Thyroid hormone stimulates the renal Na/H exchanger NHE3 by
transcriptional activation
Adriana
Cano1,2,
Michel
Baum1,3, and
Orson W.
Moe1,2
Departments of 1 Internal
Medicine and 3 Pediatrics,
University of Texas Medical Center, and
2 Department of Veterans Affairs
Medical Center, Dallas, Texas 75235-8856
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ABSTRACT |
Thyroid hormone
stimulates renal proximal tubule NaCl and
NaHCO3 absorption in part by
activating the apical membrane Na/H exchanger NHE3. We used a renal
epithelial cell line, the opossum kidney (OK) cell, to define the
mechanism by which 3,5,3'-triiodothyronine (T3) increases NHE3 activity.
T3 stimulated NHE3 activity, an effect that was blocked by inhibition of cellular transcription or
translation. The increase in activity was associated with increases in
steady-state cell surface and total cellular NHE3 protein and NHE3
transcript abundance. T3
stimulated transcription of the NHE3 gene and had no effect on NHE3
transcript stability. The transcriptional activity of the
5'-flanking region of the rat NHE3 gene was stimulated by
T3 when expressed in OK cells.
When heterologously expressed rat NHE3 transcript levels were clamped constant with a constitutive promoter in OK cells,
T3 has no effect on rat NHE3
protein abundance, suggesting the absence of regulation of NHE3 protein
stability or translation. These studies demonstrate that
T3 stimulates NHE3 activity by
activating NHE3 gene transcription and increasing NHE3 transcript and
protein abundance.
proximal tubule; acid-base balance; NaCl homeostasis; development; promoter
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INTRODUCTION |
SODIUM CHLORIDE AND
NaHCO3 absorption in the mammalian
renal proximal tubule is affected by the thyroid status of the animal (10, 11, 21, 22). The developmental maturation of proximal tubule NaCl
and NaHCO3 absorption is
temporally preceded by an increase in circulating thyroid hormone
levels (29). A significant portion of proximal tubule NaCl and
NaHCO3 transport is mediated by
apical membrane Na/H exchange (24, 25). Na/H exchange activity in renal
cortical apical membrane vesicles is increased in hyperthyroid and
decreased in hypothyroid animals (18, 19). Although the effect on
apical membrane Na/H exchange may be mediated in part by changes in
glomerular filtration rate, systemic hemodynamic and/or
neurohumoral changes (17), a direct effect of thyroid hormone on the
maximum velocity
(Vmax) of the
Na/H exchanger has been demonstrated in a cell culture model of the
proximal tubule, the opossum kidney (OK) cell (30).
With the identification of Na/H exchanger isoform cDNAs (reviewed in
Ref. 28), specific reagents are now available to address the mechanisms
of regulation of proximal tubule Na/H exchange. The predominant isoform
responsible for proximal tubule apical membrane Na/H exchange is NHE3
(1, 6), and an opossum NHE3 homologue is expressed in OK cells. Most
chronic biological effects of thyroid hormone are believed to be
mediated via activation of gene transcription (26). However, one study
in rat kidney showed that changes in the NHE3 transcript in response to
thyroid hormone are not accompanied by changes in NHE3 protein
abundance, raising doubts as to the role of increased NHE3
transcript in mediating the increased NHE3 activity (2).
In the present study, we found that thyroid hormone administration to
adult rats increases both apical membrane Na/H exchanger activity and
NHE3 protein abundance. We used OK cells that express NHE3 to further
define the mechanisms by which thyroid hormone increases NHE3 activity. We showed that thyroid hormone increases NHE3 activity by activating the promoter and transcription of the NHE3 gene, leading to increased NHE3 transcript and protein abundance.
