Estrogen and progesterone regulate α, β, and γENaC subunit mRNA levels in female rat kidney

Kidney International, Vol. 65 (2004), pp. 1774–1781

ION CHANNELS – MEMBRANE TRANSPORT – INTEGRATIVE PHYSIOLOGY

Estrogen and progesterone regulate a, b, and cENaC subunit mRNA levels in female rat kidney LORRAINE GAMBLING, SUSAN DUNFORD, CATHERINE A. WILSON, HARRY J. MCARDLE, and DEBORAH L. BAINES Development, Growth and Function Division, Rowett Institute, Aberdeen, Scotland, United Kingdom; and Department of Basic Medical Sciences, St. George’s Hospital Medical School, London, United Kingdom

Estrogen and progesterone regulate a, b, and cENaC subunit mRNA levels in female rat kidney. Background. Estrogen and progesterone regulate a, b, and c amiloride-sensitive epithelial sodium channel (ENaC) subunit mRNA levels in female rat kidney. Renal Na+ handling differs between males and females. Further, within females Na+ metabolism changes during the menstrual cycle and pregnancy. Electrolyte homeostasis and extracellular fluid volume are maintained primarily by regulated transport of Na+ via the amiloride-sensitive Na+ channel. This study examines the role of the female gender steroids in the regulation of expression of ENaC. Methods. We measured ENaC subunit mRNA levels in rat kidney using Northern blotting. Kidneys were taken from male and females at different ages and from adult ovariectomized rats treated with 17-b-estradiol benzoate (estrogen) and/or progesterone for 8 or 24 hours. Results. The abundance of a, b, and cENaC mRNA was significantly higher in female compared to male rat kidneys from 10 weeks of age (P = 0.001, P = 0.004, and P = 0.02, N = 10, respectively). These differences were abolished in ovariectomized rats. Treatment of ovariectomized rats with estrogen increased aENaC mRNA abundance in the kidney at both 8 and 24 hours (P < 0.05, N = 6; and P < 0.05, N = 7, respectively). Progesterone inhibited the effect of estrogen on aENaC mRNA at 8 hours but when given alone increased cENaC mRNA (P < 0.05, N = 3). Neither hormone, alone or in combination, had any significant effect on bENaC mRNA levels at 8 or 24 hours. Conclusion. Female gonadal steroids differentially modulate expression of ENaC subunit mRNA in the rat kidney.

The kidney is central to the maintenance of electrolyte homeostasis and extracellular fluid volume. Regulated vectorial transport of Na+ ions and water from the lumen of the distal nephron is a critical mechanism by which homeostasis is maintained (for review see [1]).

Key words: ENaC, hormone, kidney. Received August 6, 2003 and in revised form November 10, 2003 Accepted for publication December 16, 2003
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2004 by the International Society of Nephrology

The amiloride-sensitive epithelial sodium channel (ENaC), is principally located in the cells of the distal convoluted tubule, connecting tubule and cortical collecting duct (CCD) of the mammalian kidney [2–4]. Studies have shown that its activity is the rate-limiting step in Na+ reabsorption in the rabbit CCD [5]. Abnormalities that increase ENaC activity have been shown to lead to increased Na+ uptake, fluid retention and hypertension (e.g., Liddle’s syndrome) [6], whereas those that decrease channel activity can result in salt wasting and hypotension (e.g., pseudohypoaldosteronism type I) [7]. Na+ transport is dependent on both the number of ENaC channels located at the membrane of the epithelial cell and the rate at which Na+ can be transported through the channel. In the kidney, ENaC activity is modulated by a number of hormones. Aldosterone, vasopressin, insulin, and catecholamines can induce a rapid increase in Na+ transport via changes in cellular localization of subunits and a rise in ENaC channel density at the cell membrane [8, 9]. These effects are mediated by a number of second messengers, including Serum and glucocorticoidinduced kinase 1 (SGK), cyclic adenosine monophosphate (cAMP), and inositol-1,4,5 triphosphate (IP 3 ) [8, 10–13]. In contrast, adrenaline has been shown to modulate vasopressin action upon Na+ transport by inhibiting vasopressin-dependent cAMP production in the rat CCD via a G- protein mechanism [14]. Several of the hormones described above also have genomic effects that modulate the level of mRNA encoding the ENaC subunits and change cellular expression patterns [9]. The ENaC channel comprises three subunits a, b, and c [15] and the cellular abundance of mRNAs encoding these subunit proteins is an important determinant of ENaC channel activity. Aldosterone has been reported to increase moderately both aENaC mRNA and protein levels but not b or cENaC mRNA in the rat kidney [16, 17]. The glucocorticoid dexamethasone significantly increases aENaC mRNA in rat lung tissue and human lung cells lines with a concomitant increase in functional Na+ transport [18–20]. It has also

