Regulation of rENaC mRNA by dietary NaCl and steroids: organ,
tissue, and steroid heterogeneity
John B.
Stokes and
Rita D.
Sigmund
Laboratory of Epithelial Transport, Department of Internal Medicine,
University of Iowa and Department of Veterans Affairs Medical Center,
Iowa City, Iowa 52242
 |
ABSTRACT |
Rats on a low-NaCl diet have a high
Na+ channel activity in colon and
kidney. To address the mechanism of this increased activity, we
measured mRNA levels of three Na+
channel subunits in epithelial tissue (rENaC) from rats having been fed
either a low (0.13%)- or high (8%)-NaCl diet for 2-3 wk. The
size of the mRNA for each of the rENaC subunits as determined by
Northern blot was unaffected by diet. RNase protection assay showed
heterogeneity of response by organs and subunit. In lung, there was no
effect of diet on any of the three subunits. In descending colon, the
low-NaCl diet increased
- and
-rENaC mRNA, with no effect on
-rENaC mRNA. In the kidney, the response to dietary NaCl was
dependent on the region. In cortex and outer medulla, diet had no
effect on any of the subunits. Rats fed the low-NaCl diet had greater
-rENaC in inner medulla but not
- or
-rENaC mRNA. We next
asked whether acute administration of pure glucocorticoid (GC) or
mineralocorticoid (MC) hormones to adrenalectomized rats reproduced the
effects of a low-NaCl diet. Six hours after administration of GC or MC,
a somewhat different heterogeneity occurred. In lung,
-rENaC mRNA
was increased but only in response to GC. In colon, either GC or MC
increased
- or
-rENaC, and there was no effect on
-rENaC. In
kidney, either GC or MC increased
-rENaC, without an effect on
-
or
-rENaC. In contrast to the response to a low-NaCl diet, all three
regions were similarly affected by acute steroids. These results
demonstrate a striking heterogeneity in response to physiological
stimuli that regulate ENaC function. The mRNA levels of each of the
rENaC subunits can be determined by the type of steroid and by factors
unique to the organ and even to the specific region of the kidney.
kidney; kidney medulla; colon; lung; sodium channel; aldosterone; RU-28362; dietary salt
 |
INTRODUCTION |
THE DISCOVERY AND MOLECULAR CLONING of the three
homologous proteins that comprise the epithelial
Na+ channel (ENaC) have provided
investigators with a critically important tool to further explore the
mechanisms involved in Na+
homeostasis (10, 11, 27). The ENaC complex is present in the descending
colon, distal nephron, and airway epithelia, where it permits the entry
of Na+ into the cell across the
apical cell membrane (9, 20, 39). The physiological regulation of
Na+ transport by cells of the
colon and renal collecting duct is greatly influenced by the amount of
dietary Na+ and circulating levels
of aldosterone (6, 20, 39). There is general agreement that a
low-Na+ diet increases
Na+ absorption by the target
epithelial cells, at least in part, by increasing circulating
aldosterone levels. The molecular details of the effects of low dietary
NaCl and the extent to which aldosterone can account for its effects on
target epithelial cells are incompletely understood.
Most or all of the effect of aldosterone on the apical
Na+ channel requires new protein
synthesis (2, 25). However, the specific proteins involved in the
regulation of electrogenic Na+
transport are not known. A leading possibility is the ENaC complex. The
simplest hypothesis linking dietary NaCl to the magnitude of
electrogenic Na+ transport would
be as follows. Aldosterone levels increase in response to dietary NaCl
restriction (3, 33). The occupied mineralocorticoid (MC) receptors
translocate to the nucleus, where they interact with hormone response
elements to increase ENaC mRNA transcription and subsequently ENaC
subunit protein synthesis. The increased amount of one or more of the
three subunit proteins increases the assembly of functional channels in
the apical membrane. More Na+
channels in the apical membrane increase the rate of
Na+ entry and thus the magnitude
of transepithelial Na+ transport.
This simple scenario does not exclude other important events, such as
alteration in the function of the
Na+ pump, as playing additional
roles in this process.
