Endogenous and exogenous glucocorticoid regulation of ENaC mRNA expression in developing kidney and lung

Kenzo Nakamura1, John B. Stokes1, and Paul B. McCray Jr.2

Departments of 1 Internal Medicine and 2 Pediatrics, University of Iowa College of Medicine and Veterans Affairs Medical Center, Iowa City, Iowa 52242


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Lung liquid absorption at birth is crucial for the successful onset of respiration. Na absorption by the renal collecting duct plays an important role in renal fluid and electrolyte homeostasis during the early postnatal period. The epithelial Na channel (ENaC) plays a central role in mediating these functions, and its subunit expression is developmentally regulated in a temporal and tissue specific pattern. Several lines of evidence suggest that the prenatal increase in circulating glucocorticoids may play an important role in increasing ENaC expression during maturation. We tested the role of the prenatal surge using corticotropin-releasing hormone (CRH) knockout (KO) mice. Relative ENaC expression in lungs of KO mice increased at the same rate as in wild-type (WT) mice, but absolute expression was only 20-30% of WT. In contrast, relative and absolute expression of all three subunits in kidneys was not different between KO and WT mice. Dexamethasone (Dex) increased alpha -ENaC mRNA in fetal lung and kidney explants within 24 h but had different effects on beta - or gamma -ENaC. Dex increased beta - and gamma -ENaC in lung, but only after >48 h of exposure, and had no effect on kidney. The results suggest that the kidney metabolizes endogenous glucocorticoids, but the lung does not. Furthermore, the marked difference between lung and kidney responsiveness to glucocorticoids in beta - and gamma -ENaC expression suggests that factors other than steroids may be important in regulating functional ENaC expression during development.

epithelial sodium channel; dexamethasone; corticotropin-releasing hormone; mouse


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ELECTROGENIC SODIUM (Na) transport occurs across many epithelial surfaces and plays a key role in regulating salt and water absorption. The molecular pathway underlying this Na transport is the epithelial Na channel (ENaC), a multimeric complex consisting of three subunits: alpha -, beta -, and gamma -ENaC (9). Amiloride-sensitive Na transport mediated via ENaC plays a key role in regulating salt and liquid absorption in the perinatal period (21, 26, 28, 30). Lung liquid absorption at the time of birth is crucial for the successful onset of respiration, as demonstrated in mice deficient in the alpha -ENaC subunit (18). Similarly, Na absorption by collecting duct epithelia plays an important role in renal fluid and electrolyte homeostasis during the early postnatal period, as demonstrated by the development of lethal hyperkalemia in mice with defective beta - or gamma -ENaC subunits (5, 22). Immaturity of this mechanism of ion transport contributes to the salt wasting and fluid loss in preterm infants (1, 35-37, 42).

Previous in vitro and in vivo studies in a number of models show that ENaC subunit expression is developmentally regulated in the lung, kidney, and colon (reviewed in Ref. 17). Interestingly, the temporal pattern of ENaC subunit expression varies in each of these tissues. In the lung, there is a late gestational rise in alpha -ENaC expression that temporally correlates with the rise in fetal and maternal glucocorticoids (27, 44, 45, 51). Although the prenatal increase in mRNA abundance is most pronounced for the alpha -ENaC subunit, both beta - and gamma -ENaC mRNAs also increase during the same time period (44, 49, 51). In contrast to the lung, there are more gradual prenatal increases in alpha -, beta -, and gamma -ENaC expression in both the kidney and colon, with adult levels attained after the postnatal period (13, 46, 50, 51). These striking tissue-specific differences suggest that different regulatory stimuli may underlie the maturation of ENaC expression in each tissue.

One potentially important regulator of ENaC expression is glucocorticoid hormone. There are two lines of evidence supporting its role in the developmental expression of ENaC in lung. First, exogenous synthetic glucocorticoids strongly increase alpha -ENaC mRNA expression in adult lung, both in vitro (10, 48) and in vivo (34, 41, 45). When administered to mothers at 17-19 days (but not at 16 days) gestation, dexamethasone also increases alpha -ENaC expression in the fetus (27). Second, there is a strong correlation between the increase in the prenatal endogenous glucocorticoid levels and the level of alpha -ENaC mRNA (3, 13, 15, 44, 45, 47, 52). However, there is reason to suspect that endogenous glucocorticoids cannot account completely for lung ENaC development. Whereas beta -and gamma -ENaC increase in the prenatal period (albeit less dramatically than alpha -ENaC), exogenous glucocorticoids do not increase expression of these subunits in fetal lung (45) or in adrenalectomized adults (34, 41). In addition, the human fetal lung appears to have a much different pattern, with all subunits responsive to steroids in midgestation (48).