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been reported to increase aENaC mRNA levels in mouse kidney explants [21]. In contrast to aldosterone and dexamethasone, chronic treatment of rats with vasopressin did not affect aENaC mRNA levels but substantially increased expression of b and cENaC subunit mRNA protein and Na+ transport in the renal collecting duct [22]. Recently, estrogen and progesterone have been shown to modulate the mRNA and functional expression of ENaC subunits in the rat lung [23]. They also affect fluid balance, neurohypophysial hormone concentrations and the magnitude of changes in electrolyte excretion in response to neurohypophysial hormone administration [24]. It has been reported that renal Na+ handling changes in rats during the estrous cycle [25] and during pregnancy [26]. Furthermore, differences in renal Na+ handling have been described between men and women, particularly during the luteal phase of the female menstrual cycle and it has been suggested that ovarian steroids may act to promote Na+ and water retention in the body [27–29]. Therefore, we hypothesised that estrogen and/or progesterone also modulate ENaC mRNA expression levels in the kidney. To address this, we have examined the mRNA abundance of the three ENaC subunits in whole kidneys of developing rats from newborn to 16 weeks of age, with sexual maturity being reached by 10 weeks of age. In parallel, we have investigated the effect of ovariectomy and examined the effect of female sex hormone replacement (17-b-estradiol benzoate and progesterone) on the expression of ENaC subunit mRNAs in the female rat kidney. METHODS Ovariectomy and hormone administration Female Wistar rats (12 weeks) were anesthetised with halothane (3% in O 2 ) and ovariectomy was performed as previously described. After a 2-week recovery period, ovariectomized rats were subjected to treatment with hormones or vehicle control. Ovariectomized rats (average weight 300 g) were given subcutaneous injection of 17-b-estradiol benzoate (estrogen) (Sigma, Poole, UK) at 10 lg per rat or progesterone at 1 mg per rat (Sigma) or a combined injection containing both hormones. Both hormones were dissolved in corn oil and control animals were injected with corn oil alone. Animals were killed by cervical dislocation or decapitation at 8 or 24 hours after treatment and kidneys removed for preparation of RNA. Levels of cortisol, aldosterone and arginine vasopressin (AVP) (which can all modulate ENaC expression) are known to be subject to diurnal rhythms [30, 31]. Therefore, to exclude any effect from the diurnal fluctuation of these hormones in our study, estrogen and progesterone treated animals were compared directly to control ovariectomized animals and male rats at each of these time points. For the developmental study, male and

