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Central serous chorioretinopathy (CSCR) is a vision-threatening eye disease with no validated treatment and unknown pathogeny. In CSCR, dilation and leakage of choroid vessels underneath the retina cause subretinal fluid accumulation and retinal detachment. Because glucocorticoids induce and aggravate CSCR and are known to bind to the mineralocorticoid receptor (MR), CSCR may be related to inappropriate MR activation. Our aim was to assess the effect of MR activation on rat choroidal vasculature and translate the results to CSCR patients. Intravitreous injection of the glucocorticoid corticosterone in rat eyes induced choroidal enlargement. Aldosterone, a specific MR activator, elicited the same effect, producing choroid vessel dilation -and leakage. We identified an underlying mechanism of this effect: aldosterone upregulated the endothelial vasodilatory K channel KCa2.3. Its blockade prevented aldosterone-induced thickening. To translate these findings, we treated 2 patients with chronic nonresolved CSCR with oral eplerenone, a specific MR antagonist, for 5 weeks, and observed impressive and rapid resolution of retinal detachment and choroidal vasodilation as well as improved visual acuity. The benefit was maintained 5 months after eplerenone withdrawal. Our results identify MR signaling as a pathway controlling choroidal vascular bed relaxation and provide a pathogenic link with human CSCR, which suggests that blockade of MR could be used therapeutically to reverse choroid vasculopathy., Introduction Glucocorticoid hormones act by binding to the glucocorticoid receptor (GR) and also to the mineralocorticoid receptor (MR), both of which belong to the nuclear receptor superfamily. Noticeably, MR has [...]

Aldosterone classically modulates Na transport in tight epithelia such as the renal collecting duct (CD) through the transcellular route, but it is not known whether the hormone could also affect paracellular permeability. Such permeability is controlled by tight junctions (TJ) that form a size- and charge-selective barrier. Among TJ proteins, claudin-4 has been highlighted as a key element to control paracellular charge selectivity. In RCCD2 CD cells grown on filters, we have identified novel early aldosterone effects on TJ. Endogenous claudin-4 abundance and cellular localization were unaltered by aldosterone. However, the hormone promoted rapid (within 15-20 min) and transient phosphorylation of endogenous claudin-4 on threonine residues, without affecting tyrosine or serine; this event was fully developed at 10 nM aldosterone and appeared specific for aldosterone (because it is not observed after dexamethasone treatment and it depends on mineralocorticoid receptor occupancy). Within the same delay, aldosterone also promoted an increased apical-to-basal passage of [sup.125]I (a substitute for [sup.36]C1), whereas [sup.22]Na passage was unaffected; paracellular permeability to [[sup.3]H]mannitol was also reduced. Later on (45 min), a fall in transepithelial resistance was observed. These data indicate that aldosterone modulates TJ properties in renal epithelial cells. paracellular permeability; mannitol flux; occludin; iodine; sodium; kidney; RCCD2 cells

The renal collecting duct plays a key role in control of ion and fluid homeostasis. Genes encoding for ion transporters, hormone receptors, or regulatory proteins specifically expressed in the collecting duct are mutated in several genetic diseases with altered blood pressure. Suitable cellular models expressing genes in a conditional way should represent attractive systems for structure-function analyses and generation of appropriate physiopathological models of related diseases. However, generation of such systems remains laborious and quite inefficient. We adapted and improved a conditional Cre-lox-inducible system in the highly differentiated aldosterone-sensitive rat cortical collecting duct (RCCD2) cell line. The inducible MerCreMer recombinase allowed tight control and high levels of transgene expression, whereas flanking a selection marker with two loxP sites strongly improved the selection procedure. We have used this system to conditionally express an enhanced green fluorescent protein-tagged human mineralocorticoid receptor. In the future, this will allow structure-function analyses as well as mineralocorticoid receptor trafficking studies in these epithelial cells, which retain the features of the native collecting duct. Improvements in the conditional Cre-lox expression system have potentially wide applications in other epithelial or nonepithelial cell lines. Cre recombinase; rat cortical collecting duct 2 cells; mineralocorticoid receptor; aldosterone