The evidence that endogenous glucocorticoids play an important role in developmental ENaC expression in the kidney is less convincing than that of the lung. The time course of ENaC expression in neonatal kidney does not parallel the prenatal rise in endogenous glucocorticoids as it does in the lung (50, 51). Furthermore, beta -ENaC, not alpha -ENaC, is the predominant subunit in the fetal kidney. This relative abundance of subunit expression is different from that produced by administration of exogenous synthetic glucocorticoids in adult adrenalectomized rats, which increase alpha -ENaC mRNA without any effect on beta - or gamma -ENaC expression (34, 41).

At the present time, there is no explanation why the prenatal ENaC expression pattern in response to exogenous glucocorticoids varies in different tissues. One possibility is that the prenatal glucocorticoid surge is not necessary for the increased ENaC expression; the relationship may be only circumstantial. Another possibility is that glucocorticoids are necessary for ENaC expression but that other factors, produced by each target tissue, modify the response to the endogenous glucocorticoids. Yet another possibility is that some tissues might metabolize endogenous glucocorticoids (and not synthetic glucocorticoids) and thus produce tissue-specific effects.

To test the idea that the prenatal surge in glucocorticoids is important for ENaC expression in lung and kidney, we used the corticotropin-releasing hormone (CRH)-deficient mouse. CRH knockout (KO) mice have a greatly blunted ability to increase circulating corticosterone levels and, without supplementation, die in the neonatal period from respiratory insufficiency (24, 25, 47). We also used tissue explants from normal mice to evaluate in vitro responsiveness to glucocorticoids. The results demonstrate a dependency on endogenous glucocorticoid production and tissue-specific responsiveness to endogenous glucocorticoids.


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INTRODUCTION
METHODS
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Tissue Preparation

CRH KO mice were obtained from Dr. Louis Muglia at Washington University (24). A breeding colony of CRH KO mice was maintained at the University of Iowa. Females in estrus were mated with stud males. The presence of a vaginal plug on the morning after the female was introduced into the male cage was set as embryonic (E) day 0.5 (E0.5). To produce CRH KO mice for breeding, we administered corticosterone at a concentration of 30 µg/ml in the drinking water of CRH KO pregnant females beginning on E12.5 and continuing until the pups were weaned 21 days postpartum. This level of supplementation allows for lung maturation and viable offspring (24). Mothers were euthanized with pentobarbital sodium, and the fetuses were removed and dissected in a sterile petri dish on ice. Fetal kidney and lung tissues were obtained on E16.5, E17.5, and E18.5. Fetal tissues were pooled for analysis as follows: E16.5, 2 lungs and 8-12 kidneys; E17.5, 1 lung and 4-7 kidneys; and E18.5, <FR><NU>2</NU><DE>3</DE></FR> lung and 3-6 kidneys. Pooled samples were used only for a single measurement. The protocol was approved by the Animal Care and Use Committee of the University of Iowa.

Preparation of RNA

The kidney and lung tissues were rapidly removed and immersed directly in liquid nitrogen. Total RNA was isolated from tissues by using the method of Chomczynski and Sacchi (11). An amount of tissue estimated to yield 25-50 µg of total RNA was homogenized by using a 92/Polytron PT-DA 3012/2 TS probe in 1.0 ml of the guanidinium isothiocyanate solution for 3-5 s, and the RNA was extracted in phenol-chloroform acidified to pH 4.0 by the addition of 2 M sodium acetate. After precipitation with isopropanol, the RNA was rinsed with 75% ethanol and resuspended in diethyl pyrocarbonate-treated water (41).

RNase Protection Assays

Probe design. Plasmid constructs for mouse alpha - and beta -ENaC (alpha - and beta -mENaC) were generously provided by Dr. Monica Driscoll. A 451-bp fragment of alpha -mENaC was provided in the pCRII vector (Invitrogen, Carlsbad, CA). When linearized with BsmF1, an antisense riboprobe was generated by using T7 polymerase to yield unprotected and protected fragments of 192 and 125 nt, respectively. The protected fragment encompassed bp 1589-1713 (GenBank accession no. AF112185). A 1,651-bp fragment of beta -mENaC was subcloned into the pCRII vector (Invitrogen). When linearized with BsaB1, an antisense riboprobe was generated by using SP6 polymerase to yield unprotected and protected fragments of 345 and 265 nt, respectively. The protected fragment encompassed bp 1396-1660 (GenBank accession no. NM011325). A 630-bp fragment of gamma -mENaC was PCR amplified and cloned into the pKRX vector (gift of Dr. Brian Schutte). When linearized with Sty1, an antisense riboprobe was generated by using T7 polymerase to yield unprotected and protected fragments of 232 and 191 nt, respectively. The protected fragment encompassed bp 1794-1984 (GenBank accession no. NM011326).