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METHODS |
Apical membrane preparation from hyperthyroid
rats. Sprague-Dawley rats (150-200 g) with free
access to food and water were given 100 µg/kg of
3,5,3'-triiodothyronine (T3;
dissolved in 100 µl 0.1 mM NaOH and pH adjusted to 7.4 with HCl)
intraperitoneally once daily for 4 days. Control animals were given an
injection of the vehicle. Renal cortex was dissected and homogenized in buffer containing (in mM) 300 mannitol, 20 HEPES (pH 7.50), and 5 EGTA,
as well as (in µg/ml) 100 phenylmethylsulfonyl fluoride (PMSF), 2 leupeptin, 2 aprotinin, and 2 pepstatin A, in a Brinkmann Polytron. The
apical membrane vesicles were prepared from the cortical homogenate by
exposing cortical membranes to three consecutive precipitations by 15 mM MgCl2, and the final apical
membrane-enriched vesicles were pelleted from the supernatant (20,000 rpm, 40 min, 4°C; Beckman J2-21M, JA-20 rotor).
Cell culture. OK cells were maintained
in DMEM supplemented with 4.5 mg/ml glucose as well as 100 U/ml penicillin and 100 µg/ml streptomycin (penicillin-streptomycin).
Confluent monolayers on glass coverslips (NHE activity) or plastic
petri dishes (all other studies) were rendered quiescent by serum
removal for 48 h before study. T3
was dissolved in 10
2 M NaOH
that was diluted 105-fold in
culture medium during experiments (final concentration = 10
7 M). Control cells
received similar amounts of NaOH as
T3-treated cells. For studies
examining constitutively transcribed heterologous rat NHE3, rat NHE3
was cloned in pcDNA-1 (Invitrogen, San Diego, CA) and transfected into
OK cells by CaPO4 precipitation
along with pSV40Neo to confer neomycin resistance, and the
transfectants were selected (400 µg/ml) and maintained (200 µg/ml)
in G418. G418 was replaced with penicillin-streptomycin two passages
before studies.
NHE3 activity, immunoreactive protein, and
transcripts. NHE3 activity in OK cells was measured
fluorometrically by the pH-sensitive dye
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (10 µM) in the absence of
HCO3/CO2
as Na-dependent (Na concn = 138 mM) cell pH
(
ex 500/450 and
em 530 nm) recovery
(dpHi/dt;
pH units/min) after acid loading with the K/H ionophore nigericin. For
measurement of Na/H exchange activity in rat renal apical membranes,
200 µg of vesicles were acid loaded (in mM: 300 mannitol, 20 MES, pH 5.5) and exposed to 10× volume uptake solution [in mM: 300 mannitol, 20 Tris (pH 7.5), 0.1 22NaCl] to initiate
transport at 20°C for 10 s. Uptake was stopped by dilution with
stop solution (in mM: 150 NaCl, 20 Tris, pH 7.5) at 4°C, and
22Na uptake was quantified by
rapid filtration on 0.65 µM Millipore filters and by scintillation counting.
The NHE3 antigen was quantified by immunoblots. Total cellular NHE3 was
measured by lysing cells in membrane buffer [150 mM NaCl, 50 mM
Tris (pH 7.5), 5 mM EDTA, 100 µg/ml PMSF, 4 µg/ml aprotinin, and 4 µg/ml leupeptin], and the membrane fraction was pelleted
(109,000 g at
rmax
50,000 rpm for 25 min on a Beckman TLX:TLA 100.3 rotor at 4°C).