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female rats were killed (as above) immediately after birth or at 10 or 16 weeks of age and kidneys removed for analysis. All procedures were carried out in accordance with current United Kingdom legislation. Northern blot analysis Frozen kidney tissue samples were transferred directly to TRI reagent (Genetic Research Instrumentation, Braintree, UK). RNA was prepared according to the manufacturer’s instructions. Total RNA was resuspended in RNase free H 2 O and quantified by analysis of optical density (OD) at 260k by ultraviolet spectrophotometry. For the Northern analysis, 20 lg of total RNA was denatured in formamide (50%), formaldehyde (2.2 mol/L), in 3-[N-morpholino] propane sulfonic acid (MOPS) buffer [0.2 mol/L MOPS, pH 7.0, 50 mmol/L CH 3 COONa, 5 mmol/L ethylenediaminetetraacetic acid (EDTA)] for 15 minutes at 65◦ C and cooled rapidly on ice. Samples were loaded onto formaldehyde gels (1.0% agarose, 2.2 mol/L formaldehyde in MOPS buffer), electrophoresed in MOPS buffer at 1 V cm−1 for 3 to 4 hours, then transferred to a nylon membrane (Amersham Biosciences, Bucks, UK) by an electrophoretic transfer system (Amersham) and cross-linked with an ultraviolet cross-linker (UVP, Upland, CA, USA). Rat-specific a, b, and cENaC probes were prepared by reverse transcription-polymerase chain reaction (RTPCR) from rat lung RNA using standard protocols and primers designed from rat sequences. The sense and antisense primers correspond to bases 1059–1079 and 1802– 1822 of rat aENaC (Genbank accession no. X70497), 880–900 and 1232–1252 of rat bENaC (Genbank accession no. X77932) and rat cENaC (Genbank accession no. X77933). The sequences PCR products were then ligated into pGEM-T easy vector (Promega, Hamps, UK). An 18S oligonucleotide probe was used to correct for any loading variance between samples. The aENaC cDNA in pGEM-T easy vector was amplified by PCR. The resulting PCR product was labeled with a-[32 P] deoxycytidimine triphosphate (dCTP) by random priming with “Ready-to-Go” labeling beads (Amersham). Membranes were prehybridized at 42◦ C for 30 minutess in Ultrahyb (Ambion, Abingdon, UK). Hybridizations were carried out overnight at 42◦ C. The blots were then washed to high stringency in 0.05 × standard sodium citrate (SSC) + 0.1% sodium dodecyl sulfate (SDS) at 42◦ C. The b and cENaC cDNA templates in pGEM-T easy vector (Promega) were linearised by restriction digestion with SacI and NcoI, respectively. Antisense riboprobes incorporating a-[32 P] CTP were generated from the linearised vectors using T7 RNA polymerase and SP6 polymerase, respectively. The 18S deoxyoligonucleotide probe terminal phosphate was replaced with 32 P-c adenosine triphospate (ATP) using polynucleotide kinase (Promega). Prehybridization,

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hybridization and washes were carried out as described above but at a higher temperature of 68◦ C for these probes. All blots were imaged on a wire proportional counter (Packard Instant Imager, Perkin-Elmer, Bucks, UK). After hybridization and analysis of Northern blots with either aENaC or bENaC or cENaC probes, filters were stripped in boiling 0.1% SDS and allowed to cool to room temperature. Complete removal of labeled probes from the blots was confirmed using the wire proportional counter prior to reprobing with 18S. No blot was probed more than three times. The intensity (proportional to mRNA abundance) of ENaC subunit mRNA and 18S ribosomal RNA bands imaged on the Northern blots was quantified by measuring the amount of emitted radioactivity. Data presentation and statistical analysis Prior to statistical analysis samples were corrected for loading by normalizing to 18S ribosomal RNA. To correct for interblot variation due to differences in hybridization conditions and specific activity of the probes a quality control was included on each membrane. Data are expressed as percentage control and all results are presented as mean ± SEM. Where appropriate, data were analyzed by unpaired t test. Where more complex variables were included, data were analyzed by one-way analysis of variance (ANOVA) in Genstat Edition 6 (Release 6.1, Lawes Agricultural Trust, Rothamsted, Herts, UK). All pairwise comparisons were tested using Fisher’s unprotected least significant difference test. To compare treatments, the t-statistic was calculated from the ANOVA output. Significance was assumed at P < 0.05. RESULTS Development Immediately after birth, there was no significant difference in mRNA abundance of any of the ENaC subunits in the kidneys of male and female rats (Fig. 1A to C). However, by 10 weeks of age there were significantly higher levels of a, b, and cENaC mRNA in female compared to male kidneys (P = 0.001, N = 10; P = 0.004, N = 10; and P = 0.02, N = 10, respectively) (Fig. 1D to F). At 16 weeks, a, b, and cENaC mRNA levels remained significantly higher in females compared to males (P = 0.004, N = 10; P = 0.004, N = 10; and P < 0.002, N = 10) and the difference was more pronounced (Fig. 1G to I). Ovariectomy Following ovariectomy, levels of a, b, and cENaC in the kidneys were not significantly different between females and males (P = 0.9, N = 6; P = 0.2, N = 6; and P = 0.8, N = 6) (Fig. 2).