A 158-nt fragment of mouse endo B cytokeratin (CK18; homologous to human cytokeratin 18), corresponding to nt 731-888 of the coding region (GenBank accession no. M11686), was PCR amplified and cloned into the pCR- BluntII-TOPO vector (Invitrogen). The plasmid was linearized by SpeI to allow for transcription of an unprotected fragment of 263 nt and a protected fragment of 159 nt. A 613-nt fragment of mouse surfactant protein B (mSP-B; gift of Dr. Jeanne Snyder) was subcloned into pBluescriptII SK(-) (Stratagene, La Jolla, CA) using the EcoRI site. The plasmid was linearized by StyI to allow for transcription of an unprotected fragment of 460 nt and a protected fragment of 384 nt, corresponding to nt 247-629 of the coding region. To prepare a mNHE-3 construct, we designed primers to the rat NHE-3 sequence (GenBank accession no. M85300) and PCR amplified a 423-nt fragment, corresponding to nt 1129-1551 of the coding region (PCR primers for rNHE-3: forward, 5'-CTTCATGTTCCTGGGCATCT-3'; reverse, 5'-ATGGCTG- AGAGGATGTGGTC-3'). The 423-nt product was cloned into the pCR-BluntII-TOPO vector. When linearized with SpeI, transcription of this construct yielded an unprotected fragment of 528 nt and a protected fragment of 423 nt.

Antisense riboprobes for the ribonuclease protection assay (RPA) were synthesized from the appropriate constructs by using the BrightStar BIOTINscript nonisotopic in vitro transcription kit or the MAXIscript in vitro transcription kit (Ambion). The amount of biotin-labeled CTP was adjusted to give the highest possible specific activity except for the mSP-B and 18S constructs. In pilot experiments the mSP-B signal overshadowed all other signals. Therefore, the activity was decreased by adding a nonbiotinylated (cold) probe at a ratio of 24:1 (unlabeled to labeled), and we confirmed that the signal intensity was saturated under these conditions. We used an 18S antisense probe to normalize for the amount of RNA assayed. The human 18S cDNA consisted of an unprotected fragment of 110 nt. Because the abundance of the 18S mRNA is several orders of magnitude greater than that of the mENaC subunits, we constructed separate biotin-labeled and unlabeled antisense RNA probes. On the basis of extensive preliminary testing, we determined that a mixture of 1:1,000 (labeled to unlabeled, total of 1 µg of antisense RNA) saturated the 18S mRNA in the reaction and provided a signal that was easily detectable under our experimental conditions. The molar ratio of 18S probe to 18S mRNA was 5:1.

The hybridization of ~1 ng of each of the mENaC, CK18, and mNHE-3 probes, ~25 ng of mSP-B probe, and 1 µg of 18S probe with 25 µg of total RNA from each of the tissues was conducted by using the RPA II kit (Ambion). Hybridization was conducted overnight at 45°C. Digestion with RNase A and RNase T1 was conducted at 37°C for 30 min. The products were subjected to electrophoresis through a 5% denaturing polyacrylamide/8 M urea gel buffered with Tris borate for 2 h at 250 V and transferred to a nylon membrane (BrightStar Plus, Ambion) by using a semidry electroblotter (Fisher, Itasca, IL). The membrane was subsequently ultraviolet cross-linked (UV Stratalinker, Stratagene), and development of the protected RNA fragments was conducted by using the BrightStar Biodetect nonisotopic detection kit (Ambion). The developed blots were exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) for 1-45 min depending on the intensity of the product. The autoradiograms were quantitated with a scanning densitometer by using Kodak Digital Science 1D image analysis software (Eastman Kodak). The exposure time of the autoradiograms was adjusted so that the density of the majority of the bands on a given gel fell into the linear range of the instrument.

