Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry.

A highly selective, amiloride-sensitive, epithelial sodium channel from rat colon (rENaC), composed of three homologous subunits termed alpha, beta, and gamma rENaC, has been cloned by functional expression and was proposed to mediate electrogenic sodium reabsorption in aldosterone-responsive epithelia. To determine whether rENaC could account for sodium absorption in vivo, we studied the cellular localization of the sodium channel messenger RNA subunits by in situ hybridization and their cellular and subcellular distribution by immunocytochemistry in the kidney, colon, salivary, and sweat glands of the rat. In the kidney, we show that the three subunit mRNAs are specifically co-expressed in the renal distal convoluted tubules (DCT), connecting tubules (CNT), cortical collecting ducts (CCD), and outer medullary collecting ducts (OMCD), but not in the inner medullary collecting ducts (IMCD). We demonstrate co-localization of alpha, beta, and gamma subunit proteins in the apical membrane of a majority of cells of CCD and OMCD. Our data indicate that alpha, beta, and gamma subunit mRNAs and proteins are co-expressed in the distal nephron (excepting IMCD), a localization that correlates with the previously described physiological expression of amiloride-sensitive electrogenic sodium transport. Our data, however, suggest that another sodium transport protein mediates electrogenic amiloride-sensitive sodium reabsorption in IMCD. We also localized rENaC to the surface epithelial cells of the distal colon and to the secretory ducts of the salivary gland and sweat gland, providing further evidence consistent with the hypothesis that the highly selective, amiloride-sensitive sodium channel is physiologically expressed in aldosterone-responsive cells.

Distribution of 11 beta-hydroxysteroid dehydrogenase along the rabbit nephron.

It has been recently proposed that 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) is responsible for aldosterone tissue specificity. A 11 beta-OHSD deficiency has been invoked as a cause of the syndrome of apparent mineralocorticoid excess, and 11 beta-OHSD inhibition by liquorice has been invoked to explain the hypertension induced by this drug. Since the renal tubule is composed of aldosterone-sensitive and insensitive segments, we determined the distribution of 11 beta-OHSD along the rabbit tubule. Pools of tubular segments isolated by microdissection were incubated for 2 h at 37 degrees C in the presence of [3H]corticosterone (3H-B, 8.10(-9) M). Afterwards, the amounts of 3H-B and of the metabolite 11-dehydrocorticosterone (3H-A) were determined using HPLC analysis. In the proximal tubule, in either its convoluted or straight portion, and in the medullary thick ascending limb, the amount of 3H-A was 19.6 +/- 3.8% (n = 12), 17.9 +/- 3.4 (n = 8), and 15.0 +/- 2.2 (n = 4), respectively, of the sum of 3H-A + 3H-B. In the cortical ascending limb and the collecting tubule in its cortical and medullary parts, it was 74.7 +/- 6.8% (n = 4), 74.1 +/- 4.9 (n = 9) and 64.6 +/- 14.1 (n = 3), respectively. In both proximal and cortical collecting tubule, addition of carbenoxolone 8.10(-4) M, an inhibitor of 11 beta-OHSD, almost completely inhibited the conversion of 3H-B to 3H-A. Thus, 11 beta-OHSD activity was high in the aldosterone-sensitive segments, and low in the aldosterone-insensitive segments. These results strongly favor the hypothesis that 11 beta-OHSD is a key enzyme in mineralocorticoid tissue specificity along the rabbit nephron. They reinforce the notion that a defect in 11 beta-OHSD plays a major role in the syndrome of apparent mineralocorticoid excess and liquorice-induced hypertension.

Vasopressin potentiates mineralocorticoid selectivity by stimulating 11 beta hydroxysteroid deshydrogenase in rat collecting duct.

Arginine vasopressin (AVP) and corticosteroid hormones are involved in sodium reabsorption regulation in the renal collecting duct. Synergy between AVP and aldosterone has been well documented, although its mechanism remains unclear. Both aldosterone and glucocorticoid hormones bind to the mineralocorticoid receptor (MR), and mineralocorticoid selectivity depends on the MR-protecting enzyme 11 beta hydroxysteroid deshydrogenase (11-HSD), which metabolizes glucocorticoids into derivatives with low affinity for MR. We have investigated whether the activity of 11-HSD could be influenced by AVP and corticosteroid hormones. This study shows that in isolated rat renal collecting ducts, AVP increases 11-HSD catalytic activity. This effect is maximal at 10(-8) M AVP (a concentration clearly above the normal physiological range of AVP concentrations) and involves the V2 receptor pathway, while activation of protein kinase C or changes in intracellular calcium are ineffective. The stimulatory effect of AVP on 11-HSD is largely reduced after adrenalectomy, and is selectively restored by infusion of aldosterone, not glucocorticoids. We conclude that this synergy between AVP and aldosterone in controlling the activity of 11-HSD is likely to play a pivotal role in resetting mineralocorticoid selectivity, and hence sodium reabsorption capacities of the renal collecting duct.

