Mechanisms of inactivation of the action of aldosterone on
collecting duct by TGF-
Russell F.
Husted,
Rita D.
Sigmund, and
John B.
Stokes
Department of Internal Medicine, University of Iowa, and
Department of Veterans Affairs Medical Center, Iowa City, Iowa
52242
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ABSTRACT |
The
purpose of these experiments was to investigate the mechanisms whereby
transforming growth factor-
(TGF-
) antagonizes the action of
adrenocorticoid hormones on Na+ transport by the rat inner
medullary collecting duct in primary culture. Steroid hormones
1) increased Na+ transport by three- to fourfold,
2) increased the maximum capacity of the
Na+-K+ pump by 30-50%, 3)
increased the steady-state levels of the
1-subunit of
the Na+-K+-ATPase by ~30%, and 4)
increased the steady-state levels of the
-subunit of the rat
epithelial Na+ channel (
-rENaC) by nearly fourfold.
TGF-
blocked the effects of steroids on the increase in
Na+ transport and the stimulation of the
Na+-K+-ATPase and pump capacity. However, there
was no effect of TGF-
on the steroid-induced increase in mRNA levels
of
-rENaC. The effects of TGF-
were not secondary to the decrease
in Na+ transport per se, inasmuch as benzamil inhibited the
increase in Na+ transport but did not block the increase in
pump capacity or Na+-K+-ATPase mRNA. The
results indicate that TGF-
does not inactivate the steroid receptor
or its translocation to the nucleus. Rather, they indicate complex
pathways involving interruption of the enhancement of pump activity and
activation/inactivation of pathways distal to the steroid-induced
increase in the transcription of
-rENaC.
sodium transport; inner medullary collecting duct; epithelial
sodium channel; sodium-potassium-adenosinetriphosphatase; benzamil; glucocorticoid; mineralocorticoid; Northern blot; ribonuclease
protection assay; electrophysiology
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INTRODUCTION |
THE MOLECULAR ACTIONS OF STEROID hormones that produce
an increase in electrogenic Na+ transport by
high-resistance epithelia remain poorly understood, despite extensive
investigation over several decades. Recently, we reported that
transforming growth factor-
(TGF-
) can render the rat inner
medullary collecting duct (IMCD) unresponsive to the natriferic effects
of mineralocorticoid and glucocorticoid hormones (13, 14). The unique
features of this action of TGF-
include 1) no effect on
Na+ transport within 30 min of TGF-
exposure, 2)
no effect on the capacity for Cl
secretion in
response to cAMP, and 3) prolonged resistance to steroids for
>2 days after removal of TGF-
from the media.
Increasing evidence points to the possible physiological relevance of
the effects of TGF-
on IMCD function in vivo. First, there is a
considerable amount of TGF-
1 in the inner medulla normally. The amount of TGF-
mRNA is much higher in the inner medulla than in the cortex of normal rats (20), and the amount of
TGF-
protein is greater in the inner medulla than in the cortex, as
assessed by immunocytochemistry (24). Second, recent evidence indicates
that dietary NaCl can regulate the amount of TGF-
in the kidney: a
high-salt diet increases TGF-
production and excretion (42).
Finally, TGF-
production is increased in several disorders of renal
function. One of the best studied is obstructive uropathy, where acute
and chronic obstruction produces time-dependent and regional changes in
TGF-
(6, 21, 24). This increased production of TGF-
may play a
role in the well-described phenomenon of aldosterone resistance in some
cases of urinary obstruction.
These results suggest that TGF-
can antagonize the action of
aldosterone in vivo. This interaction may play an important role in the
low level of Na+ absorption by IMCD dissected from rodents
treated with mineralocorticoid hormone (16, 25, 30). The reason for the
relatively high amounts of TGF-
in the inner medulla is not clear.
One possibility is to downregulate the actions of aldosterone on the
IMCD in the salt-replete state. In this regard, it is important to note
that a major difference between the cortical collecting duct (CCD), where there is little TGF-
, and the IMCD is the capacity for K+ secretion. Whereas the CCD responds to aldosterone by
secreting large amounts of K+, the IMCD does not possess an
intrinsic capacity for active K+ secretion (31). It seems
possible that varying the responsiveness of the collecting duct to
aldosterone within regions of the kidney might effect regional
differences in Na+ absorption. Thus TGF-
might play a
role in regulating Na+ balance by modulating regional
responsiveness to aldosterone.