Culture of embryonic lung and kidney explants. Fetal lung and kidney tissues were isolated at E15.5 from timed pregnant wild-type C57BL/6 mice. Harvested tissues were rinsed in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 nutrient (F-12) containing antibiotics (penicillin and streptomycin) and antimycotics (amphotericin B). Lung tissue explants were minced into 2- to 4-mm cubes and then cultured on 0.4-µm polyethylene terephthalate membranes (Falcon) in six-well multiwell plates (Costar). Lung and kidney tissues were cultured in a 1:1 mixture of DMEM and F-12 supplemented with 1 µg /ml bovine serum albumin and 10 µg/ml transferrin with antibiotics (penicillin and streptomycin) (32, 54). Approximately six to seven lung tissue explants and eight to nine whole kidneys were placed on the single membrane and cultured in 5% CO2 at 37°C. Culture medium was changed every 48 h. Explants were harvested at 24, 48, 72, and 96 h as indicated, immersed in liquid nitrogen, and stored at -80°C. Total RNA was isolated from the explants as described in Probe design. Each isolation (4-6 lung explants, 15-18 kidney explants) yielded ~25 µg of total RNA for use in a single assay. To normalize the signal between assays, we extracted RNA from E18.5 mouse lung and kidney and pooled sufficient amounts to run a normalization lane in each assay. Thus each measured mRNA level was normalized to the 18S probe intensity and the amount of ENaC in the 18.5-day controls.

Statistical Analysis

Values are reported as means ± SE. Statistical analysis was conducted by using paired or unpaired t-test or ANOVA with subsequent one-sample t-test as indicated. Statistical significance was concluded when P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Influence of CRH KO Phenotype on Ontogeny of mENaC mRNA Expression in Embryonic Lung and Kidney Tissues

Figure 1 shows a representative RPA contrasting ENaC expression in wild-type and CRH KO mice in lung and kidney at E16.5, E17.5, and E18.5, corresponding to periods of peak corticosterone levels in developing mice (6, 14). In both wild-type and CRH KO mice there was a gestation-dependent increase in ENaC expression in both lung and kidney. Preliminary examination of these data suggested tissue-specific differences in expression, because the levels of lung ENaC expression in CRH KO mice appeared to be lower than in wild-type mice.


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Fig. 1.   RNase protection assay (RPA) for mouse epithelial Na channel alpha -, beta -, and gamma -subunits (alpha -, beta , and gamma -mENaC) in lung and kidney at embryonic days 16.5, 17.5, and 18.5 (E16.5, E17.5, and E18.5). This representative autoradiograph demonstrates increases in expression with development and contrasts expression in wild-type and corticotropin-releasing hormone knockout (CRH KO) mice.

Quantitative analysis of replicate experiments is shown in Fig. 2. For this analysis we expressed the relative abundance of alpha -, beta -, and gamma -mENaC in lung and kidney normalized to the level of expression at E18.5 for both wild-type and CRH KOs. In both lung and kidney, the mRNA expression for the alpha - and gamma -mENaC subunits in both wild-type and CRH KO mice significantly increased with time. The increase in beta -mENaC was significantly less and only reached significance in lung; in the kidney beta -mENaC failed to increase in either group. These results demonstrate that the absence of CRH does not prevent the alpha - and gamma -mENaC mRNA abundance from increasing with time. The relative increases in mRNA abundance seem to be comparable in the two groups of mice.


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Fig. 2.   Relative abundance of alpha -, beta -, and gamma -mENaC for CRH KO and wild-type mice normalized to 18S RNA and to the abundance at E18.5. Data for lung and kidney are shown. ** Increase in expression for both wild-type and CRH KO mice (P < 0.05). The results were the same whether the comparisons were made by 2-way ANOVA comparing E16.5 and E17.5 or by one-sample t-test comparing E16.5 and E18.5 (wild type, n = 5; CRH KO, n = 6).

The initial analysis of ENaC subunit expression in wild-type and CRH KO mice (Figs. 1 and 2) suggested that although there were increases in alpha -and gamma -mENaC expression with time, the absolute amounts might be different. Because these experiments were not designed to make direct comparisons, we conducted another set of experiments to compare the two groups at E18.5. We included additional genes, surfactant protein B for the lung (29) and NHE-3 for the kidney (17), because these genes are regulated by glucocorticoids. As shown in Fig. 3A, the relative abundance of alpha -, beta -, and gamma -mENaC and SP-B were all significantly reduced in the lung of CRH KO mice to ~18-33% of wild-type levels at E18.5. This marked reduction in message abundance implies that there is a requirement for the increase in glucocorticoid production in the maturation of mENaC expression in the lung. In contrast to the results in the lung, Fig. 3B shows that there were no significant differences in mRNA abundance between wild-type and CRH KO mice for alpha -, beta -, gamma -mENaC or NHE-3 in the kidney at E18.5.