Like the [gamma]-subunit of Na-K-ATPase, the corticosteroid hormone-induced factor (CHIF) is a member of the FXYD family of one-transmembrane-segment proteins. Both CHIF and two splice variants of [gamma], [[gamma].sub.a] and [[gamma].sub.b], are expressed in the kidney. Immunolocalization experiments demonstrate mutually exclusive expression of CHIF and [gamma] in different nephron segments. Specific coimmunoprecipitation experiments demonstrate the existence in kidney membranes of the complexes [alpha]/[beta]/[[gamma].sub.a], [alpha]/[beta]/[[gamma].sub.b], and [alpha]/[beta]/CHIF and exclude mixed complexes such as [alpha]/[beta]/[[gamma].sub.a]/[[gamma].sub.b] and [alpha]/[beta]/[gamma]/CHIF. CHIF has been expressed in HeLa cells harboring the rat [[alpha].sub.1]-subunit of Na-K-ATPase. [sup.86]Rb flux experiments demonstrate that CHIF induces a two-to threefold increase in apparent affinity for cytoplasmic Na ([K'.sub.Na]) but does not affect affinity for extracellular K (Rb) ions ([K'.sub.K]) or [V.sub.max]. Measurements of Na-K-ATPase using isolated membranes show similar but smaller effects of CHIF on [K'.sub.Na], whereas [K'.sub.K] and [K'.sub.ATP] are unaffected. The functional effects of CHIF differ from those of [gamma]. An implication of these findings is that other FXYD proteins could act as tissue-specific modulators of Na-K-ATPase. corticosteroid hormone-induced factor; sodium-potassium-adenosine 5'-triphosphatase; tissue-specific modulator

Puttini, Stefania, Ahmed T. Beggah, Antoine Ouvrard-Pascaud, Christine Legris, Marcel Blot-Chabaud, Nicolette Farman, and Frederic Jaisser. Tetracycline-inducible gene expression in cultured rat renal CD cells and in intact CD from transgenic mice. Am J Physiol Renal Physiol 281: F1164-F1172, 2001. First published August 30, 2001; 10.1152/ajprenal.00360.2000.--The renal collecting duct (CD) plays a key role in the control of ion and fluid homeostasis. Several genetic diseases that involve mutations in genes encoding for ion transporters or hormone receptors specifically expressed in CD have been described. Suitable cellular or transgenic animal models expressing such mutated genes in an inducible manner should represent attractive systems for structure-function relationship analyses and the generation of appropriate physiopathological models of related diseases. Our first goal was to develop a CD cell line that allows inducible gene expression using the tetracycline-inducible system (Tet-On). We designed several strategies aimed at the development of a tight and highly inducible system in RCCD1 cells, a rat cortical collecting duct (CCD) cell line exhibiting several properties of the native CCD. Analysis of reporter gene expression demonstrated that the Tet-On system is suitable for inducible gene expression in these cells. In a second step, we have tested whether transgenic Tet-On mice expressing the tetracycline transactivator under the control of the human cytomegalovirus promoter were suitable for inducible gene expression in tubule epithelial cells. The results indicate that, in vivo, the inducible expression of the lacZ reporter gene appeared to be restricted to the CD. This particular strain of transgenic mice may therefore be useful for the expression of genes of interest in an inducible manner in the collecting duct. transgenic; inducible systems; conditional systems; kidney; physiopathology Received 31 December 2000; accepted in final form 30 July 2001

Gene expression and localization of the channel inducing factor (CHIF) was investigated in rat tissues by in situ hybridization. CHIF mRNA was detected in cells at the distal parts of the nephron and in the epithelial cells of the distal colon. Exposure of the cells in the colon to corticosteroid hormones, sodium restriction, low-potassium diet and metabolic acidosis markedly increased CHIF mRNA expression, whereas metabolic acidosis increased CHIF expression in the kidney.

Alterations in cell volume has a significant influence on both the stimulation incited by rise in intracellular Na concentration (Na-i) and Na(+)-K(+)-ATPase pump recruitment in cortical collecting duct (CCD). Cell swelling is capable of enhancing stimulation of pump recruitment without being affected by Na load. Image analysis aids the measurement of cell volume, and specific ouabain binding is used to measure basolateral pumps.

A study of the effects of adrenalectomy, with or without glucocorticoid replacement, on mRNA expression encoding alpha subunit isoforms of Na ion-K ion adenosine triphosphatase, show a prominent increase in the mRNA expression of alpha 3 subunit in dentate gyrus granule cells and in some hippocampal neurons. Corticosteroid upregulate the expression of alpha1 isoform in transporting epithelia, despite variation in receptor specificity.