The present studies were conducted to gain insight into the actions
produced by aldosterone and the mechanisms whereby TGF-
antagonizes
these actions. We focused on the apical membrane entry pathway, the
epithelial Na+ channel (ENaC), and the basolateral
Na+-K+ pump, because these molecules play the
dominant role in regulating the steroid-induced effects (34). We
conducted the studies in three phases: 1) the actions of
steroids alone, 2) the actions of steroids on IMCD cells, where
Na+ entry was inhibited with the amiloride analog benzamil,
and 3) the action of aldosterone in the presence of TGF-
.
The results provide insights into molecular mechanisms whereby
Na+ transport by the collecting duct might be regulated.
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METHODS |
Preparation of monolayers.
Primary cultures of IMCD cells were prepared from 4- to 5-wk-old Wistar
rats by the hypotonic lysis isolation method, as previously described
by this laboratory (11, 12, 18). The inner medulla was dissected,
minced, and incubated in an isotonic solution containing 0.1%
collagenase for 2-3 h. The solution was made hypotonic by addition
of two volumes of distilled water containing 10 µg/ml DNase, and
cells were recovered after two centrifugation steps. This isolation
procedure usually yielded 20-40 12-mm monolayers from 6 kidneys.
Cells were seeded onto collagen-coated Millicell PCF filters
(Millipore). Seeding density was 20 µg DNA/12-mm filter or 140 µg
DNA/30-mm filter (~350,000 cells/cm2). Cells were grown
for 3 days in a medium composed of a 1:1 mixture of DMEM-Ham's F-12
supplemented with 50 µg/ml gentamicin, 20 µg/ml norfloxacin, 5 pM
triiodothyronine, 50 nM cortisol, 5 µg/ml transferrin, 5 µg/ml
bovine insulin, 10 nM sodium selenite, and 1% (wt/vol) bovine albumin.
On the 3rd day the medium was changed to one from which cortisol,
norfloxacin, and albumin were omitted. After 24 h in the steroid-free
medium, the monolayers were confluent, as evidenced by a transmonolayer
electrical resistance (RT) of >100
/cm2. Where indicated, each isolation was randomized to
a treatment group by a Latin square procedure according to the
short-circuit current (Isc).
Monolayers were exposed to 100 nM dexamethasone and 10 µM
spironolactone, a mineralocorticoid receptor antagonist (GC), 100 nM
aldosterone and 10 µM RU-38486, a glucocorticoid receptor antagonist (27) (MC), or vehicle (control, ethanol) for 24 h. These treatments provide nearly complete occupancy of the glucocorticoid and
mineralocorticoid receptors with minimal crossover occupancy (12).
Electrical measurements.
RT and Isc were initially
measured under sterile conditions by placement of 12-mm Millicell
filters into modified Ussing chambers (Jim's Instruments, Iowa City,
IA). Measurements were made in media without additives at 37°C with
a University of Iowa voltage clamp (11, 12, 18). A positive
Isc indicates a flow of positive charges from the
apical to the basal surface. Electrical measurements for pump current
were made in nonsterile chambers designed to accommodate Millicell PCF
filters. Amphotericin B (30 µM) was added to the apical solution to
permeablize the apical membrane to monovalent ions (15). The solution
used to measure pump current contained (in mM) 25 NaCl, 120 tetramethylammonium hydroxide, 90 gluconic acid, 5 sodium
HEPES, 5 acid HEPES, 1.5 CaCl2, 1.0 MgCl2, 5 BaCl2, and 5 D-glucose, pH 7.4. After steady
state was achieved, ouabain (2 mM) was added to the basolateral
solution. The ouabain-sensitive current was taken as a measure of the
maximum capacity of the pump to transport Na+, as
previously described (15).
Northern blot analysis of the
1- and
1-subunits of the
Na+-K+-ATPase.
