Departments of 1 Internal Medicine and 2 Pediatrics, University of Iowa College of Medicine and Veterans Affairs Medical Center, Iowa City, Iowa 52242
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ABSTRACT |
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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 -ENaC mRNA in fetal lung and kidney explants within 24 h but had different effects on
- or
-ENaC. Dex increased
- and
-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
- and
-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
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INTRODUCTION |
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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: -,
-, and
-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
-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
- or
-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 -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
-ENaC
subunit, both
- and
-ENaC mRNAs also increase during the same
time period (44, 49, 51). In contrast to the lung, there
are more gradual prenatal increases in
-,
-, and
-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 -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
-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
-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
-and
-ENaC increase in the prenatal period
(albeit less dramatically than
-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, -ENaC, not
-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
-ENaC mRNA without any
effect on
- or
-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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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,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 - and
-ENaC (
- and
-mENaC)
were generously provided by Dr. Monica Driscoll. A 451-bp fragment of
-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
-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
-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).
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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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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Quantitative analysis of replicate experiments is shown in Fig.
2. For this analysis we expressed the
relative abundance of -,
-, and
-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
- and
-mENaC subunits in both wild-type and CRH KO mice significantly
increased with time. The increase in
-mENaC was significantly less
and only reached significance in lung; in the kidney
-mENaC failed
to increase in either group. These results demonstrate that the absence of CRH does not prevent the
- and
-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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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
-and
-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
-,
-, and
-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
-,
-,
-mENaC or NHE-3 in the kidney at E18.5.
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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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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 -,
-, and
-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
-mENaC and SP-B mRNAs, whereas the
induction for
- and
-mENaC subunits took longer than 24 h.
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In E15.5 kidney tissues grown under the same conditions as the lung
tissues, treatment with Dex increased -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
- or
-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 - and
-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
-,
-,
-mENaC, and SP-B mRNA and
normalized to CK18 abundance. As shown in Fig.
6, there were significant increases in
-,
-,
-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
-mENaC and NHE-3 mRNA expression. Thus Dex
increases
-mENaC mRNA within 24 h in both lung and kidney
explants. In contrast, steroids have a large effect on
- and
-mENaC in lung explants at 72 h and no effect on
- and
-mENaC in kidney explants. The observation that the Dex effects on
lung
- and
-mENaC require more than 24 h for their onset
suggests that the mechanism of this effect is different from the more
rapid response for
-mENaC.
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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 -mENaC and
SP-B mRNAs were detected at 48 and 96 h of Dex treatment (Fig. 7). After 96 h of Dex treatment, the
- and
-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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One implication of the data presented in Figs. 5-7 is that the
induction of lung - and
-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,
-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
-ENaC expression. Second,
24 h of steroid exposure was sufficient to increase
-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
-mENaC expression to increase, but a 48-h
exposure did. When measured immediately after 48 h of steroid
exposure,
-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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DISCUSSION |
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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 -mENaC message could be induced by Dex within 24 h
in vitro, but
- and
-mENaC were unresponsive to glucocorticoid stimulation. Whereas
-mENaC message in cultured E15.5 lung explants was also induced by 24 h of Dex in vitro, increases in
- and
-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 - and
-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 -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 -ENaC was stimulated by Dex
in the kidney explants in a similar fashion as in the lung (Fig. 5).
The time course of
-ENaC responsiveness in lung is most consistent
with direct effects of steroid receptor activation on transcription.
Analysis of the 5'-flanking sequence of the
-ENaC gene supports this
idea (31, 38). The mechanisms responsible for regulating
- and
-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 -ENaC expression in kidney because
-ENaC is clearly responsive
to exogenous glucocorticoids, but
- and
-ENaC expression are not
(Fig. 5B).
The third, and in our view the most likely, explanation is that the
differences in -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 11
-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 11
-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
11
-hydroxysteroid dehydrogenase activity in colon and kidney from
late-gestation rabbits and rats was oxidative and consistent with type
2 11
-hydroxysteroid dehydrogenase. In both the rat and rabbit kidney
there was a progressive increase in type 2 11
-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
-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
-mENaC mRNA abundance in lung. However, the lack of
glucocorticoid responsiveness by renal
- and
-mENaC cannot be
explained by metabolism of the steroid by type 2 11
-hydroxysteroid
dehydrogenase because Dex, a nonmetabolized glucocorticoid, increased
only
-mENaC and had no effect on
- or
-mENaC. This result
suggests a fundamental lack of glucocorticoid inducibility for these
subunits in the fetal kidney, regardless of gestational age.
The regulation of - and
-ENaC expression in the fetal lung is
significantly different from that of the kidney. In the lung, the
time-dependent increases in
- and
-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
- and
-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
- or
-mENaC mRNA expression, whereas both
-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 - and
-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
- and
-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
-
and
-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
-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 17
-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 -mENaC
induction are distinct from those for
- and
-mENaC induction.
Steroids may induce factors that subsequently increase lung
- and
-ENaC expression. In the kidney, glucocorticoids increased
-mENaC
expression in vitro but had no effect on
- and
-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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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