Twenty micrograms of either OK cell or rat renal cortical apical
membrane were fractionated by SDS-PAGE and transferred to
nitrocellulose filters. To measure cell surface NHE3, surface proteins
were labeled with biotin [10 mM triethanolamine (pH 7.4), 2 mM
CaCl2, 150 mM NaCl, and 1.5 mg/ml
sulfosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate; Pierce,
Rockford, IL] for 30 min at 4°C. After quenching
[in mM: 140 Na2HPO4
(pH 7.4), 0.1 CaCl2, 1 MgCl2, 100 glycine], cells
were lysed in RIPA buffer [150 mM NaCl, 50 mM
Tris · HCl (pH 7.4), 0.5 mM EDTA, 80 mM NaF, 25 mM
sodium pyrophosphate, 1 mM sodium orthovanadate, 1% Triton X-100
(vol/vol), 0.5% deoxycholate (wt/vol), 0.1% SDS (wt/vol), 250 µg/ml
PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 20 µg/ml
pepstatin], and an aliquot of the whole cell lysate was saved for
simultaneous measurement of changes in whole cell NHE3 in the same
cells. The remainder of the lysate was equilibrated with
streptavidin-agarose at 4°C on a rotary mixer overnight. After
being washed with RIPA, biotinylated surface proteins were released
from the biotin-streptavidin/agarose complex by 100 mM dithiothreitol,
and the liberated proteins were retrieved as a supernatant after
centrifugation. Anti-OK NHE3 antiserum (no. 5683, raised against a
fusion protein of maltose binding protein and OK NHE3 amino acids
484-839) was used at 1:500 dilution. The specificity of the
antisera was confirmed by complete blockade of labeling by the
immunogenic fusion protein but not maltose binding protein (data not
shown). Several anti-rat NHE3 antisera were used: no. 1568 (against
epitope DSFLQADGPEEQLQ at 1:1,000 dilution), no. 1566 (against epitope
YSRHELTPNEDEKQ at 1:1,000 dilution), and no. 1313 (against a fusion
protein of maltose binding protein and rat NHE3 amino acids
405-831 at 1:400 dilution). After incubation with horseradish
peroxidase-coupled mouse anti-rabbit secondary antibody, signals were
detected by enhanced chemiluminescence (Amersham, Arlington Heights,
IL) and quantified by densitometry.
NHE3 transcript abundance was measured by RNA blots. Monolayers were
lysed in GTC solution [4 M guanidinium thiocyanate, 0.1 M
2-mercaptoethanol, 0.025 M sodium citrate (pH 7.0), 0.5%
(wt/vol) N-lauroylsarcosine],
and RNA was extracted with phenol-chloroform, precipitated with
ethanol, size fractionated with formaldehyde gel electrophoresis,
transferred to a nylon membrane, and hybridized with a
32P- labeled full-length coding OK NHE3
cDNA (0.3-kb 5'UTR + 2.5-kb coding) or a 1.2-kb
Pst I fragment of the rat NHE3 cDNA.
Hybridization conditions were exactly as previously described (4).
NHE3 transcript half-life and transcription
rate. To determine the effect of
T3 on the half-life of NHE3 mRNA,
OK cells were treated with T3 or
vehicle for 16 h, and then
10
5 M actinomycin was added
to completely inhibit transcription. Preliminary studies have
documented that this dose of actinomycin inhibits transcription in OK
cells by 98% (data not shown). Cells were harvested at the designated
time points for NHE3 transcript quantification by RNA blots. The in
vitro transcription rate was measured by nuclear run-on assay as
previously described (4). Briefly, after treatment with
T3 or vehicle for 4 h, cells were scraped, washed in PBS at 4°C, and lysed [10 mM NaCl, 2 mM
MgCl2, 10 mM
Tris · HCl (pH 7.4), and 0.5% (vol/vol) Nonidet
P-40]. Nuclei were pelleted (500 g for 5 min at 45°C), washed,
resuspended in reaction buffer (140 mM KCl, 20 mM
Tris · HCl, pH 7.5, 10 mM
MgCl2, 13.4 mM 2-mercaptoethanol,
1 mM MgCl2, 0.9 mM of each NTP, 10 mM phosphocreatine, 10 µg phosphocreatine kinase, and 250 µCi [32P]UTP), and in
vitro transcription was allowed to proceed at 30°C for 30 min.
Nuclei were then lysed by shearing in high-salt buffer (in mM: 500 NaCl, 2 CaCl2, 50 MgCl2, 10 Tris · HCl, pH 7.5) and treated with RNase-free DNase
(20 units at 30°C for 30 min), and the nuclear nascent RNA was
recovered by phenol-chloroform extraction and LiCl precipitation and
was resuspended in NETS buffer (in mM: 200 NaCl, 10 EDTA, 10 Tris · HCl, pH 7.4). After brief NaOH hydrolysis, the
radiolabeled RNA was hybridized for 48 h to linearized full-length
coding OK NHE3 cDNA, along with pBluescript and
-actin controls
immobilized on nylon filters by slot blotting (20 µg DNA/slot).