Total RNA was prepared from IMCD monolayers grown on 30-mm filters by
the acid guanidinium thiocyanate-phenol-chloroform extraction method,
as described previously (4, 37). Each 30-mm filter yielded
~20-25 µg of RNA. This RNA was dissolved in water, denatured, and subjected to electrophoresis through a 1.5% agarose-6.6%
formaldehyde gel at 100 V for 2 h at 18°C. The RNA was transferred
by capillary electrophoresis to Hybond N nylon membrane (Amersham,
Arlington Heights, IL) and cross-linked (UV Stratalinker, Stratagene,
La Jolla, CA).
The rat
1 and
1 cDNA clones were a kind
gift from Jerry Lingrel (29). We used actin or glyceraldehyde
3-phosphate dehydrogenase (GAPDH) to normalize for loading. We also
determined loading by densitometry of the ribosomal bands on the
agarose gel by ethidium bromide staining. None of the treatments
affected GAPDH or actin mRNA abundance. The cDNA was excised from the
cloning vector by use of the appropriate restriction endonuclease and
gel purified using Geneclean (Bio 101, Vista, CA). The probes were
radiolabeled with [
-32P]dCTP (3,000 Ci/mmol)
with use of a random prime DNA labeling kit (Boehringer-Mannheim). The
hybridization procedure proceeded as described previously (5, 37).
After hybridization, the bands were quantitated using scanning
densitometry with Quantity One software (PDI, Huntington Station, NY).
The exposure of the autoradiograms was adjusted so that the density of
each of the bands fell into the linear range of the instrument.
RNase protection assay.
The constructs used for the RNase protection assay (RPA) were those
previously described for
-,
-, and
-subunits of rat ENaC
(rENaC) and for GAPDH (37) and modified for RPA (32). The
-rENaC
construct was a 422-nt segment, the
-rENaC construct was a 249-nt
segment, and the
-rENaC construct was a 190-nt segment. The
-rENaC fragment was shortened from that previously reported (37) by
using the BsrF1 restriction site. The rat GAPDH construct was a 140-nt
segment extending from the translation start site to the first
Sty I restriction site. All the probes represented unique
sequences directed against segments within the open reading frame.
Antisense probes for the RPA were synthesized from the appropriate
constructs with 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, 453, and 220 nt for
-,
-, and
-rENaC and GAPDH, respectively.
The hybridization of ~1 ng of each of these probes with RNA from a
single 30-mm filter (~25 µg total RNA) was conducted using the
RPAII RPA kit (Ambion). After RNase treatment, 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) with use of a semidry electroblotter (Fisher, Itasca, IL). The membrane was subsequently ultraviolet cross-linked (UV Stratalinker, Stratagene), and the protected RNA fragments were developed using the BrightStar Biodetect nonisotopic detection kit (Ambion) with minor modifications. To reduce
background, 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
-rENaC probe 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.
Statistics.
Data were analyzed by paired analysis or by ANOVA, as indicated. When
data were found to be inhomogeneous by Bartlett's test, analyses were
conducted on logarithmically transformed values. When multiple
comparisons were made, a Bonferroni correction was used. Significance
was assumed at P < 0.05.
Materials.
Culture medium was obtained through the University of Iowa Diabetes and
Endocrinology Research Center. Benzamil was a generous gift from Merck
(West Point, PA). TGF-
1 was purchased from R & D Systems
(Minneapolis, MN). All other chemicals and reagents were purchased from
Sigma Chemical (St. Louis, MO).
 |
RESULTS |
Effect of steroids on
Na+-K+-ATPase.
Figure 1 shows the effect of GC or MC
treatment for 24 h on the Na+ transport rates and maximum
pump capacity. Steroids increased Isc, a measure of
Na+ transport by intact monolayers (12, 18), by 3.3-fold
(Fig. 1A). Steroids also increased the maximum capacity of the
Na+ pump by 30-50% (Fig. 1B). In the steady
state the rate of Na+ transport across the apical membrane
(through the Na+ channel) must be equal to the rate of exit
through the basolateral membrane Na+ pump. It is clear that
steroids increased the transcellular Na+ transport to a
greater extent than they increased the maximum capacity of the pump.