Portions of this hypothesis have begun to be examined. Elevated
steady-state mRNA levels for
- and
-subunits of rat ENaC (rENaC)
have been detected in colons from animals eating a low-NaCl diet (3,
34). Steroids have been implicated in ENaC mRNA regulation in lung, but
there is some disagreement about whether the target is the
-subunit
alone (38) or all three subunits (34). The effects of dietary NaCl and
steroids on ENaC mRNA in the kidney are unclear (3, 34).
The purpose of these studies was to test the idea that acute steroid
administration to adrenalectomized (ADX) rats would reproduce the
effects of chronic dietary NaCl restriction on steady-state mRNA levels
in lung, colon, and kidney. Because the collecting duct in the cortex,
outer medulla, and inner medulla serves somewhat different functions
(37), we also sought to determine the response to these maneuvers in
these three regions of the kidney. Finally, we designed these studies
to separate the acute effects of pure glucocorticoid (GC) hormone from
pure MC hormone.
 |
METHODS |
Preparation of rats.
For the studies comparing low- and high-NaCl diets, normal Wistar rats
of either sex were purchased from Harlan Sprague Dawley (Indianapolis,
IN) at the age of ~5 wk. On arrival at the University of Iowa, the
shipments were randomly divided into those to receive a low
(0.13%)-NaCl diet (ICN, Nutritional Biochemicals) or an otherwise
identical diet but high in NaCl (8%). After 2-3 wk, some rats
were placed in a metabolic cage, and their 24-h
Na+ and
K+ excretion rates were measured.
Rats eating the low-NaCl diet excreted 264 ± 18 µmol
Na+ and 1,207 ± 288 µmol
K+. Rats on the high-NaCl diet
excreted 6,481 ± 862 µmol
Na+ and 1,834 ± 242 µmol
K+.
Rats to be given steroids were obtained from Harlan Sprague Dawley 1 wk
after undergoing ADX. They were fed 0.9% NaCl solution to drink and a
normal rat diet (1.3% NaCl). ADX rats were maintained for 2-3
days at the University of Iowa Animal Care unit before being killed. On
the morning of the study, the rats weighed ~220 g and received either
the pure GC RU-28362 (1.0 mg/kg) or a combination designed to activate
MC receptors consisting of aldosterone (1.5 mg/kg) plus the GC
antagonist RU-38486 (1.8 mg/kg). Control animals received ethanol
vehicle only (400 µl/kg ip). These protocols were chosen because this
dose of RU-28362 saturates ~50% of the GC receptors, and this dose
of aldosterone plus RU-38486 saturates ~90% of the MC receptors
without crossover binding to the GC receptor (30). Organs from these
rats were harvested 6 h after steroid administration.
Preparation of RNA.
Rats were anesthetized with methoxyflurane and decapitated, and the
lung, descending colon, and kidneys were rapidly removed. Lungs were
immersed in liquid nitrogen. The descending colon was rinsed with
ice-cold PBS, and the most distal 1.5 cm were trimmed and frozen. The
kidneys were chilled and dissected into cortex, outer medulla, and
papilla (inner medulla). Small pieces (<0.25 cm3) of cortex and outer medulla
and the entire inner medulla were rapidly frozen in liquid nitrogen.
Total RNA was isolated from tissues by using the method of Chomczynski
and Sacchi (13). An amount of tissue estimated to yield 25-50 µg
total RNA was homogenized 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
addition of 2 M sodium acetate. After precipitation with isopropanol,
the RNA was rinsed with 75% ethanol and resuspended in diethyl
pyrocarbonate-treated water. RNA from the inner medulla was subjected
to an additional step by centrifugation through RNeasy (Qiagen,
Chatsworth, CA) to reduce contaminating materials that seem to be
unique to this tissue. This technique permits recovery of >90% of
starting RNA and greatly facilitates electrophoresis.
RNA analysis.
Northern blot analysis was conducted on lung, ascending and descending
colon, and each kidney region as previously described (40).
Approximately 25 µg total RNA was fractionated by electrophoresis through a 1.5% agarose-6.6% formaldehyde gel buffered with 10 mM
NaH2PO4
(pH 6.5) using a Hoefer HE-99 horizontal submarine unit (Hoefer
Instruments, San Francisco, CA). RNA was transferred onto nylon (Hybond
N, Amersham, Arlington Heights, IL) by capillary transfer in 10×
standard saline citrate and ultraviolet (UV) cross-linked (UV
Stratalinker, Stratagene, La Jolla, CA). The blots were prehybridized and hybridized in a buffer containing 1% BSA, 1 mM EDTA, 500 mM NaH2PO4
(pH 7.2), and 7% SDS according to Church and Gilbert (14).