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Fig. 3.   A: relative abundance of alpha -, beta -, gamma -mENaC and SP-B in lung at E18.5 in CRH KO mice compared with wild type. B: relative abundance of kidney alpha -, beta -, gamma -mENaC and NHE-3 at E18.5 in CRH KO mice compared with wild type. Values are means ± SE; n = 6 for alpha -, beta -, and gamma -mENaC, n = 3 for SP-B and NHE-3. * P < 0.01 (by one-sample t-test).

These results can be explained by either 1) a reduction of ENaC and SP-B mRNA abundance in lung epithelial cells (and not in kidney distal nephron cells) or 2) a failure of CRH-deficient mice to reduce the lung interstitial cell population normally. Because the mRNA abundance (in both lung and kidney) was normalized to total 18S RNA, changes in the relative proportion of epithelial cells would result in parallel changes in relative amounts of ENaC and SP-B mRNA. The latter possibility seemed plausible given the lung histology of the CRH KO mice (24, 25).

To address these possibilities directly, we measured the abundance of the mENaC subunit and SP-B mRNA together with CK18, an epithelium-specific gene product (53), in the lungs of control and CRH KO mice. Figure 4A shows a representative RPA from these experiments and Fig. 4B shows the quantitation. Whereas the reduction in mENaC and SP-B gene expression in the CRH KO mice is clear, CK18 mRNA abundance was unaffected. These data indicate that the relative proportion of epithelial cells is similar in normal and CRH KO mice. They imply that the diminution in ENaC and SP-B expression is due to a reduction in the amount of the expression in epithelia.


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Fig. 4.   A: representative RPA for alpha -, beta -, gamma -mENaC, SP-B, cytokeratin 18 (CK18), and 18S mRNAs in lung at E18.5. B: abundance of alpha -, beta -, gamma -mENaC, SP-B, and CK18 in lung at E18.5. Values are means ± SE; n = 5 wild type, n = 4 CRH KO. * P < 0.05 (by unpaired t-test).

Glucocorticoid Actions on Fetal Lung and Kidney Explants

The marked difference between the ENaC maturation in lung and kidney between normal and CRH KO mice indicates that the prenatal surge in circulating glucocorticoids is essential for full expression in the lung but not in the kidney. One possible explanation for this difference is that the fetal kidney metabolizes corticosterone to an inactive metabolite whereas the fetal lung does not. To test this hypothesis, we incubated tissue explants from E15.5 fetuses with dexamethasone (Dex), a relatively nonmetabolizable synthetic glucocorticoid.

As shown in Fig. 5A, lung tissues exposed to Dex showed an approximately two- to fourfold increase in alpha -, beta -, and gamma -mENaC mRNA expression by 96 h in culture. Interestingly, the glucocorticoid effect on SP-B was smaller, only 1.5-fold increased at 96 h. The glucocorticoid effect was evident within 24 h for alpha -mENaC and SP-B mRNAs, whereas the induction for beta - and gamma -mENaC subunits took longer than 24 h.


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Fig. 5.   A: effect of 100 nM dexamethasone (Dex) on the abundance of alpha -, beta -, gamma -mENaC and SP-B in wild-type fetal lung explants dissected and cultured at E15.5. Data are normalized to E18.5 wild-type lung prepared from pooled samples and run with each individual assay. * P < 0.05, Dex- vs. Dex+ (by unpaired t-test; n = 6 for Dex- and Dex+). B: effect of 100 nM Dex on the abundance of alpha -, beta -, gamma -mENaC and NHE-3 in fetal kidney explants dissected and cultured at E15.5. Data are normalized to E18.5 wild-type kidney. * P < 0.05, Dex- vs. Dex+ (by unpaired t-test; n = 3 for Dex- and Dex+).

In E15.5 kidney tissues grown under the same conditions as the lung tissues, treatment with Dex increased alpha -mENaC mRNA abundance (Fig. 5B), which remained significantly greater than in control tissues for the duration of the experiment. Dex treatment had no significant effect on the abundance of beta - or gamma -mENaC. Kidney explants treated with Dex showed only a trend toward an increase in NHE-3 mRNA.