Therefore, steroids increased the fraction of the maximal capacity at
which the pump functioned from 21% to ~50% (Fig. 1C). The
effect of GC was not different from that of MC. These results are
similar to those we reported for IMCD monolayers isolated from Dahl
salt-sensitive and -resistant rats (15) and imply that steroids
increase the entry of Na+ across the apical membrane to a
greater extent than they increase the capacity of the Na+
pump.

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Fig. 1.
Effect of 24 h of glucocorticoid (GC) or mineralocorticoid (MC)
treatment on Na+ transport by inner medullary collecting
duct (IMCD) monolayers. A: effect on Na+ transport
across intact monolayers. B: effect on maximum capacity of
Na+ pump (after apical membrane had been treated with
amphotericin B to greatly increase its permeability). C:
fraction of maximal capacity at which Na+ pump operates in
intact monolayers (i.e., ratio of individual values in A to
individual values in B). Values are means ± SE; n = 32 monolayers from 6 isolations for each treatment. ** P < 0.01 compared with no steroid. There is no difference between GC and MC
treatment.
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We measured the effect of GC and MC on the mRNA abundance of the
1- and
1-subunits of the
Na+-K+-ATPase by Northern blot analysis. Figure
2 shows an example of a series where MC and
GC effects were examined at 3, 8, and 24 h. Inasmuch as the maximal
effect of steroids on Na+ transport and mRNA abundance
seemed to occur at ~24 h, we continued the analyses using this time
point. As shown in Fig. 3, both steroid treatments produced a modest (27-39%) but significant increase in
the abundance of the
1-subunit of
Na+-K+-ATPase mRNA. There was a tendency for
steroid treatment to increase the abundance of the
1-subunit of Na+-K+-ATPase mRNA,
but the effect was not statistically significant.

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Fig. 2.
Northern blot demonstrating effect of GC or MC on amount of
1-subunit of Na+-K+-ATPase mRNA.
RNA was isolated at 3, 8, or 24 h after steroid exposure. C, no steroid
treatment; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
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Fig. 3.
Effects of 24 h of treatment with GC or MC on abundance of
1- (A) and 1-(B) subunits
of Na+-K+-ATPase mRNA. Dashed lines, abundance
of RNA in monolayers not treated with steroids. All values were
normalized to GAPDH or actin. Values are means ± SE; n = 10 sets ( 1) and 12 sets ( 1). ** P < 0.01 compared with no steroid by paired (ratio) analysis.
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Interaction of steroids and blockade of
Na+ entry.
In analyzing the action of an agent that inhibits Na+
transport on the intermediate steps leading to alteration of transport, it is important to assess the effects of steroids under conditions where the increase in the rate of Na+ transport is
prevented by other means. Hence, we examined the effect of steroids on
IMCD monolayers that had been treated with 1 µM benzamil to inhibit
Na+ transport.
Figure 4A shows the effect of 24 h
of benzamil treatment with and without steroid treatment. In the
absence of benzamil, GC and MC had their usual stimulatory effect on
Na+ transport. Benzamil produced a reduction in
Isc in all groups (~80%). The magnitude
of Isc is larger in monolayers treated with
steroids and benzamil than in monolayers treated with benzamil alone.
We are not certain of the reason for this difference. We intentionally
used a low concentration of benzamil (1 µM) to reduce the possibility
of benzamil producing effects other than inhibition of the
Na+ channels. It is possible that benzamil may be partially
metabolized by these cells over the 24 h of treatment and thus may have
less active product available for inhibition of the Na+
channel.1 We think it unlikely
that steroids are stimulating Cl
secretion, which
would produce a benzamil-insensitive current, inasmuch as we previously
showed that steroids do not enhance this current in IMCD cells (14). In
any event, treatment with benzamil produced what we had planned: a
large reduction in the magnitude of Na+ transport in
steroid-treated monolayers.

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Fig. 4.