The probes used for the Northern blots were those previously described
for
-,
-, and
-rENaC and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (40). These constructs were modified for the
RNase protection assay (RPA) as follows. The
-rENaC construct was a
422-nt segment subcloned into the pKS(
) vector (Stratagene) using restriction sites EcoR I and
Pst I. The
-rENaC construct was a
249-nt segment subcloned into the vector pCR-Script SK(+) (Stratagene)
using restriction sites Pst I and
Sac I. The
-rENaC construct was a
675-nt segment subcloned into the pCRII-TA vector (Invitrogen,
Carlsbad, CA) using restriction sites
Xba I and
Bst XI. The rat GAPDH construct was a
140-nt segment extending from the translation start site to the first
Sty I restriction site. All of the
probes were directed against segments within the open reading frame.
Antisense probes for the RPA were synthesized from the appropriate
constructs using the BrightStar BIOTINscript nonisotopic in vitro
transcription kit (Ambion, Austin, TX). The amount of biotin-labeled
CTP was adjusted to give the highest possible specific activity. The
lengths of the biotin-labeled, unprotected fragments were 480, 280, 750, and 220 nt for
-,
-,
-rENaC, and GAPDH, respectively
(Fig. 1).

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Fig. 1.
Northern blots of RNA from regions of kidney, colon, and lung from rats
fed a high (H)- or a low (L)-NaCl diet for 2 wk. Each blot hybridized
twice, first with rat epithelial
Na+ channel (rENaC) probe and
again with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe. KC,
kidney cortex; KOM, outer medulla; KIM, inner medulla; Asc Colon,
ascending colon; Des Colon, descending colon.
|
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The hybridization of ~1 ng of each of these probes with 25 µg total
RNA from each of the tissues was conducted using the RPAII RNase
protection assay kit (Ambion). RNase A and RNase T1 were diluted to 250 and 10,000 U/l, respectively, and the reaction was incubated for 18 h.
The products were subjected to electrophoresis through a 5% denaturing
polyacrylamide-8 M urea gel buffered with Tris borate for 2.5 h at 250 V and transferred to a nylon membrane (BrightStar Plus, Ambion) using a
semidry electroblotter (Fisher, Itasca, IL). The membrane was
subsequently UV cross-linked, and development of the protected RNA
fragments was conducted using the BrightStar Biodetect nonisotopic
detection kit (Ambion) with minor modifications. To reduce background
noise, the wash times after incubation with the streptavidin-alkaline
phosphatase conjugate solution were increased threefold. The developed
blots were exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY)
for 1-45 min depending on the intensity.
The
- and
-rENaC probes occasionally showed degradation products
when the amount of the respective mRNA was large. The magnitude of
these products was <10% of the completely protected fragment, and
the shorter fragments did not interfere with the ability to quantitate
any of the other bands of interest. Therefore all quantitation was
conducted on the major protected fragments.
The autoradiograms were quantitated with a PDI scanning densitometer
using Quantity One software (Huntington Station, NY). The exposure time
of the autoradiograms was adjusted so that the density of each of the
bands on a given gel fell into the linear range of the instrument. In
general, gels were arranged so that organs from randomly paired rats
were analyzed together. In this way, we could appreciate the
quantitative differences in the amount of each subunit in each of the
tissues as well as between treatment groups. An additional set of three
pairs of inner medullae from rats fed high- and low-NaCl diets were
analyzed without other tissues.
Statistical analysis was conducted by use of the Student's paired
t-test comparing treatment groups
analyzed in the same assay. All values were normalized to the GAPDH
band (exposed for a shorter period to account for considerably larger
amount of mRNA). In general, the GAPDH bands were exposed for 1.0 min
and all other bands for 15 min. The experimental conditions were
designed to test for increases in mRNA produced by a low-NaCl diet or
steroids; therefore a one-tailed analysis was used.
P < 0.05 was considered significant.
Materials.
Unless otherwise specified, chemicals were purchased from Sigma (St.