Estrogen and progesterone Replacing estrogen (10 lg per rat) in ovariectomized females significantly increased kidney aENaC mRNA levels compared to male and untreated ovariectomized rats 8 hours after injection (P < 0.05, N = 6; and P < 0.05, N = 6, respectively), (Fig. 3A). Progesterone inhibited this stimulation (Fig. 3A). Twenty-four hours after injection, levels were still significantly increased in animals injected with estrogen alone and progesterone did not inhibit this rise (Fig. 3A). In contrast to aENaC, neither estrogen, progesterone, or combined treatment of ovariectomized rats had any significant effect on bENaC mRNA levels after 8 hours or 24 hours (N = 7) (Fig. 3B). Replacing estrogen had no effect on cENaC levels in the kidneys of ovariectomized rats after 8 hours or 24 hours. Combined treatment of estrogen and progesterone also had no effect. However, progesterone alone significantly increased cENaC mRNA levels compared to untreated ovariectomized animals at 24 hours (P < 0.05, N = 3) but did not quite reach significance at 8 hours (P = 0.057, N = 3) (Fig. 4). DISCUSSION In this paper, we have shown that the mRNA levels of ENaC subunits in the female rat kidney are higher than in the male. This difference is not evident at birth but becomes apparent during development (up to 16 weeks) suggesting that hormones associated with gender and sexual maturity are involved. In support of this, ovariectomy abolishes the differences between the sexes. Such a phenomenon has been described in other tissues, such as the lung, where female ovarian hormones were shown to upregulate a and cENaC subunits as well as the cystic fibrosis transmembrane regulator (CFTR) mRNA level [23]. Importantly, our data also indicate that steroid hormones regulate the three subunits differently. Estrogen increased aENaC mRNA levels and progesterone, when administered in combination, appeared to inhibit this effect. In contrast, progesterone had no effect when administered alone on a and bENaC mRNA levels but increased cENaC mRNA. Increases in aENaC mRNA levels induced by estrogen and antagonized by progesterone have not, to our knowledge, been previously described. However, there is a precedent for such an effect. In guinea pig endometrial epithelial cells, CFTR mRNA levels were also increased by estrogen and decreased by estrogen plus progesterone [32]. Our findings contrast with those described in lung, where only a combined treatment of estrogen and progesterone, but neither hormone alone, increased aENaC and interestingly, CFTR mRNA levels [23]. This disparity most likely reflects differential responses to steroids between tissues. For example, corticosteroids have little effect in the distal

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Fig. 2. Amiloride-sensitive epithelial sodium channel (ENaC) subunit mRNA abundance after ovariectomy. The graphs show mRNA abundance (as % control) (see Methods section) for aENaC (A), bENaC (B), and cENaC (C) in male (M) and ovariectomized (OVX) female rats. The graphs represent data from six samples. There was no significant difference in mRNA abundance of the ENaC subunits in ovariectomized female compared to male rats. Above each graph is a representative Northern blot of the ENaC subunit mRNA product together with the corresponding 18S ribosomal RNA bands from the same sample.