Effect of 24 h of treatment with 1 µM benzamil on Na+
transport by IMCD monolayers with or without steroids. A:
benzamil inhibits magnitude of Na+ transport by ~80% in
all groups. # P < 0.0001. B: effect of benzamil
and steroids on pump current. Benzamil produced a small inhibitory
effect in all groups (# P < 0.01) but did not prevent
steroids from stimulating pump current (** P < 0.0001).
There was no interaction between benzamil and steroids by 2-way ANOVA.
Values are means ± SE; n = 12 monolayers from 3 isolations
for each treatment.
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Figure 4B shows the effect of these treatments on the magnitude
of the pump current. There are three points. First, benzamil did not
prevent steroids from stimulating the pump current (P < 0.0001). There was no difference in the ability of MC or GC to
stimulate pump current with or without benzamil. Second, benzamil modestly reduced the maximum pump current (by ~14%) irrespective of
whether the monolayers were treated with steroids (P < 0.01). Third, there was no interaction between steroids and benzamil by
two-way ANOVA. Thus benzamil had a small inhibitory effect on maximum
pump current but did not interfere with the ability of steroids to
produce stimulation.
The effect of benzamil on the abundance of the
Na+-K+-ATPase subunit mRNA is shown in Fig.
5. In general, benzamil had no effect on
the abundance of the
1- and
1-subunits of
Na+-K+-ATPase mRNA. However, there was a single
exception. Benzamil increased the abundance of the
1-subunit of Na+-K+-ATPase mRNA
in monolayers treated with MC, but not in those treated with GC. The
same tendency was seen for the
1-subunit of
Na+-K+-ATPase mRNA, but the effect did not
reach statistical significance. These results suggest that the increase
in the maximum capacity of the pump produced by steroids is not
secondary to an increase in Na+ transport. This effect
appears to be a direct steroid effect.

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Fig. 5.
Effect of 1 µM benzamil (Benz) on abundance of 1- and
1-subunits of Na+-K+-ATPase
mRNA. Values represent ratio of each steroid group treated with
benzamil to same steroid group without benzamil. Dashed line, identity.
* P < 0.05. Values are means ± SE; n = 8 monolayers for each 1 analysis and 7 assays for each
1 analysis.
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Interaction of TGF-
1 and steroid
treatment of IMCD monolayers.
Figure 6 shows the effect of
TGF-
1 on Na+ transport by intact IMCD
monolayers. When monolayers were treated with MC and
TGF-
1 together, the increase in Na+
transport produced by MC was largely prevented (Fig. 6A). This result is similar to that which we previously reported (13). We did not
study the effect of TGF-
1 in monolayers not treated with
steroids (in this group of experiments), inasmuch as the effect is
small or negligible (13).

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Fig. 6.
Effect of 24 h of treatment with MC hormone and transforming growth
factor- 1 (TGF- 1) on Na+
transport by IMCD monolayers. A: MC stimulated Na+
transport 3.9-fold. ** P < 0.01. Monolayers treated with
MC and TGF- 1 (10 ng/ml) had Na+ transport
values not different from group without steroid. B: MC
stimulated maximum capacity of Na+ pump (* P < 0.05), and TGF- 1 prevented MC stimulation. C:
fraction of maximum capacity at which pump operates in intact cells is
increased by MC. This increase is eliminated by simultaneous treatment
with TGF- 1. ** P < 0.01. Values are
means ± SE; n = 12 monolayers from 3 isolations.
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As shown in Fig. 6B, whereas MC stimulated maximum pump current
by ~30%, monolayers treated with both MC and TGF-
1
had pump currents not different from those without steroid. The ratio
of the Isc to the maximum capacity, the fraction of
the maximal capacity at which the pump operates in intact monolayers
(Fig. 6C), was increased by MC, and the increase was eliminated
by TGF-
1.
The effect of steroid and TGF-
1 treatments on
Na+-K+-ATPase subunit abundance is shown in
Fig. 7. We combined the results of experiments with GC or MC treatments, inasmuch as there were no differences. Steroid treatment increased the abundance of the
1-subunit of Na+-K+-ATPase mRNA
by 33%, whereas there was no effect on the abundance of the
1-subunit of Na+-K+-ATPase mRNA
(similar to Fig. 3). TGF-
1 in the absence of steroid had
no effect on the abundance of either subunit. However,
TGF-
1 prevented the steroid-induced increase in the
abundance of the
1-subunit of
Na+-K+-ATPase mRNA.