We also tested whether the observed differences in Dex responsiveness between lung and kidney explants were due to tissue-specific effects on cell abundance. The specific question was similar to the one we addressed in the native lung: whether the increase in beta - and gamma -mENaC mRNA seen at the late time points in lung might represent a steroid-induced reduction in the number of nonepithelial cells. We cultured E15.5 lung explants with or without Dex for 72 h and quantitated expression of alpha -, beta -, gamma -mENaC, and SP-B mRNA and normalized to CK18 abundance. As shown in Fig. 6, there were significant increases in alpha -, beta -, gamma -mENaC and SP-B mRNAs in Dex-treated lung explants when normalized to CK18. In contrast, Dex-treated E15.5 kidney explants only showed increases in alpha -mENaC and NHE-3 mRNA expression. Thus Dex increases alpha -mENaC mRNA within 24 h in both lung and kidney explants. In contrast, steroids have a large effect on beta - and gamma -mENaC in lung explants at 72 h and no effect on beta - and gamma -mENaC in kidney explants. The observation that the Dex effects on lung beta - and gamma -mENaC require more than 24 h for their onset suggests that the mechanism of this effect is different from the more rapid response for alpha -mENaC.


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Fig. 6.   Effects of Dex on gene expression in cultured lung and kidney explants. Tissues were harvested at E15.5 and cultured for 72 h. Quantitation was normalized to CK18 for lung and 18S mRNA for kidney. * P < 0.05 (by paired t-test; n = 3 for Dex- and Dex+).

Because tissue responsiveness to steroids may be dependent on gestational age (45), a similar experimental protocol was followed for E13.5 lung tissues. Significant increases in alpha -mENaC and SP-B mRNAs were detected at 48 and 96 h of Dex treatment (Fig. 7). After 96 h of Dex treatment, the beta - and gamma -mENaC mRNAs showed a trend toward an increase, but this reached only marginal statistical significance. Thus ENaC induction in fetal lung of earlier gestational age was less glucocorticoid responsive. These results demonstrate that both the time in culture and the gestational age of the explant are important variables in assessing developmental regulation of ENaC.


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Fig. 7.   Effects of Dex on gene expression in cultured lung explants. Tissues were harvested at E13.5 and cultured for up to 96 h. Quantitation was normalized to 18S mRNA for each lane and for E18.5 lung for each assay. * P < 0.05 (n = 3-4 for Dex- and Dex+).

One implication of the data presented in Figs. 5-7 is that the induction of lung beta - and gamma -ENaC requires Dex exposure for a time period >24 h. To directly address this issue, we exposed wild-type E15.5 lung explants to 100 nM Dex for 24, 48, or 87 h. In the groups treated for 24 or 48 h, the media were changed to a steroid-free medium for the remainder of the 87 h incubation. As shown in Fig. 8, the pattern of ENaC expression was significantly different from that determined when expression was measured immediately following steroid exposure (Fig. 5A). There are several key features. First, alpha -mENaC expression was enhanced by steroids even after steroids had been withdrawn for 63 h. Thus sustained steroid exposure does not appear to be necessary to maintain elevated alpha -ENaC expression. Second, 24 h of steroid exposure was sufficient to increase gamma -mENaC expression (measured at 87 h) when an increase was clearly not evident at 24 h (Fig. 5A). Third, this same 24-h treatment did not cause beta -mENaC expression to increase, but a 48-h exposure did. When measured immediately after 48 h of steroid exposure, beta -mENaC expression was not increased (Fig. 5A). Finally, expression of mSP-B was not elevated unless steroids were present continuously. This result implies that withdrawal of steroids might result in a decline in SP-B expression, because 24 h of steroid exposure does increase expression (Fig. 5A).


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Fig. 8.   Effect of duration of Dex exposure on ENaC mRNA expression in E15.5 lung explants. E15.5 lung explants were exposed to 100 nM Dex for 24, 48, or 87 h, and ENaC mRNA expression was measured by RPA at 87 h. * P < 0.05 vs. no Dex (by 1-way ANOVA, followed by Student-Newman-Keuls test; n = 2).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present studies demonstrate a marked developmental heterogeneity in the sensitivity of mENaC mRNA expression to the effects of prenatal glucocorticoids in lung and kidney. Using complementary in vivo and in vitro models, we found that fetal glucocorticoid deficiency in CRH KO mice is associated with a significant decrease in the mRNA abundance for all ENaC subunits in late gestation lung, although relative increases in abundance with time were similar. In contrast to the findings in lung, there was no change in the prenatal maturation of kidney mENaC expression in the CRH KO mice. Kidney alpha -mENaC message could be induced by Dex within 24 h in vitro, but beta - and gamma -mENaC were unresponsive to glucocorticoid stimulation. Whereas alpha -mENaC message in cultured E15.5 lung explants was also induced by 24 h of Dex in vitro, increases in beta - and gamma -mENaC mRNA were delayed 48-72 h in their onset. These results show that mENaC expression is developmentally regulated in a temporal and tissue-specific manner, suggesting different, subunit-specific regulatory mechanisms.