colon, but consistently up-regulate aENaC mRNA levels in kidney and lung [17, 21, 33]. Glucocorticoids elicit more pronounced effects in the lung than in the kidney, where mineralocorticoid responses prevail [34]. It is also possible that differences in methodology between the studies could account for the contrasting findings. We injected 17-b-estradiol benzoate, which is less easily metabolized and thus physiologically active for a longer period time than the 17-b-estradiol used in the lung study. In our study progesterone and estrogen were injected at a ratio of 100:1. This ratio has been widely used in the study of steroid regulation of gonadotrophin release and female sexual behavior [35] and is closer to the ratios determined in humans (females in the follicular phase show ratios of ∼37:1 rising to ∼150:1 in luteal phase of the menstrual cycle) [29]. While the profile of hormone levels may change as the hormones diffuse out of the oil, it is possible that the higher ratios used by Sweezy et al [23] of 250:1 or greater (progesterone:estrogen) suspended in saline could induce a different response. Levels of bENaC mRNA were higher in females compared to males in both kidney and lung [23]. However, although ovariectomy abolished these differences in the

kidney, neither estrogen nor progesterone nor a combination of the hormones had any significant effect on mRNA expression over the time course studied. It is possible that the regulation of this subunit may involve other (possibly female-derived) factors. Interestingly, we found that cENaC mRNA levels were increased by progesterone alone but not by combined treatment of progesterone and estrogen (100:1). Such an effect has not previously been described. In the lung, cENaC mRNA levels were elevated only when estrogen was present with a 500:1 or greater excess of progesterone, which may relate to the differences in ratio effects described above. These results are intriguing as progesterone is a mineralocorticoid antagonist. However, the kidney has been described as a progesterone-metabolizing and androgen-producing organ [36]. While some metabolites of progesterone retain antagonist properties, others can act as agonists (e.g., 11-b-hydroxyprogesterone stimulated Na+ absorption in mouse mpk CCD cells) [37]. Furthermore, progesterone has been reported to inhibit renal 11-b-hydroxysteroid dehydrogenase type 2 thus allowing steroids, such as cortisol, access to the mineralocorticoid receptor [38].

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Fig. 1. Amiloride-sensitive epithelial sodium channel (ENaC) subunit mRNA abundance during development. The graphs show mRNA abundance (as% control) (see Methods section) for aENaC (A, D, and G), bENaC (B, E, and H), and cENaC (C, F, and I) in male (M) and female (F) rat kidney at birth (A to C), 10 weeks (D to F) and 16 weeks of age (G to I). The graphs represent data from ten samples. mRNA abundance of all ENaC subunits was significantly higher in female compared to male rats at 10 and 16 weeks. ∗ P < 0.05; ∗∗ P < 0.01. Above each graph is a representative Northern blot of the ENaC subunit mRNA product together with the corresponding 18S ribosomal RNA bands from the same sample.

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Fig. 3. a and b amiloride-sensitive epithelial sodium channel (ENaC) subunit mRNA abundance after treatment with female gonadal hormones. The graphs show mRNA abundance (as % control) (see Methods section) for aENaC (A) and bENaC (B) in ovariectomized female rats treated with vehicle (OVX), estrogen (O), or estrogen + progesterone (O + P) for 8 hours and 24 hours. The graphs represent data from six to seven samples. Treatment with estrogen significantly increased aENaC mRNA abundance at both time points. ∗ P < 0.05. There was no significant effect of any treatment on bENaC mRNA abundance. Above each graph is a representative Northern blot of the ENaC subunit mRNA product at 24 hours, together with the corresponding 18S ribosomal RNA bands from the same sample.