Hybridization and washing conditions were exactly as previously
described (4).
Activity of NHE3 5'-flanking region.
Promoter-reporter constructs containing 2.2 or 0.82 kb of the
5'-flanking region of the rat NHE3 gene cloned upstream to the chloramphenicol acetyltransferase (CAT) gene in the vector pBLCAT3 were
used for these studies. Transfections were performed by
CaPO4 precipitation along with a
pTK-gal plasmid to normalize for transfection efficiency. Forty hours
posttransfection, cells were treated with 10
7 M
T3 or vehicle for 4 h, washed in
PBS, and lysed by shearing (250 mM Tris · HCl, pH
7.8). Lysate supernatant (5,000 g for
10 min at 4°C) expressing equivalent amounts of
-galactosidase
activity was incubated in CAT reaction buffer (500 mM
Tris · HCl, pH 7.8, 0.53 mM acetyl-CoA, 0.1 µCi
[14C]chloramphenicol),
and chloramphenicol and its acetylated products were extracted by ethyl
acetate, resolved by TLC [19:1 (vol/vol) chloroform-methanol], and identified by autoradiography.
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RESULTS |
T3 increases apical membrane Na/H
activity and NHE3 protein abundance in rat cortex.
Four days of T3 administration
increased apical membrane NHE activity by 45% (means ± SE in
pmol · mg
protein
1 · 10 s
1: euthyroid, 521 ± 27, n = 3; hyperthyroid, 728 ± 45, n = 3;
P < 0.05, t-test). NHE3 antigen was quantified
in apical membranes from euthyroid and hyperthyroid animals with three
independent antisera, and the result using antiserum no. 1568 is shown
in Fig. 1. Apical membrane NHE3 antigen was
increased by 83% (P < 0.05, t-test; control,
n = 3;
T3,
n = 4) in hyperthyroid animals. Antisera nos. 1566 and 1313 revealed similar results (data not shown).
We next examined the mechanism of this stimulation in more detail in a
cell culture model.

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Fig. 1.
Effect of 3,5,3'-triiodothyronine
(T3) on NHE3 protein abundance
in rat cortical apical membranes. Rats rendered hyperthyroid with 100 µg · kg 1 · day 1
T3 for 4 days were compared with
euthyroid controls given vehicle (Con). NHE3 antigen in 20 µg renal
cortical apical membrane protein was quantified by immunoblot using
antiserum no. 1568. Mobility (in kDa) is shown at
right.
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Stimulation of NHE3 activity by T3 is
dependent on intact RNA and protein synthesis in OK cells.
As shown previously by Yonemura and co-workers (30),
addition of T3 directly stimulated
NHE3 activity in OK cells (Fig. 2,
A and
B). In the presence of either
actinomycin or cycloheximide, T3
had no effect on NHE3 activity, suggesting that the stimulation is
dependent on intact transcription and translation. However, sensitivity
to actinomycin and cycloheximide does not necessarily indicate that
transcription and translation of NHE3 mediate the stimulation of NHE3
activity by T3.

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Fig. 2.
Effect of T3 on NHE3 activity in
opossum kidney (OK) cells: dependence on translation and transcription.
Confluent quiescent OK cells were treated with
10 7 M
T3 or vehicle for 24 h, and NHE3
activity was measured fluorometrically as Na-dependent cell pH recovery
after an acid load
(dpHi/dt).
The T3 effect was studied in the
absence or presence of 100 µM cycloheximide
(A) or 10 µM actinomycin
(B).
* P < 0.05 (control vs.
T3, unpaired
t-test).
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T3 increases NHE3 mRNA and immunoreactive
protein abundance.