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Fig. 7.
Effect of steroid hormone and TGF- 1 on abundance of
1- and 1-subunits of
Na+-K+-ATPase mRNA. A:
TGF- 1 eliminated increase in abundance of
1-subunit of Na+-K+-ATPase mRNA
produced by steroids. * P < 0.05 compared with all other
groups. Values are means ± SE; n = 6 monolayers for
each 1 analysis and 10 monloayers for each
1 analysis (B).
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The effect of MC and TGF-
1 on rENaC mRNA abundance is
shown in Figs. 8 and
9. As we showed previously using a
Northern blot analysis (37), MC increased the abundance of
-rENaC
mRNA but had no effect on
- or
-rENaC mRNA. TGF-
1
in the absence of steroid had no effect on the abundance of the mRNA of
any of the subunits. In contrast to its effect on the
1-subunit of Na+-K+-ATPase mRNA,
TGF-
1 had no effect on the ability of MC to increase
-rENaC mRNA abundance.

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Fig. 8.
RNase protection assay of -, -, and -subunits of rat
epithelial Na+ channel (rENaC) mRNA demonstrating effect of
no steroid or TGF- 1 (Control), mineralocorticoid (MC)
hormone, and TGF- 1 on IMCD cells. RNA was isolated after
24 h of exposure. Left, length of undigested (Undig) probes;
right, length of digested probes.
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Fig. 9.
Effect of MC and TGF- 1 on -, -, and -rENaC
subunit mRNA abundance. ** P < 0.02 compared with
no-steroid group. Values are means ± SE; n = 8 experiments.
TGF- 1 had no effect on mRNA abundance of any subunit.
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DISCUSSION |
The present results begin to address the mechanisms involved in the
resistance of distal tubular epithelia to the action of steroid
hormones. To determine how steroid hormone action might be antagonized,
we first needed to establish the actions of steroid hormone on primary
cultures of IMCD cells. Steroids increase Na+ transport by
1) increasing the maximum capacity of the
Na+-K+ pump (Fig. 1), 2) increasing the
abundance of the
1-subunit of
Na+-K+-ATPase mRNA modestly without changing
the abundance of the
1-subunit of
Na+-K+-ATPase mRNA (Fig. 3), and 3)
increasing the abundance of the
-rENaC mRNA but not the
- or
-rENaC mRNA levels (Fig. 9).
Steroid hormones and the
Na+-K+
pump.
These effects of GC and MC on the pump are somewhat different from
those reported for other model systems. In A6 cells, aldosterone produces a greater increase in the
1-subunit of
Na+-K+-ATPase mRNA, as well as a two- to
threefold increase in the
1-subunit of
Na+-K+-ATPase mRNA (35, 36). Studies in vivo
have shown that adrenalectomy reduces the abundance of the
1-subunit, but not the
1-subunit, of
Na+-K+-ATPase mRNA in the distal nephron and
colon (7, 8, 33). Glucocorticoid hormone, but not aldosterone, acutely
increases the
1-subunit of
Na+-K+-ATPase mRNA levels in colon of normal
rats (9), and aldosterone infusion restores the modest reduction in the
1-subunit of Na+-K+-ATPase mRNA
produced by adrenalectomy (7). A low-NaCl diet increases modestly the
mRNA abundance of the
1- and
1-subunits of Na+-K+-ATPase in colon (40). Neither
adrenalectomy nor aldosterone has more than a modest effect on
Na+-K+-ATPase mRNA of the CCD (7). In contrast
to Na+-transporting epithelia, glucocorticoid produces a
40-fold increase in the
1-subunit of
Na+-K+-ATPase mRNA in a liver cell line while
producing a less than twofold effect on the abundance of the
1-subunit of Na+-K+-ATPase mRNA
(3). It is clear that the effects of steroids on
Na+-K+-pump mRNA levels are cell and tissue specific.
