thrive due to the resistance towards the action of aldosterone. Aldosterone levels and plasma renin activity are extremely elevated and aldosterone binding sites in peripheral mononuclear leukocytes have regularly shown to be reduced or absent. Sporadic as well as familial cases have been identified and an autosomal dominant as well as an autosomal recessive mode of inheritance has been described. A defect in the aldosterone receptor has been postulated, however, molecular genetic analysis in selected patients has not revealed a mutation in the sequence of the coding region of the cDNA of the mineralocorticoid receptor gene. In the present study we have used a fluorescence-labeled antibody to detect possible receptor expression in monocytes from patients with various clinical forms of pseudohypoaldosteronism. Patients with the sporadic as well as with the autosomal dominant form were clearly immunopositive despite being negative in terms of aldosterone receptor binding. In contrast in two patients with the autosomal recessive form there was no detectable receptor protein, consistent with the results obtained in the aldosterone binding studies. These results suggest that the pathogenesis of pseudohypoaldosteronism is heterogeneous not only regarding the mode of inheritance but also in terms of receptor binding. Thus, in a subgroup of patients the inability of the receptor to bind ligand may be due to a defect involving other, probably cellular factors rather than a deficiency or a defect in the mineralocorticoid receptor system itself.     

Nongenomic actions of aldosterone and progesterone revisited

After almost 30 years of research, the existence of nongenomic steroid actions is no longer disputed. Yet, there is still a debate on the nature of receptors involved, and answers to the inherent questions are important for translational activities. In the case of aldosterone, the existence of receptors different from the classic mineralocorticoid receptors (MR) had been postulated 25 years ago as the pharmacology of about 50% of rapid actions of aldosterone reported so far is incompatible with MR involvement (insensitivity to classic MR antagonists). Candidates proposed as alternatives to MR were protein kinase C, sodium-potassium ATPase or aberrant forms of MR, none of which supported convincing evidence to represent 'the aldosterone membrane receptor'. Early in 2011, data on GPR30 showed its involvement in rapid aldosterone action, and major pharmacological aspects of this action are compatible with the landmark deviations from MR pharmacology mentioned above. GPR30, therefore, may be a receptor candidate for nongenomic aldosterone action. Similarly, a variety of promising candidates mediating rapid progesterone action has been described, including progesterone receptor membrane component 1 (PGRMC1) which seems to be associated with tumor proliferation, and membrane progesterone receptor (mPR) originally identified in fish with potential linkage to reproductive processes. So far, no candidate was unanimously convincing. In 2010, two independent groups reported that CatSper, a calcium channel, is a strong receptor candidate for the rapid action of progesterone on sperm fertilization. With these novel receptors cloned, translational activities ultimately leading to new drugs for cardiovascular protection (in the case of aldosterone) or fertilization benefits (for progesterone) are much more promising.     

Common Polymorphisms in Genes Encoding the Human Mineralocorticoid Receptor and the Human Amiloride-sensitive Sodium Channel

We have examined the human mineralocorticoid receptor gene and the genes encoding the three subunits of the human amiloride-sensitive epithelial sodium channel. Eight new common polymorphisms were identified in these genes which may be useful in genotyping and linkage analysis.     

Sodium pumps,ouabain and aldosterone in the brain: A neuromodulatory pathway underlying salt-sensitive hypertension and heart failure

Accumulating evidence obtained over the last three decades has revealed a neuroendocrine system in the brain that mediates long term increases in blood pressure. The system involves distinct ion transport pathways including the alpha-2 isoform of the Na,K pump and epithelial sodium channels, as well as critical hormone elements such as angiotensin II, aldosterone, mineralocorticoid receptors and endogenous ouabain. Activation of this system either by circulating or central sodium ions and/or angiotensin II leads to a cascading sequence of events that begins in the hypothalamus and involves the participation of several brain nuclei including the subfornical organ, supraoptic and paraventricular nuclei and the rostral ventral medulla. Key events include heightened aldosterone synthesis and mineralocorticoid receptor activation, upregulation of epithelial sodium channels, augmented synthesis and secretion of endogenous ouabain from hypothalamic magnocellular neurons, and sustained increases in sympathetic outflow. The latter step depends upon increased production of angiotensin II and the primary amplification of angiotensin II type I receptor signaling from the paraventricular nucleus to the rostral ventral lateral medulla. The transmission of sympathetic traffic is secondarily amplified in the periphery by increased short- and long-term potentiation in sympathetic ganglia and by sustained actions of endogenous ouabain in the vascular wall that augment expression of sodium calcium exchange, increase cytosolic Ca²⁺ and heighten myogenic tone and contractility. Upregulation of this multi-amplifier system participates in forms of hypertension where salt, angiotensin and/or aldosterone are elevated and contributes to adverse outcomes in heart failure.     

Dissecting mineralocorticoid receptor structure and function

The molecular mechanisms by which aldosterone regulates epithelial sodium transport in the distal colon and the distal nephron remain to be fully elucidated. Aldosterone acts via the mineralocorticoid receptor (MR) to induce the expression of genes whose products are involved in sodium transport. The structural basis of MR interactions with aldosterone has been examined by creating chimeras of the MR and the closely related glucocorticoid receptor; we have exploited differences in ligand-binding specificity to determine the region(s) of the MR that confer aldosterone-binding specificity. These findings have been related to a three-dimensional model of the MR based on the crystal structure of the progesterone receptor. These studies have been extended to include the characterisation of interactions between the N- and C-termini of the MR. We have characterised six genes that are regulated in vivo in the distal colon and/or kidney of the rat that are directly and acutely regulated by aldosterone administration: the three subunits of the epithelial sodium channel, serum and glucocorticoid-induced kinase, channel-inducing factor and K-ras2A. These studies provide insights into the molecular pathways that mediate aldosterone-induced amiloride-sensitive epithelial sodium transport.     

Antimineralocorticoids.

A number of chemical modifications in the spironolactone molecule were attempted over the last decade to synthesize ligands with high affinity for the mineralocorticoid receptor (MCR), and for possible use in the clinical control of the hypertensive disease. ZK 91587 has been commercialized as the 'ideal' ligand for the MCR, replacing the natural hormone aldosterone. None of the derivatives was retained for possible clinical use as an improvement over Canrenone or Spironolactone. No apparent correlation could be drawn between affinity for the MCR in vitro and biological potency in vivo. Such considerations challenge classical notions regarding the receptor mediated hormone action.