In both brush-border vesicles and OK cells,
T3 activates Na/H exchange by
increasing its
Vmax (18, 30), a
kinetic effect that is compatible with increased transporter protein
abundance. To further define the mechanisms of the increase, we
examined steady-state NHE3 mRNA and protein levels in response to
T3 in OK cells. A typical
experiment of the time course of the
T3 effect on NHE3 mRNA is shown in
Fig. 3. An increase in NHE3 mRNA was detectable as early as 4 h and persisted to 24 h (% increases, means ± SE, n = 4 for each time point: 4 h, 80 ± 15%, P < 0.05; 8 h, 125 ± 31%, P < 0.05; 12 h, 180 ± 25%; 24 h, 175 ± 75%, P < 0.05). As shown in Fig. 4, increased NHE3 protein
was evident at 8 h and further increased at 24 h (% increases, mean ± SE, n = 4 for each time point: 4 h, 5 ± 15%, not significant; 8 h, 110 ± 35%,
P < 0.05; 24 h, 230 ± 86%,
P < 0.05). Next, the effect of
T3 on cell surface and total
cellular NHE3 was simultaneously measured in the same cells. In this
set of experiments, T3 increased surface NHE3 by 110 ± 14% (n = 4, P < 0.05) and total
cellular NHE3 by 207 ± 43% (n = 4, P < 0.05) in the same cells (Fig.
5).

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Fig. 3.
Effect of T3 on NHE3 mRNA levels
in OK cells. Confluent quiescent OK cells were treated with
10 7 M
T3 or vehicle (C) for the
designated period of time, and NHE3 transcript abundance in 20 µg
total RNA was quantified by RNA blot. Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) served as loading control. Four independent
experiments showed similar results. NHE3 transcript, 9.5 kb; GAPDH
transcript, 1.9 kb.
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Fig. 4.
Effect of T3 on NHE3 antigen
abundance in OK cells. Confluent quiescent OK cells were treated with
10 7 M
T3 for stated period of time, and
NHE3 antigen abundance in 20 µg of cell membranes was quantified by
immunoblot. Mobility (in kDa) is shown on
right. Four independent experiments
showed similar results.
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Fig. 5.
Effect of T3 on surface and total
cellular NHE3 antigen in OK cells. Confluent quiescent OK cells were
treated with 10 7 M
T3 for 24 h, and surface and
total cellular proteins were retrieved separately as the biotinylated
protein fraction and total cell lysate, respectively. NHE3 antigen
abundance was quantified by immunoblot. Mobility (in kDa) is shown on
right. Four independent experiments
showed similar results.
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T3 stimulates NHE3 transcription without
affecting transcript half-life.
Increased steady-state NHE3 mRNA can be due to increased gene
transcription, stabilization of transcripts, or both. In certain thyroid hormone-responsive genes, message stability has been proposed as a mechanism of regulation of increased transcript abundance (9, 15,
23, 27). We next addressed these two possibilities by directly
measuring the effect of T3 on NHE3
transcript half-life and transcription rate. Figure
6 shows that, although
T3 increased NHE3 transcript
abundance, it had no effect on the rate of decay of NHE3 mRNA when
transcription was completely inhibited. This suggests that the increase
in NHE3 mRNA abundance is likely due to an increase in transcription.
An in vitro transcription assay confirmed that
T3 treatment increased the NHE3
transcription rate by about twofold (207 ± 35%,
n = 3, P < 0.05; Fig.
7).

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Fig. 6.
Effect of T3 on NHE3 mRNA
half-life. OK cells were treated with
10 7 M
T3 or vehicle for 4 h, and NHE3
mRNA decay was determined by RNA blots at indicated times in the
presence of 10 µM actinomycin to inhibit transcription. Graph
quantifies time-dependent decay of NHE3 transcripts expressed as
percentage of level before addition of actinomycin. Circles and error
bars depict means ± SE from 3 independent experiments.
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Fig. 7.
Effect of T3 on NHE3 transcription
rate. Nuclei were isolated from OK cells treated with
T3 or vehicle for 4 h, and in
vitro transcription rates were determined by nuclear run-on assay.
Transcription rates for NHE3 and GAPDH are shown along with the
background control with plasmid pBluescript (pBS). Three independent
experiments showed similar results.