Louis, MO). Methoxyflurane was purchased from Mallinckrodt Veterinary
(Mundelein, IL) and BSA from Boehringer-Mannheim (Indianapolis, IN).
RU-28362 and RU-38486 were gifts from Roussel-UCLAF (Romainville, France), and the rat GAPDH construct was a gift from Dr. Christie Thomas (University of Iowa).
 |
RESULTS |
Effects of dietary NaCl on rENaC mRNA.
We initially used Northern blot analysis to assess the effect of
dietary NaCl on mRNA from lung, ascending and descending colon, kidney
cortex, and outer and inner medullae. An example is shown in Fig. 1. We
reached two preliminary conclusions. First, dietary NaCl intake had no
detectable effect on the size of
-,
-, or
-rENaC mRNA for any
of the organs. Second, there appeared to be a greater abundance of
rENaC mRNA in the descending colon and inner medulla of rats fed a
low-NaCl diet. We therefore proceeded to quantitate the abundance of
the
-,
-, and
-rENaC subunits by RPA.
Figure 2 shows a representative RPA of
-,
-, and
-rENaC mRNA from lung, descending colon, and three
regions of the kidney taken from rats fed either a low- or high-NaCl
diet. We did not perform extensive analysis on the ascending colon
because the amount of each transcript seemed to be too small to
quantitate reliably (and amount was variable). Table
1 displays the calculated ratios for all
RPAs factored for the GAPDH signal. There was no effect of dietary NaCl
on GAPDH mRNA (based on loading approximately same amount of RNA in
each lane). There was no effect of diet on any rENaC mRNA abundance in
lung. In descending colon,
- and
-rENaC were much greater in rats
fed the low-NaCl diet, but
-rENaC mRNA abundance was not different.
The dietary NaCl effects on the kidney depended on region. There was no
effect on any subunit in renal cortex or outer medulla. There was also
no effect of diet on
- or
-rENaC abundance in inner medulla.
However, rats fed the low-NaCl diet had greater
-rENaC mRNA
abundance in the inner medulla.

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Fig. 2.
Example of an RNase protection assay (RPA) for -, -, and
-rENaC and GAPDH. Rats were fed either H- or L-NaCl diet for 2 wk.
Labels on left show position of
protected fragments. Labels on right
show position and length of unprotected fragments. All such
autoradiograms were quantitated by scanning densitometry. Intensity of
GAPDH probe was measured from shorter exposures. DC, descending colon;
other abbreviations as in Fig. 1.
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Table 1.
Relative abundance of -, -, and
-rENaC mRNA in colon, lung, and kidney region in
rats fed either a low- or high-NaCl diet
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Table 1 also shows the relative tissue abundance of
-,
-, and
-rENaC mRNA factored for GAPDH expression. For rats fed a high-NaCl
diet, the abundance of
-,
-, and
-rENaC followed the general
rank order lung > descending colon > cortex > outer medulla > inner medulla. For rats fed a low-NaCl diet, there was the
same rank order for
-rENaC. However, in these rats, the descending colon had the lowest relative amount of
- and
-rENaC mRNA.
The relative amounts of
-,
-, and
-rENaC mRNA are graphically
shown in Fig. 3 for the two tissues in
which we detected an effect. The difference was most dramatic in
-
and
-rENaC mRNA from descending colon, where the low- to high-NaCl
ratio was 6.1 ± 2.5 for
-rENaC and 28 ± 12 for
-rENaC.
Interestingly, the pattern for the inner medulla was quite different.
Although there was no significant effect on
- or
-rENaC,
-rENaC mRNA abundance was 4.3 ± 1.1-fold higher in the low-NaCl
group.

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Fig. 3.
Mean ratios of -, -, or -rENaC mRNA from descending colon
(A) and inner medulla
(B) taken from rats fed either L- or
H-NaCl diet. Values are from RPA. All ratios factored for GAPDH
expression. Primary data in Table 1. In this analysis, L and H results
were analyzed in pairs according to each assay.
* P < 0.01 by paired
analysis.
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Acute effects of steroid hormones on rENaC mRNA.
We performed at least one Northern blot analysis on each rENaC subunit
for each tissue from untreated ADX rats and from ADX rats treated with
GC or MC. A sample of the results is shown in Fig.