Effect of cell sodium on Na+/K(+)-ATPase-dependent sodium efflux in cortical collecting tubule of rabbits under different aldosterone status.

Aldosterone increased the tubular volume in cortical collecting tubules (CCD) of rabbit kidney. It modulated the rate of cell sodium accumulation, under condition of ATPase inhibition (4 degrees C, in the absence of K+). In contrast, the relationship between Na+/K(+)-ATPase-dependent Na+ extrusion rate and intracellular Na+ concentration (Nai+) was similar in control, adrenalectomized, and aldosterone-treated adrenalectomized animals: Na+ extrusion rate increased with Nai+, up to 70 mM Nai+, and then plateaued. This indicates that aldosterone does not modify the characteristics of Nai(+)-dependent Na+ extrusion rate by the Na+/K(+)-ATPase pump in CCD.

Characteristics and regulation of 11 beta-hydroxysteroid dehydrogenase of proximal and distal nephron.

Enzymatic properties of the enzyme 11 beta-hydroxysteroid dehydrogenase (11-HSD), which confers mineralocorticoid selectivity, have been explored in the aldosterone-sensitive collecting duct (CCD) and the aldosterone-insensitive Pars Recta (PR) of the rat kidney. After incubation of freshly isolated tubular segments with [3H]corticosterone (3H-B) or [3H]dehydrocorticosterone (3H-A), the rate of transformation of 3H-B into 3H-A (dehydrogenase activity), or the reverse reaction (reductase activity) were measured by HPLC, Vmax for dehydrogenase activity was found to be 8- to 10-fold higher in CCD than PR. The enzyme functions over a very wide range (0.1-5000 nM) of corticosterone concentration. In CCD, enzyme kinetics suggest either the presence of two 11-HSD forms, differing by their affinity for corticosterone, or complex kinetics. Addition of NAD or NADP to permeabilized tubules revealed that dehydrogenase activity is NAD-dependent in CCD and NADP-dependent in PR. Cofactor addition was ineffective in intact tubules. CCD exhibited an exclusive dehydrogenase activity, whereas in PR dehydrogenase and reductase activity were found. No regulation of dehydrogenase activity could be evidenced in adrenalectomized rats receiving or not aldosterone, corticosterone or dexamethasone, for 2 h, 3 days or 4 days. We conclude that 11-HSD in the CCD and PR differs by its Vmax and cofactor dependence. Corticosteroid hormones do not influence 11-HSD activity.

Autoradiographic evidence of nuclear binding of spironolactone in rabbit cortical collecting tubule.

Previous biochemical studies indicated that the spirolactone-mineralocorticoid receptor complexes are unable to translocate into the nucleus. The present study was designed to reinvestigate the intracellular distribution of spirolactone-binding sites, using autoradiography. For this purpose, rabbit kidney pyramids were incubated at 30 C with tritiated SC9420 or aldosterone. Thereafter, aldosterone-sensitive cortical collecting tubules were microdissected and processed for dry film autoradiography. The concentration was 2 nM for both steroids. Non-specific labeling was determined by incubations with tritiated steroids plus a 100-fold excess of unlabeled steroids. Results show the presence of specific nuclear labeling for both [3H] aldosterone and [3H]SC9420. Specific cytoplasmic labeling was very low for both [3H]aldosterone and [3H]SC9420. The nuclear labeling by [3H]SC9420 was equally and almost completely displaced by a 100-fold excess of unlabeled aldosterone or SC9420 (91% and 87%, respectively). We conclude that spironolactone-receptor complexes migrate into the nucleus. The difference between these results and those of previous studies with biochemical techniques, which failed to detect specific nuclear binding of spirolactone, may be due to methodological reasons.

Tissue-specific expression of alpha and beta messenger ribonucleic acid isoforms of the human mineralocorticoid receptor in normal and pathological states.