The present demonstration of an increase in the functional capacity of
the Na+-K+ pump with steroid treatment (Fig. 1)
is consistent with our previous results demonstrating an increase in
ouabain binding after GC or MC exposure (18). Similar increases in
ouabain binding occur in the colon of rabbits fed a low-NaCl diet (40)
or in aldosterone-treated A6 cells (2). The results support a general
model where steroids increase transcription of one or more of the
Na+-K+-ATPase subunits (albeit modestly),
increase pump synthesis (10), and insert more pumps on the basolateral membrane.
Interactions of Na+ entry
and steroids on the
Na+-K+
pump.
The precise molecular mechanisms that lead to the increase in pump
activity resulting from steroid stimulation of Na+
transport are poorly understood. Considerable evidence suggests that
intracellular Na+ concentration can influence this process
(1, 17, 41). There is also evidence that the increase in transcription
of the Na+-K+-ATPase subunits in A6 cells does
not require an increase in protein synthesis or Na+
transport (35, 36). Our results generally support the idea that
steroids can increase pump capacity without a concomitant increase in
intracellular Na+ concentration, because GC or MC increases
pump capacity in the presence or absence of benzamil (Fig. 4). Although
blocking Na+ entry with benzamil had a modest effect on the
basal pump capacity, the change in pump capacity produced by GC or MC
is the same with or without benzamil (Fig. 4). This result is in marked
contrast to those reported by Palmer et al. (23) for the rat CCD. They found that amiloride caused a marked reduction in the magnitude of the
pump current that was stimulated by aldosterone. The reasons for this
difference are not clear, but we suggest two possibilities. First, the
use of cultured vs. native cells might influence the nature of the
response. Second, the IMCD and CCD respond quite differently to dietary
NaCl restriction with respect to ENaC mRNA levels (32). We therefore
suspect that fundamental biological differences in cell machinery may
be responsible for effecting aldosterone's action on IMCD and CCD
cells. The difference in response to amiloride may be one manifestation
of these differences.
The effect of steroids on Na+-K+ pump mRNA
levels is sufficiently modest in our system that some limitations are
encountered in analyzing the factors that modify the response.
Nevertheless, limiting Na+ entry with benzamil does not
reduce the amount of the
1- or
1-subunit
of Na+-K+-ATPase mRNA induced by GC or MC (Fig.
5). Thus these data support the notion that steroids increase the
amount of the
1-subunit of
Na+-K+-ATPase mRNA directly and do not require
an increase in Na+ entry. However, the scenario may be more
complicated. Benzamil actually increases the abundance of the
1-subunit of Na+-K+-ATPase mRNA
in monolayers treated with MC but not in those treated with GC (Fig.
5). These results, taken together with our previous findings (18),
suggest that the action of GC on Na+-K+ pump
activity may be influenced by the magnitude of Na+ entry,
whereas the action of MC is not.
Interactions of TGF-
1 and MC on the
Na+-K+
pump.
We focused our investigation on the interactions of MC and
TGF-
1, because MC is more physiologically important in
regulating IMCD Na+ transport than is GC and the effects of
TGF-
1 on GC- and MC-treated IMCD cells are the same
(13). In contrast to benzamil, TGF-
completely eliminates the MC
stimulation of pump capacity as well as the MC stimulation of
Na+ transport by intact cells (Fig. 6). In addition,
TGF-
1 also eliminates the increase in the
1-subunit of Na+-K+-ATPase mRNA
produced by MC without having any effect on the levels in cells not
exposed to MC (Fig. 6).
The ratio of the magnitude of Na+ transport by intact
monolayers to that by the same monolayers after their apical membrane is permeablized with amphotericin is an estimate of the fraction of the
capacity at which the pump operates during normal (steady-state) Na+ transport in intact cells. This analysis permits
inferences regarding the relative extent of the effects of agents on
the Na+ channel and Na+-K+ pump. GC
and MC increase the rate of Na+ transport in intact cells
to a greater degree than they increase the capacity of the pump (Fig.