4; treatments did not alter the apparent
length of the mRNA for any of the transcripts in any tissue. We
therefore proceeded with the quantitative RPA.

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Fig. 4.
Examples of Northern blots of RNA from regions of kidney, colon, and
lung from adrenalectomized (ADX) rats treated for 6 h with vehicle or
glucocorticoid (GC) or mineralocorticoid hormone (MC) as described in
METHODS.
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|
Table 2 shows the ratio of the mRNA
abundance for each of the subunits factored for GAPDH expression. In
contrast to the effects of low-NaCl diet, every tissue responded to at
least one steroid hormone. The mean ratios of GC to ADX and MC to ADX
for all tissues and subunits are shown in Fig.
5. It is clear that the most dramatic
effect is seen in the descending colon. GC and MC increased
-rENaC
mRNA 20- and 13-fold, respectively, and increased
-rENaC mRNA 12- and 5-fold, respectively. Neither GC nor MC had an effect on
-rENaC
mRNA.
In the lung, GC treatment but not MC treatment increased
-rENaC mRNA
(by 2.7-fold); there was no effect of either GC or MC on
- or
-rENaC mRNA. Each region of the kidney responded to GC and to MC,
but only
-rENaC mRNA, and not
- or
-rENaC, was increased. The
magnitude of the increase in the three regions of the kidney was
similar to the increase in the lung and smaller than the increase in
- or
-rENaC mRNA in descending colon. In contrast to the lung,
-rENaC mRNA in kidney was increased by both GC and MC, although the
magnitude of the stimulation by MC was consistently smaller than the
stimulation by GC.
 |
DISCUSSION |
These results demonstrate that dietary NaCl and acute steroids produce
a striking heterogeneous effect in the rENaC mRNA levels in lung,
colon, and kidney. The distal colon responds to a low-NaCl diet and
acute GC or MC exposure by greatly increasing the abundance of
- and
-rENaC but not
-rENaC mRNA. The inner medulla responds in a
completely different fashion. Dietary NaCl restriction or acute GC or
MC increases
-rENaC but not
- or
-rENaC mRNA. The responses in
the lung, kidney cortex, and outer medulla show variations on the
pattern of the inner medulla.
Colon.
There is now excellent agreement that dietary NaCl restriction or MC
agonists increase the abundance of
- and
-rENaC mRNA in colon,
with little or no effect on
-rENaC mRNA (3, 26, 34). Other
investigators have demonstrated a good temporal correlation between the
increase in Na+ current and the
magnitude of
-rENaC mRNA levels (26). Taken together, these results
lend strong support to the idea that a major mechanism of
aldosterone's action to increase electrogenic Na+ transport is mediated through
this increase in mRNA abundance.
The fact that GC administration had an effect on rENaC (in colon)
similar to MC is somewhat surprising. It is well known that large doses
of dexamethasone can increase electrogenic
Na+ transport (32) and
- and
-rENaC mRNA levels (34). However, this action of dexamethasone on
Na+ transport is mediated via the
MC receptor and thus is not strictly a GC effect (4). GC hormones,
administered to ADX rats in doses that allow binding exclusively to GC
(type II) receptors, stimulate electroneutral not electrogenic
Na+ transport (5, 7). In this
setting, pure GCs actually inhibit electrogenic
Na+ transport (8). Taken together,
these results suggest that an increase in
- and
-rENaC mRNA in
colonic epithelia will not inevitably cause an increase in electrogenic
Na+ transport. Clearly,
posttranscriptional events must play an important role in modulating
the steroid effects leading to altered
Na+ transport.
Lung.
The importance of the rENaC complex, and specifically the
-rENaC
subunit, to lung function is dramatically illustrated in the mouse with
a homozygous deletion of the
-ENaC gene. These mice are born
normally but die within 48 h because they fail to absorb fluid from
their lungs (22). The magnitude of
-rENaC mRNA detected by our RPA
is consistent with the amount detected by in situ hybridization in
airway epithelial cells (19, 29). The lack of change in rENaC abundance
by dietary NaCl restriction (Table 1) is consistent with that reported
by others (34).