Expression of the mineralocorticoid receptor (MR) is restricted to some sodium-transporting epithelia and a few nonepithelial target tissues. Determination of the genomic structure of the human MR (hMR) revealed two different untranslated exons (1alpha and 1beta), which splice alternatively into the common exon 2, giving rise to two hMR mRNA isoforms (hMR alpha and hMR beta). We have investigated expression of hMR transcripts in renal, cardiac, skin, and colonic tissue samples by in situ hybridization with exon 1alpha and 1beta specific riboprobes, using an exon 2 probe as internal control. Specific signals for either exon 1alpha- and 1beta-containing mRNAs were detected in typically hMR-expressing cells in all tissues analyzed. hMR alpha and hMR beta were present in distal tubules of the kidney, in cardiomyocytes, in enterocytes of the colonic mucosa, and in keratinocytes and sweat glands. Interestingly, although both isoforms appear to be expressed at approximately the same level, the relative abundance of each message compared with that of exon 2-containing mRNA strikingly differs among aldosterone target tissues, suggesting the possibility of other tissue-specific transcripts originating from alternative splicing. Finally, functional hypermineralocorticism was associated with reduced expression of hMR beta in sweat glands of two patients affected by Conn's and Liddle's syndrome, whereas normal levels of hMR isoforms were found in one case of pseudohypoaldosteronism. Altogether, our results indicate a differential, tissue-specific expression of hMR mRNA isoforms, hMR beta being down-regulated in situations of positive sodium balance, independently of aldosterone levels.

Human skin as target for aldosterone: coexpression of mineralocorticoid receptors and 11 beta-hydroxysteroid dehydrogenase.

The expression of mineralocorticoid receptors (MR) and 11 beta-hydroxysteroid dehydrogenase (11HSD) activity has been investigated in the epidermis and appendages of the human skin. Aldosterone binds to MR and regulates sodium transport in tight epithelia. Mineralocorticoid selectivity is achieved through coexpression of MR and 11HSD, which prevents permanent MR occupancy by glucocorticoids. Some forms of hypertension may involve abnormalities of MR and/or 11HSD. However, their direct assessment in humans remains difficult in the kidney or colon. This led us to explore this system in human skin easily accessible to biopsy. In situ hybridization with specific MR complementary ribonucleic acid probes and immunohistochemistry using three different anti-MR antibodies showed that MR was expressed at both the messenger ribonucleic acid and protein levels in the keratinocytes of the epidermis, in the sweat and sebaceous glands, and in the hair follicles. A significant 11HSD activity was found in isolated sweat gland ducts (5 fmol/3-mm length.10-min incubation with 10 nmol/L corticosterone as substrate) and was very low in the epidermis. In both structures, reductase activity was 10 times lower than that of dehydrogenase. Studies on the cofactor specificity of the enzyme showed a nicotinamide-adenine-dinucleotide preference in sweat glands, contrasting with a nicotinamide-adenine-dinucleotide phosphate dependence in epidermis. Human skin appears as a new target for aldosterone because it coexpresses MR and 11HSD. Our findings present the possibility to explore the functionality of the MR system in human tissue and its implications in various physiopathological situations.

Vasopressin stimulates long-term net chloride secretion in cortical collecting duct cells.

The classical short-term effect (within minutes) of arginine vasopressin (AVP) consists in increasing sodium, chloride and water transport in kidney cells. More recently, long-term actions (several hours) of the hormone have been evidenced on water and sodium fluxes, due to transcriptional enhancement in the expression of their transporters. The present study demonstrates that AVP is also responsible for a long-term increase in net chloride secretion. In the RCCD(1) rat cortical collecting duct cell line, 10(-8) M AVP induced, after several hours, an increase in net (36)Cl(-) secretion. This delayed effect of AVP was inhibited by basal addition of 10(-4) M bumetanide and apical addition of 10(-4) M glibenclamide, suggesting chloride entry at the basal membrane through a Na(+)/K(+)/2Cl(-) and apical secretion through a chloride conductance. An original acute cell permeabilization method was developed to allow for entry of antibodies directed against the regulatory region (R) of the cystic fibrosis transmembrane regulator (CFTR) into the cells. This procedure led to a complete and specific blocking of the long-term net chloride secretion induced by AVP. Finally, it was observed that CFTR transcripts steady-state level was significantly increased by AVP treatment. Besides the well-documented short-term effect of AVP on chloride transport, these results provide evidence that in RCCD(1) cells, AVP induces a delayed increase in transepithelial net chloride secretion that is mediated by a Na(+)/K(+)/2Cl(-) co-transporter and CFTR.

Regulation of sodium transport by steroid hormones.