1). Monolayers treated with MC or GC have a rate of Na+
transport that causes the pump to operate at ~50% of its maximal capacity. The pumps in monolayers not treated with steroids operate at
~20% of maximal capacity (Fig. 1). These values are similar to those
we reported using IMCD primary cultures from Dahl salt-sensitive and
-resistant rats (15). The straightforward interpretation of these
results is that steroids increase the entry of Na+ across
the apical membrane to a greater extent than they increase the capacity
of the Na+-K+ pump to extrude Na+.
In so doing, steroids cause the Na+ concentration in the
cell to increase by an estimated 2 mM (15).
TGF-
effects on
Na+ entry and on the
Na+-K+
pump.
TGF-
1 inhibits the action of MC to increase
Na+ entry across the apical membrane. This statement is
true, because the steady-state Na+ transport rate is
reduced to values near those of monolayers not treated with steroid.
However, in contrast to benzamil, which inhibits only the entry of
Na+, TGF-
1 also decreases the effects of MC
on the pump (Figs. 6 and 7). The fact that monolayers treated with MC + TGF-
1 have a pump that operates at the same capacity as
monolayers not treated with MC suggests that TGF-
1
inactivates the responsiveness to steroids. Such a scenario might
suggest an action of TGF-
1 to block the
mineralocorticoid and glucocorticoid receptor activation.
However, the interaction of TGF-
1 and MC seems to be
much more complicated than simply blocking steroid receptors. One major action of steroids on IMCD cells is the enhancement of the amount of
-rENaC mRNA (Fig. 9) (37). However, TGF-
1 did not
alter this action (Fig. 9). These results are most consistent with the idea that treatment with TGF-
1 does not alter the MC
actions to bind to the mineralocorticoid receptor, activate and
translocate to the nucleus (28), and increase the transcription of
-rENaC (26). Rather, they suggest that TGF-
1 induces
a posttranscriptional modification of rENaC, such that its full
expression is prevented.
There is little precedent for such an action. Perhaps it is because we
understand relatively little about the mechanisms of aldosterone's
action that we do not understand how they can be counterregulated.
There is one situation, however, where some analogy might be drawn.
Aldosterone acts on the CCD to increase the activity of ENaC (22).
However, alterations in dietary NaCl that alter circulating aldosterone
concentrations and ENaC activity do not alter
-rENaC mRNA levels in
renal cortex (32). In contrast, the same maneuvers do produce a change
in
-rENaC mRNA levels in the inner medulla (32). These results
suggest that increasing the activity of ENaC may be more complex than
simply increasing
-rENaC transcription, protein production, and
apical membrane delivery. The diversity of tissue and organ responses
to dietary NaCl and steroids (19, 32) and the differences in the
developmental responses in colon, lung, kidney, and bladder (38, 39)
support the notion that regulation of ENaC expression and function is complex.
In conclusion, we have found that TGF-
1 produces
antagonism to the action of aldosterone on IMCD cells by complex
actions that include preventing the increase in mRNA levels of the
1-subunit of Na+-K+-ATPase and
preventing the aldosterone-induced increase in pump capacity. Its
antagonism is not manifest at the level of rENaC mRNA, but
TGF-
1 does prevent aldosterone from increasing the activity of ENaC. Taken together, these results suggest that
aldosterone and TGF-
1 can act in parallel at sites
independent of transcription of ENaC to produce counteracting effects
on Na+ channel activity.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by National Institutes of Health
Grants DK-52617 and HL-55006 and by the Department of Veterans Affairs.
Support was also received from the University of Iowa Diabetes and
Endocrinology Research Center.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
1
We tested this hypothesis on M1 cells grown
under similar conditions. In this cell line, long-term (24-h)
incubation with benzamil is less effective in reducing Na+
transport than it is in IMCD cells. Benzamil at 1 µM produced only
22% of the inhibitory capacity of fresh benzamil (1 µM) applied to
the same monolayers. It appears that collecting duct cells may have a
capacity to metabolize benzamil.
Address for reprint requests and other correspondence: J. B. Stokes,
Dept. of Internal Medicine, E300GH, University of Iowa, Iowa City, IA
52242 (E-mail: john-stokes{at}uiowa.edu).
Received 11 May 1999; accepted in final form 20 October 1999.
 |
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