Why are the rENaC mRNA subunits not regulated by dietary NaCl? A
teleological answer to this question would be that conservation of
Na+ and fluid is not a major
responsibility of the lung but, rather, of the kidney and colon. The
molecular explanation must incorporate our observations that neither
chronically elevated levels of aldosterone (from low-NaCl diet) nor
acute MC administration increases lung rENaC mRNA but that acute GC
administration does (Tables 1 and 2). The simplest explanation for the
lack of response of rENaC mRNA to aldosterone is that lungs do not have
MC receptors. However, MC receptors have been demonstrated in rat lung
tissue (1, 24), and aldosterone can hyperpolarize the membrane voltage in cultured type II alveolar cells (12).
How can we reconcile the presence of MC receptors in the lung with the
failure of aldosterone to produce an effect on rENaC mRNA abundance?
There are three possible explanations. First, the MC receptors might be
confined to cells that make little or no rENaC mRNA. In this regard, we
note that
-rENaC mRNA, the lung subunit responsive to GC (Table 2),
is present in nearly every epithelial cell (19, 29). However, there is
no information to date on the localization of MC receptors in lung
tissue. A second possibility is that the responses to MC receptor
occupancy in lung cells do not include activation of the system
responsible for increasing
-rENaC mRNA levels. This explanation
would invoke an unprecedented degree of diversity between the actions
of GC and MC receptors in the same cell. Finally, the amount of MC
receptor protein in lung tissue is small (24). Thus there may be too few receptors in airway epithelial cells to mount a significant physiological response to NaCl restriction.
The observation that lung ENaC mRNA does not respond to the MC actions
of aldosterone is not discrepant with the previous report of
aldosterone's action in cultured type II alveolar cells. Champigny et
al. (12) found that high concentrations of aldosterone increased
-rENaC mRNA and hyperpolarized membrane voltage. The effect of
aldosterone, at least on the mRNA levels, was inhibited by RU-38486, an
antagonist of the GC receptor (12). Thus, in these lung cells as in
others (36), aldosterone can activate GC receptors when used in high
enough concentrations. The failure of aldosterone to produce an effect
in the presence of a GC antagonist is consistent with the
interpretation that there are few MC receptors expressed in lung.
The acute response to GC infusion is consistent with an important role
for GCs in regulating
-rENaC mRNA. Other investigators (12, 31, 38)
have documented an upregulation of lung
-rENaC mRNA in response to
GCs. Longer exposure (2 days) to GCs in ADX rats appears to increase
levels of
- and
-rENaC mRNA as well (34). The most important
physiological role for these steroid effects is probably the induction
of Na+ channels at the time of
birth (31, 38). The correlation between the increase in production of
fetal steroids (28) and the increase in
-rENaC mRNA and surfactant
proteins (41) makes a compelling argument for a concerted effect of GCs
on lung maturation.
Kidney.
There is disagreement about the effect of aldosterone on rENaC mRNA
subunits in kidney cortex. Asher et al. (3) found that aldosterone or
dexamethasone infused into normal rats for 3 days increased
-rENaC
but not
- or
-rENaC mRNA. On the other hand, Renard et al. (34)
found that neither a low-NaCl diet nor 2 days of dexamethasone infusion
into normal rats produced a change in any rENaC mRNA abundance. The
failure to detect an effect of dietary NaCl or aldosterone on rENaC
mRNA in rat kidney cortex is surprising in view of the well-studied
effect of these maneuvers on electrogenic
Na+ transport by rat cortical
collecting duct (CCD), one of the major targets for aldosterone action
in the kidney (33, 35).
Our results indicate that there is no effect of chronic dietary NaCl
restriction on
-,
-, or
-rENaC mRNA abundance in renal cortex
or outer medulla. In contrast, there is an increase in
-rENaC, but
not
- or
-rENaC mRNA, from inner medulla (Table 1, Fig. 2). These
results uncover unexpected intrarenal heterogeneity in rENaC mRNA
regulation.
The observation that dietary NaCl restriction increases
-rENaC and
not
- or
-rENaC mRNA in inner medulla is consistent with our
previous reports demonstrating that aldosterone increases electrogenic
Na+ transport and
-rENaC mRNA
(but not
- or
-rENaC mRNA) of inner medullary collecting duct
(IMCD) cells in primary culture (23, 40). Thus the responses of the
intact inner medulla and the target cells for aldosterone's action in
this region of the kidney are similar.