The main mechanisms involved in the regulation of sodium transport by steroid hormones are briefly reviewed. The respective roles of the apical epithelial sodium channel, which is likely to be the limitant step of steroid-regulated transepithelial sodium transport, and Na,K-ATPase are described. Regulation of these ion transporting proteins by aldosterone and glucocorticoid hormones, probably via a two step mechanism (rapid activation of channels or pumps by unknown regulators, and modulation of the transcription/translation rate of these transporters), is discussed. The mechanisms of mineralocorticoid selectivity, that is, the integrated process allowing a specific action of aldosterone, in spite of high concentrations of glucocorticoids that crossbind with aldosterone to the mineralocorticoid receptor (MR), are explained, as is the role of the enzyme 11 beta-hydroxysteroid dehydrogenase and the differential interactions of MR with steroid ligands and hormone responsive elements of DNA. Finally, synergism between aldosterone and antidiuretic hormone for the stimulation of sodium transport is evoked.

Expression of 11 beta-OHSD along the nephron of mammals and humans.

The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) plays a major role in the protection of the mineralocorticoid receptor (MR). This cellular mechanism of aldosterone selectivity relies on the coexpression of MR and 11 beta-OHSD in the same cells. Localization of renal 11 beta-OHSD along the nephron is reviewed; comparison of data contained in different species is made; and original data is presented to show that the catalytic activity of the enzyme in tubules from human kidney is the highest in the mineralocorticoid-sensitive distal nephron.

[Binding of aldosterone to its receptors along the nephron of the normal and spontaneously hypertensive rat]. / Liaison de l'aldostérone à ses récepteurs le long du néphron de rat normal et spontanément hypertendu.

The specific binding of aldosterone has been investigated using an autoradiographic method, on isolated tubular segments of kidneys from spontaneously hypertensive (SHR) and normotensive (KWR) Okamoto rats, and normotensive Wistar (NWR) rats. A low, mineralocorticoid, concentration of aldosterone (2 X 10(-9) M) was used. In proximal tubules, almost no specific labelling was found in the three groups. In the cortical ascending limb, a specific nuclear labelling was present and equivalent in the three groups. In the distal (DCT) and cortical collecting (CCT) tubule, the specific labelling was double in SHR and KWR than in NWR. This higher binding capacity occurs in target segments for aldosterone, and coincides with the increased sodium reabsorption in the distal structures of Okamoto rats, previously reported by our laboratory. The relations between these observations and the altered membrane permeability described in hypertensive disease is discussed.

Identification and role of aldosterone receptors in the cardiovascular system.

The cardiovascular system is now recognized as an important mineralocorticoid target. All -components required for specific and selective aldosterone effects are present in the cardiovascular system. Mineralocorticoid receptors (MR) are expressed in the heart and large blood vessels together with the 11 B-hydroxysteroid dehydrogenase type II, which ensures the enzymatic protection of MR against glucocorticoids. The recent description of local vascular and cardiac aldosterone biosynthesis strongly supports an autocrine/paracrine hormonal action. Establishment of transgenic mice models of targeted overexpression of the mineralocorticoid receptor should facilitate new insights into the molecular and cellular mechanisms of aldo-sterone actions in the cardiovascular system.

[A new regulatory mechanism of the action of corticosteroid hormones: cellular 11beta-hydroxycorticosteroid dehydrogenase]. / Un nouveau mécanisme de régulation de l'action des hormones corticostéroïdes: la 11 beta-hydroxycorticostéroïde déshydrogénase cellulaire.

Eleven-beta-hydroxysteroid dehydrogenase (11 beta OHSD) protects the aldosterone receptor (MR) against its occupancy by glucocorticoid hormones. We examined the intrarenal distribution of 11 beta OHSD, as compared to that of MR. MR were localized in histological sections from rabbit kidney, using immunohistochemical methods with an anti-MR monoclonal antibody. 11 beta OHSD activity was measured in isolated tubular segments from rabbit, rat and mouse kidneys. Tubules were incubated in the presence of tritiated corticosterone (3H-B:2 x 10(-8)M). Then the rate of degradation of 3H-B into 3H-11-dehydrocorticosterone (3H-A) was determined by HPLC. MR was immunodetected in the distal tubule and the collecting duct. No positive staining was present in the proximal tubule. The conversion rate of 3H-B into 3H-A was high (approximately 80%) in the distal and collecting tubule. It was low in the proximal tubule (less than 15%) except in the rat (approximately 50%). These results indicate that MR and 11OHSD are colocalized along the mammalian nephron. This colocalization constitutes a strong argument in favor of the MR-protective role of 11 beta OHSD, and of a role of a defect of this enzyme in the genesis of some types of arterial hypertension.