During fetal life there is a late-gestational surge in systemic glucocorticoids in mammals including rats, mice, and humans (6, 12, 14, 15, 52). This late-gestational increase in glucocorticoids is temporally correlated with an increase in transcription of many gene products, including SP-A, SP-B, and NHE-3 (3). The failure of CRH KO mice born to CRH KO mothers to successfully transition from fetal to neonatal life due to respiratory problems suggests an absolute requirement for steroids for normal lung development (24). Recent work by Venihaki et al. (47) with the CRH KO model shows that this requirement can be met with steroids of fetal or maternal origin. The observed ontogeny of ENaC subunits in mouse kidney was similar to that previously reported in the rat (17, 46, 50). In general, ENaC maturation in the kidney is more gradual than in the lung, with peak levels occurring postnatally (17, 46, 50). Our finding that mENaC mRNA levels in E18.5 CRH KO and wild-type kidney are equivalent suggests that there is little requirement for the late-gestational increase in corticosterone for the prenatal ENaC maturation in the kidney. The pattern of ENaC expression in the developing mouse lung is similar to that previously reported in the rat by O'Brodovich and colleagues (27, 45) and by our group (51). Of note, these comparative studies in normal and CRH KO mice indicate a glucocorticoid dependence for beta - and gamma -ENaC expression that contrasts with the findings of Tchepichev et al. (45). The present studies in the CRH KO model support the notion that this increase in steroids is required for the appropriate maturation of mENaC mRNA expression in the fetal lung but not in the kidney.

What is the mechanism for the differences in responsiveness of alpha -ENaC to glucocorticoid induction in lung and kidney? We have considered three possible explanations: 1) resistance of the kidney to glucocorticoids, 2) quantitative differences in the responsiveness of the lung and kidney to steroids, and 3) metabolism of glucocorticoids by kidney and not lung.

First, it is possible that the prenatal kidney is resistant to glucocorticoids. However, an intrinsic fetal kidney resistance to glucocorticoids seems unlikely because alpha -ENaC was stimulated by Dex in the kidney explants in a similar fashion as in the lung (Fig. 5). The time course of alpha -ENaC responsiveness in lung is most consistent with direct effects of steroid receptor activation on transcription. Analysis of the 5'-flanking sequence of the alpha -ENaC gene supports this idea (31, 38). The mechanisms responsible for regulating beta - and gamma -ENaC transcription are less well understood.

Second, there may be quantitative differences in the requirements for steroid-induced ENaC expression. The lower levels of endogenous corticosterone (in CRH KO mice) did not have any effect on the abundance of any ENaC mRNA in the kidney (Fig. 3). Because the CRH KO mice do not have a complete absence of glucocorticoid production (47), it seems possible that low levels of endogenous steroids play a permissive role in renal ENaC expression. This scenario would require that low levels of glucocorticoids play a selective role in alpha -ENaC expression in kidney because alpha -ENaC is clearly responsive to exogenous glucocorticoids, but beta - and gamma -ENaC expression are not (Fig. 5B).

The third, and in our view the most likely, explanation is that the differences in alpha -ENaC maturation in vivo are caused by tissue-specific differences in the metabolism of endogenous glucocorticoids. In aldosterone-responsive tissues such as renal collecting duct and colon, corticosterone (and cortisol) is oxidized and inactivated by 11beta -hydroxysteroid dehydrogenase type 2 (7, 23). The observed differential effects of corticosterone on fetal tissues in the CRH KO mice may well reflect the activity of this metabolizing enzyme in kidney as early as E15.5. There is evidence to support the notion that 11beta -hydroxysteroid dehydrogenase type 2 activity is greater in fetal kidney than in lung in both mice (8) and humans (16, 20, 39, 40). Hundertmark et al. (19) recently reported that the predominant 11beta -hydroxysteroid dehydrogenase activity in colon and kidney from late-gestation rabbits and rats was oxidative and consistent with type 2 11beta -hydroxysteroid dehydrogenase. In both the rat and rabbit kidney there was a progressive increase in type 2 11beta -hydroxysteroid dehydrogenase activity in late gestation (19). Thus the activity of this enzyme in the fetal kidney could explain the lack of effect of circulating corticosterone on alpha -mENaC expression. Conversely, the lack of activity of this enzyme in lung might explain the correlation between the increase in circulating corticosterone and the rise in alpha -mENaC mRNA abundance in lung. However, the lack of glucocorticoid responsiveness by renal beta - and gamma -mENaC cannot be explained by metabolism of the steroid by type 2 11beta -hydroxysteroid dehydrogenase because Dex, a nonmetabolized glucocorticoid, increased only alpha -mENaC and had no effect on beta - or gamma -mENaC. This result suggests a fundamental lack of glucocorticoid inducibility for these subunits in the fetal kidney, regardless of gestational age.