Why is the response to dietary NaCl restriction different in the
regions of the kidney? In the cortex, rENaC mRNA is localized to both
the distal convoluted tubule (DCT) and the CCD (15, 18). It seems
possible that a large constitutive expression of rENaC mRNA in the DCT
could render a small but important effect of aldosterone in the CCD
undetectable in the entire tissue. Given the magnitude of the increase
in Na+ transport by the CCD in
response to NaCl restriction or to aldosterone (33, 35), such a
possibility seems unlikely. Furthermore, this heterogeneity does not
apply to the outer medulla, where NaCl restriction also had no effect.
It seems more likely that the differences in response to NaCl
restriction relate to the underlying function of ENaC in the cortex and
inner medulla.
In our view, a more plausible explanation for the different responses
in the cortex and inner medulla relates to the regional differences in
the role of rENaC in Na+ and
K+ balance. The CCD and DCT are
the major sites from which K+ is
secreted. K+ secretion requires
Na+ absorption through ENaC (37).
The IMCD does not share with the CCD the capacity for
K+ secretion (17, 23). Because
dietary NaCl restriction does not alter the requirements for
K+ secretion, it seems possible
that rENaC mRNA subunits in the DCT and CCD might be more directly
influenced by factors related to regulation of
K+ balance rather than
Na+ balance.
In contrast to the CCD, Na+
transport by the IMCD is related more to Na(Cl) balance than
K+ balance. From this teleological
perspective, one might suppose that dietary NaCl could have more of an
effect on rENaC mRNA in IMCD than in CCD. However, this functional
difference does not explain why CCD segments taken from animals with
high circulating MC levels absorb more
Na+ in vitro than do segments from
normal rats. The conclusion we draw from the aggregate of these results
is that an increase in rENaC mRNA abundance is not necessary for
aldosterone to increase Na+
transport by CCD. Therefore aldosterone must be producing other effects
to enhance Na+ transport by CCD.
Despite the difference in the regional response to chronic changes in
circulating aldosterone levels, all regions of the kidney are able to
respond to steroid hormones acutely, at least in ADX rats, in which the
basal level of circulating steroids is low (Table 2). These results,
taken together, suggest that basal levels of aldosterone and/or
corticosterone are necessary to produce sufficient rENaC mRNA to
respond to needs relating to K+
balance (in DCT and CCD) and that chronically elevated circulating levels of aldosterone produce different effects dependent on the specific target tissue.
General deductions.
These results reveal several levels of heterogeneity in the tissue
response to NaCl restriction and steroid hormones. The most striking
heterogeneity is the opposite responses of the descending colon and
inner medulla to NaCl restriction (Fig. 2). The intrarenal heterogeneity in the response to NaCl restriction but not to acute steroid administration provides evidence for another layer of complexity. The dissociation between rENaC mRNA levels and magnitude of
electrogenic Na+ transport in
response to steroids (in colon and kidney cortex) suggests that
posttranscription events are important in modulating the steroid
effects on Na+ transport.
The levels of heterogeneity might not be limited to tissues of the rat.
There is evidence that responses might be species dependent. For
example, cultured rabbit CCD cells respond to aldosterone by increasing
-rENaC mRNA (16), a response not detected in rat cortex (Fig. 3). In
the chicken, the intestinal response to dietary NaCl restriction
includes an increase in
-rENaC mRNA (21), a response quite different
from that of the rat colon (Fig. 2).
Despite the complexities of steroid hormone action on ENaC mRNA
abundance, the demonstration that a critically important mRNA can be
regulated by physiological maneuvers provides a valuable opportunity to
further explore the cellular mechanisms responsible for regulating
electrogenic Na+ transport.
 |
ACKNOWLEDGEMENTS |
We appreciate the assistance of Russ Husted, Chong Zhang, and Ken
Volk in preparing the tissue samples.
 |
FOOTNOTES |
This work was supported in part by National Institutes of Health Grants
HL-55006 and DK-52617 and a Merit Award from the Department of Veterans
Affairs.
Address for reprint requests: J. B. Stokes, Dept. of Internal Medicine,
University of Iowa College of Medicine, Iowa City, IA 52242.
Received 28 February 1997; accepted in final form 23 February
1998.
 |
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