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The full significance of changes in cellular ENaC subunit mRNA levels is still poorly understood. It is generally accepted that the a subunit can form a Na+ conducting channel alone whereas the b and c subunits augment channel conduction [15]. Furthermore, altering the relative levels of a, b, and c subunit mRNA available in the cell can lead to channels of different regulatory characteristics being produced [39, 40]. These include differences in ion selectivity, conductance, amiloride sensitivity, and regulation. Certainly, raised aENaC mRNA levels in lung epithelial cells after treatment with either estrogen and progesterone or dexamethasone were associated with increased channel function and Na+ transport [19, 20, 23]. Thus, the estrogen and progesterone induced changes in a and cENaC mRNA levels we describe in the rat kidney could lead to functional changes in Na+ transport. The differences in renal Na+ handling in females during the menstrual cycle and pregnancy have been proposed to lead to the peripheral edema and weight gain in the luteal phase of the menstrual cycle and to be important in the process of plasma expansion during pregnancy [27, 29]. It has also been reported that there are differences in Na+ handling during the estrous cycle in rats [25]. Es-

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Fig. 4. c amiloride-sensitive epithelial sodium channel (ENaC) subunit mRNA abundance after treatment with female gonadal hormones. The graph shows mRNA abundance (as% control) (see Methods section) for cENaC in ovariectomized female rats treated with vehicle (OVX), estrogen (O), or estrogen + progesterone (O + P) or progesterone (P) for 8 hours and 24 hours. The graph represents data from three to seven samples. Treatment with progesterone significantly increased cENaC mRNA abundance compared to untreated ovariectomized animals at 24 hours but did not quite reach significance at 8 hours. ∗ P < 0.05. Above the graph is a representative Northern blot of the ENaC subunit mRNA product at 24 hours, together with the corresponding 18S ribosomal RNA bands from the same sample.

trogen has been shown to increase Na+ uptake in distal tubules from the rabbit kidney in vitro [41], induce renal Na+ retention in dogs [42], and enhance the Na+ retaining effects of vasopressin and oxytocin in ovariectomized rats [24]. As ENaC is the primary route for Na+ absorption in the mammalian distal nephron we speculate that the changes in ENaC mRNA we describe in response to female gonadal steroids underlie these differences in renal Na+ handling. We recognize that estrogen and progesterone can also modify the activity of other renal hormones [24, 43, 44]. Thus, further work will be required to fully elucidate the effect of female sex hormones on expression of ENaC subunit mRNA, protein and function of amiloride-sensitive Na+ channels in the kidney. ACKNOWLEDGMENTS We are grateful to Biomathematics and Statistics Scotland (BioSS) for assistance with the statistical analysis and Professor M. Forsling, King’s College London, for her help and advice. This work was supported by SEERAD and the European Union FPV. Reprint requests to Deborah L. Baines, Department of Basic Medical Sciences, St. George’s Hospital Medical School, London SW17 0RE, England, United Kingdom. E-mail. [email protected]