The regulation of beta - and gamma -ENaC expression in the fetal lung is significantly different from that of the kidney. In the lung, the time-dependent increases in beta - and gamma -ENaC mRNA were less in the CRH KO mice than in wild-type controls, whereas there was no difference in kidney (Fig. 3). The in vitro studies in lung explants revealed a 24- to 48-h lag in the Dex responsiveness of beta - and gamma -mENaC mRNA expression (Fig. 5A), whereas Dex had no such effect on renal explants (Fig. 5B). In E13.5 lung explants, treatment for up to 96 h caused only a marginal increase in beta - or gamma -mENaC mRNA expression, whereas both alpha -mENaC and SP-B mRNA induction occurred within 48 h. These results are consistent with the idea, proposed by Tchepichev et al. (45), that the responsiveness of lung ENaC to steroids is dependent on a certain degree of maturation.

Why is there a time lag for the lung beta - and gamma -mENaC subunit induction in vitro? The observation that their mRNA abundance did not increase until 48 or more hours after steroid exposure (in vitro, Fig. 5A) makes it highly likely that glucocorticoids do not directly enhance transcription. Rather, it seems more likely that in the fetal lung other genes must be activated for glucocorticoids to stimulate ENaC mRNA expression. This idea is supported by the finding that beta - and gamma -mENaC are only marginally induced in E13.5 lung explants after 96 h of Dex treatment. This lack of effect might be caused by a failure of the appropriate complementary gene products to be induced. Additional evidence for other factors being involved comes from the steroid withdrawal experiments (Fig. 8). When E15.5 lungs were treated for only 24-48 h and the steroid was withdrawn, lung beta - and gamma -ENaC mRNA was increased at 87 h. However, this induction must have occurred between 48 and 87 h, because 24-48 h of steroid exposure did not produce a similar increase (Fig. 5A). The alpha -ENaC mRNA showed a similar tendency to display sustained elevated levels even after steroid removal. This pattern of response is markedly different from SP-B, which is clearly induced by steroids with 24 h of Dex (Fig. 5A) but upon withdrawal of the steroid does not demonstrate sustained elevations at 87 h (Fig. 8). These results are consistent with the idea that steroids induce the lung to produce some factors, the production of which is sustained even without the continued presence of steroids. In this regard, we note that other factors may exert permissive effects on the induction of ENaC, such as oxygen tension (2, 4, 33), progesterone, and 17beta -estradiol (43).

In summary, these results indicate remarkable, tissue-specific glucocorticoid requirements for the prenatal induction of ENaC subunit expression. In the lung, all subunits are glucocorticoid responsive. Furthermore, the findings indicate that the pathways for lung alpha -mENaC induction are distinct from those for beta - and gamma -mENaC induction. Steroids may induce factors that subsequently increase lung beta - and gamma -ENaC expression. In the kidney, glucocorticoids increased alpha -mENaC expression in vitro but had no effect on beta - and gamma -mENaC induction. Thus, in contrast to the fetal lung, the kidney either lacks the posited steroid-inducible regulatory factors or steroids do not regulate these factors in kidney.


    ACKNOWLEDGEMENTS

We thank Louis Muglia and Joseph Majzoub for generously providing the CRH KO mice. We thank Andrea Penisten and Rita Sigmund for excellent technical assistance.


    FOOTNOTES

This work was supported in part by The O'Brien Kidney Disease Center, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52617 (J. B. Stokes and P. B. McCray, Jr.), the Children's Miracle Network Telethon (P. B. McCray, Jr.), National Heart, Lung, and Blood Institute Grant HL-55006 (J. B. Stokes), and a Department of Veterans Affairs Merit Award (J. B. Stokes). Services were also provided by University of Iowa Diabetes and Endocrinology Research Center Grant DK-25295.

Address for reprint requests and other correspondence: P. B. McCray, Jr., Dept. of Pediatrics, Univ. of Iowa College of Medicine, Iowa City, IA 52242 (E-mail: paul-mccray{at}uiowa.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

June 5, 2002;10.1152/ajpcell.00029.2002

Received 20 January 2002; accepted in final form 29 April 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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