Shi, Haikun, Rivi Levy-Holzman, Francoise Cluzeaud, Nicolette Farman, and Haim Garty. Membrane topology and immunolocalization of CHIF in kidney and intestine. Am J Physiol Renal Physiol 280: F505-F512, 2001.--Corticosteroid hormone-induced factor (CHIF) is an aldosterone-induced gene, the function of which is yet unknown. It is specifically expressed in kidney collecting duct (CD) and distal colon and is upregulated by either [Na.sup.+] deprivation or [K.sup.+] loading. Hence, it may play a role in epithelial electrolyte transport. Previous studies have characterized regulation and tissue distribution of CHIF mRNA but provided no information on the protein itself. The present paper addresses this issue by using Western blotting, immunochemistry, and in vitro translation. CHIF is an ~8-kDa membranal protein, and protease digestion experiments suggest that its COOH tail faces the cell interior. The protein is abundant in distal colon, kidney medulla, and papilla but cannot be detected in a variety of other tissues. Confocal immunocytochemistry demonstrates that CHIF is present in the basolateral membrane of CD principal cells and distal colon surface cells, with occasional intracellular staining. Dexamethasone and low [Na.sup.+] intake increase the abundance of CHIF. Unlike previous Northern data, induction of CHIF protein by low-[Na.sup.+] intake was apparent not only in the distal colon but also in the kidney. corticosteroid hormone-induced factor; phospholemman; gamma-sodium-potassium-adenosine 5'-triphosphatase; collecting duct; membrane topology; aldosterone

Location and function of the epithelial Na channel in the cochlea. Am J Physiol Renal Physiol 280: F214-F222, 2001.--In the cochlea, endolymph is a K-rich and Na-poor fluid. The purpose of the present study was to check the presence and to assess the role of epithelial Na channel (ENaC) in this organ. [Alpha]-, and [Beta]-, [Gamma]-ENaC subunit mRNA, and proteins were detected in rat cochlea by RT-PCR and Western blot. [Alpha]-ENaC subunit mRNA was localized by in situ hybridization in both epithelial (stria vascularis, spiral prominence, spiral limbus) and nonepithelial structures (spiral ligament, spiral ganglion). The [Alpha]-ENaC-positive tissues were also positive for [Beta]-subunit mRNA (except spiral ganglion) or for [Gamma]-subunit mRNA (spiral limbus, spiral ligament, and spiral ganglion), but the signals of [Beta]- and [Gamma]-subunits were weaker than those observed for [Alpha]-subunit. In vivo, the endocochlear potential was recorded in guinea pigs under normoxic and hypoxic conditions after endolymphatic perfusion of ENaC inhibitors (amiloride, benzamil) dissolved either in K-rich or Na-rich solutions. ENaC inhibitors altered the endocochlear potential when Na-rich but not when K-rich solutions were perfused. In conclusion, ENaC subunits are expressed in epithelial and nonepithelial cochlear structures. One of its functions is probably to maintain the low concentration of Na in endolymph. endolymph; stria vascularis; in situ hybridization; endocochlear potential; hypoxia

Multiple aspects of mineralocorticoid selectivity. Am J Physiol Renal Physiol 280: F181-F192, 2001.--Aldosterone regulates renal sodium reabsorption through binding to the mineralocorticoid receptor (MR). Because the glucocorticoid receptor (GR) is expressed together with the MR in aldosterone target cells, glucocorticoid hormones bound to GR may also intervene to modulate physiological functions in these cells. In addition, each steroid can bind both receptors, and the MR has equal affinity for aldosterone and glucocorticoid hormones. Several cellular and molecular mechanisms intervene to allow specific aldosterone regulatory effects, despite the large prevalence of glucocorticoid hormones in the plasma. They include the local metabolism of the glucocorticoid hormones into inactive derivatives by the enzyme 11[Beta]-hydroxysteroid dehydrogenase; the intrinsic properties of the MR that discriminate between ligands through differential contacts; the possibility of forming homo- or heterodimers between MR and GR, leading to differential transactivation properties; and the interactions of MR and GR with other regulatory transcription factors. The relative contribution of each of these successive mechanisms may vary among aldosterone target cells (epithelial vs. nonepithelial) and according to the hormonal context. All these phenomena allow fine tuning of cellular functions depending on the degree of cooperation between corticosteroid hormones and other factors (hormonal or tissue specific). Such interactions may be altered in pathophysiological situations. aldosterone; glucocorticoid hormones; corticosteroid receptors; kidney; 11[Beta]-hydroxysteroid dehydrogenase; sodium transport