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REFERENCES 1. SCHAFER JA: Abnormal regulation of ENaC: Syndromes of salt retention and salt wasting by the collecting duct. Am J Physiol 283:F221–F235, 2002 2. SCHMITT R, ELLISON DH, FARMAN N, et al: Developmental expression of sodium entry pathways in rat distal nephron. Am J Physiol 276:F367–F381, 1999 3. LOFFING J, LOFFING-CUENI D, MACHER A, et al: Localization of epithelial sodium channel and aquaporin-2 in rabbit kidney cortex. Am J Physiol 278:F530–F539, 2002 4. BINER HL, ARPIN-BOTT MP, LOFFING J, et al: Human cortical distal nephron: Distribution of electrolyte and water transport pathways. J Am Soc Nephrol 13:836–847, 2002 5. PETTY KJ, KOKKO JP, MARVER D: Secondry effect of aldosterone on Na-KATPase activity in the rabbit cortical collecting tubule. J Clin Invest 68:1514–1521, 1981 6. SHIMKETS R, WARNOCK DG, BOSITIS CM, et al: Liddles’s syndrome: Hertitable human hypertension caused by mutations in the b subunit of the epithelial sodium channel. Cell 79:407–414, 1994 7. CHANG SS, GRUNDER S, HANUKOGLU A, et al: mutations in subunits of the epithelial sodium channels cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nat Genet 12:248–253, 1996 8. STAUB O, GAUTSCHI I, ISHIKAWA T, et al: Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination. EMBO 16:6325–6336, 1997 9. LOFFING J, ZECEVIC M, FERAILLE E, et al: Aldosterone induces rapid apical translocation of ENaC in early portion of renal collecting system: possible role of SGK>. Am J Physiol 280:F675–F682, 2001 10. BHARGAVA A, FULLERTON MJ, MYLES K, et al: The serum- and glucocorticoid-induced kinase is a physiological mediator of aldosterone action. Endocrinology 142:1587–1594, 2001 11. KAMYNINA E, STAUB O: Concerted action of ENaC, Nedd4-2, and Sgk1 in transepithelial Na(+) transport. Am J Physiol 283:F377– F387, 2002 12. SCHAFER JA, TROUTMAN SL: cAMP mediates the increase in apical membrane Na+ conductance produced in the rat CCD by vasopressin. Am J Physiol 259:F823–F831, 1990 13. BLAZER-YOST BL, ESTERMAN MA, BUTTERWORTH MB, VLAHOS CJ: Localisation of ENaC and PI3-kinase in A6 cells: Effect of insulin. FASEB J 14:A96, 2000 14. HAWK CT, KUDO LH, ROUCH AJ, SCHAFER JA: Inhibition by epinephrine of AVP-and cAMP-stimulated Na+ and water transport in Dahl rat CCD. Am J Physiol 265:F449–F460, 1993 15. CANESSA CM, SCHILD L, BUELL G, et al: Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367:463–467, 1994 16. MASILAMANI S, KIM GH, MITCHELL C, et al: Aldosterone-mediated regulation of ENaC a, b and c subunit proteins in rat kidney. J Clin Invest 104:R19–R23, 1999 17. STOKES JB, SIGMUND RD: Regulation of rENaC mRNA by dietry NaCl and steroids: Organ tissue and steroid heterogeneity. Am J Physiol 274:C1699–C1707, 1998 18. TCHEPICHEV S, UEDA J, CANESSA C, et al: Lung epithelial Na channel subunits are differentially regulated during development and by steroids. Am J Physiol 269:C805–C812, 1995 19. SAYEGH R, AUERBACH SD, LI X, et al: Glucocorticoid induction of epithelial sodium channel expression in lung and renal epithelia occurs via trans-activation of a hormone response element in the 5 flanking region of the human epithelial sodium channel a subunit gene. J Biol Chem 274:12431–12437, 1999 20. RAMMINGER SJ, INGLIS SK, OLVER RE, WILSON SM: Hormone modulation of Na+ transport in rat fetal distal lung epithelial cells. J Physiol 544.2:567–577, 2002 21. NAKAMURA K, STOKES JB, MCCRAY PB JR.: Endogenous and exogenous glucocorticoid regulation of ENaC mRNA expression in developing kidney and lung. Am J Physiol 283:C762–C772, 2002 22. NICCO C, WITTNER M, DI STEFANO A, et al: Chronic exposure to vasopressin upregulates ENaC and sodium transport in the rat renal collecting duct and lung. Hypertension 38:1143–1149, 2001

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23. SWEEZY N, TCEPICHEV S, GAGNON S, et al: Female gender hormones regulate mRNA levels and function of the rat lung epithelial Na channel. Am J Physiol 274:C379–C386, 1998 24. ECKERT T, FORSLING ML, SCHWARZBERG H: The effect of combined oestrogen and progesterone replacement on the renal responses to oxytocin and vasopressin in ovariectomised rats. Eur J Endocrinol 141:297–302, 1999 25. HARTLEY DE, FORSLING ML: Renal response to arginine vasopressin during the oestrous cycle in the rat: Comparison of glucose and saline infusion using physiological doses of vasopressin. Exp Physiol 87:9–15, 2002 26. BARRON WM: Volume homeostasis during pregnancy in the rat. Am J Kidney Dis 9:296–302, 1987 27. PECHERE-BERTSCHI A, MAILLARD M, STALDER H, et al: Renal segmental tubular response to salt during the normal menstrual cycle. Kidney Int 61:425–431, 2002 28. BOYCE N, WARD J, ROSSER M, et al: The effect of reproductive status on the renal response to desmopressin (DDAVP) in normal women. J Physiol 531P:164P, 2001 29. STACHENFELD NS, SPLENSER AE, CALZONE WL, et al: Sex differences in osmotic regulation of AVP and renal sodium handling. J Appl Physiol 91:1893–1901, 2001 30. BLIGH-TYNAN ME, BHAGWAT SE, CASTONGUAY TW: The effect of chronic cold exposure on diurnal corticosteroid and aldosterone levels in Sprague-Dawley rats. Physiol Behav 54:363–367, 1993 31. BELLAMY D, BOULDING J, GRIFFITHS JW: Diurnal rhythms in sodium and potassium excretion in a highly inbred rat: an evaluation of the role of aldosterone. J Endocrinol 44, 1969 32. MULARONI A, BECK L, SADIR R, et al: Down-regulation by progesterone of CFTR in endometrial epithelial cells: A study by competative PCR. Biochem Biophys Res Commun 217:1105–1111, 1995 33. FULLER PJ, BRENNAN FE, BURGESS JS: Acute differential regulation by corticosteroids of epithelial sodium channel subunit and Nedd4 ¨ mRNA levels in the distal colon. Pflugers Arch 441:94–101, 2000 34. FARMAN N, BOCCHI B: Mineralocorticoid selectivity: Molecular and cellular aspects. Kidney Int 57:1364–1369, 2000 35. WILSON CA, JAMES MD, GRIERSON JP, Hole DR: Involvement of catecholaminergicsystems in the zona incerta in the steroidal control of gonadotrophin release and female sexual behaviour. Neuroendocrinology 53:113–123, 1991 36. QUINKLER M, BUMKE-VOGT C, MAYER B, et al: The human kidney is a progesterone-metabolizing and androgen producing organ. J Clin Endocrinol Metab 88:2803–2809, 2003 37. RAFESTIN-OBLIN ME, FAGART J, SOUQUE A, et al: 11 betahydroxyprogesterone acts as a mineralocorticoid agonist in stimulating Na+ absorption in mammalian principal cortical collecting duct cells. Mol Pharmacol 62:1306–1313, 2002 38. QUINKLER M, MAYER B, OELKERS W, DIEDERICH S: Renal inactivation, mineralocorticoid generation and 11 beta hydroxysteroid dehydrogenase inhibition ameliorate the antimineralocorticoid effect of progesterone in vivo. J Clin Endocrinol Metab 88:3767–3772, 2003 39. MCNICHOLAS CM, CANESSA CM: Diversity of channels generated by different combinations of epithelial sodium channel subunits. J Gen Physiol 109:681–692, 1997 40. JAIN L, CHEN XJ, RAMOSEVAC S, et al: Expression of highly selective sodium channels in alveolar type II cells is determined by culture conditions. Am J Physiol 280, 2001 41. BRUNETTE MG, LECLERC M: Effect of estrogen on calcium and sodium transport by the nephron luminal membranes. J Endocrinol 170:441–450, 2001 42. JOHNSON JA, DAVIS JO: The effect of estrogens on renal sodium excretion in the dog. Perspect Nephrol Hypertens 5:239–248, 1976 43. RAFESTIN-OBLIN ME, FAGART J, SOUQUE A, et al: 11betahydroxyprogesterone acts as a mineralocorticoid agonist in stimulating Na+ absorption in mammalian principal cortical collecting duct cells. Mol Pharmacol 62:1306–1313, 2002 44. FRONIUS M, REHN M, ECKSTEIN-LUDWIG U, CLAUSS W: Inhibitory non-genomic effects of progesterone on Na+ absorption in epithelial cells from Xenopus kidney (A6). J Comp Physiol 171:377–386, 2001