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T3
trans-activates the 5'-flanking
region of the NHE3 gene.
Most, although not all, transcriptional activation of genes is mediated
by regulatory sequences 5' to the coding region. We have isolated
fragments of the rat NHE3 5'-flanking region that are
transcriptionally competent in
cis with reporter genes
in OK cells (8). We next tested the effect of
T3 on two fragments of the
5'-flanking region of the rat NHE3 gene in OK cells. Figure 8 shows that
T3 activates both of these
promoter fragments in OK cells.

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Fig. 8.
Effect of T3 on 5'-flanking
region of the NHE3 gene. Two chloramphenicol acetyltransferase (CAT)
reporter constructs containing either 2.2 or 0.82 kb of the
5'-flanking region of the rat NHE3 gene were transiently
transfected into OK cells along with a -galactosidase-expressing
plasmid. OK cells were treated with
10 7 M
T3 or vehicle for 4 h, and CAT
activity was assayed in cell lysates with equivalent amounts of
-galactosidase activity. Four (for 0.82 kb) and 2 (for 2.2 kb)
independent experiments showed similar results.
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T3 does not affect NHE3 protein half-life
or translation.
The data thus far unequivocally establish that
T3 activates NHE3 gene
transcription. The possibility remains that
T3 may have an additional effect
on NHE3 protein translation or stability. In the presence of an
increased pool of translatable NHE3 mRNA, it is difficult to ascertain
whether T3 directly activates
translation of NHE3. To address this possibility, we expressed rat NHE3
in OK cells driven by a constitutive cytomegalovirus
promoter that clamps the transcript of the transfected gene constant.
If T3 increases NHE3 protein
stability or translation, protein abundance of the transfected rat NHE3
should be increased by T3. Figure 8 shows that, in the absence of increased rat NHE3 mRNA,
T3 has no effect on rat NHE3
protein abundance. The native opossum NHE3 message and protein serve as
positive controls (Fig. 9). This suggests
that the T3-induced increase in
NHE3 protein abundance is mediated solely by increased NHE3
transcription.

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Fig. 9.
Effect of T3 on native and
transfected NHE3 protein and transcript abundance. Rat NHE3 was
expressed in OK cells driven by a constitutive cytomegalovirus promoter
to secure a constant level of transcripts.
T3 was added for 24 h, and RNA and
immunoblot analyses were performed for native opossum and heterologous
rat NHE3 with species-specific cDNA probes and anti-NHE3 antisera. Four
independent experiments showed similar results.
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DISCUSSION |
Thyroid hormone exerts a multitude of effects on extrarenal systems
that can secondarily influence renal function (17). Even within the
kidney, alterations in structural and absorptive capacity in the
proximal tubule in hypothyroid and hyperthyroid animals may be
partially accounted for by the reduction and augmentation of the
glomerular filtration rate, respectively (10, 11, 17, 21, 22). The
first unequivocal demonstration of a direct tubular effect was shown by
Yonemura and co-workers (30), using the OK cell culture model. The
first study that attempted to address the molecular mechanism of
increased apical membrane Na/H exchange was in adult rats (2). In this
study, although NHE3 mRNA abundance was significantly higher in
hyperthyroid compared with euthyroid and hypothyroid rats, there was no
difference in renal cortical membrane NHE3 protein abundance in
hypothyroid, euthyroid, and hyperthyroid rats. This finding casts
doubts on the role of increased NHE3 gene expression in mediating
increased Na/H exchange activity. Indeed, one study had suggested that
the apparent increase in Na/H exchange in apical membrane vesicles in
hyperthyroid animals can be accounted for by increased parallel Na and
H conductances (20).
In the current study, we found that
T3 increased both NHE3 activity
and protein in both rat renal cortex and OK cells. In OK cells, the
time profile of the T3-induced
increase in NHE3 protein in this study is almost identical to that of
increased Na/H exchange activity as defined by Yonemura and co-workers
(30). However, the increase in total NHE3 protein abundance was much larger than the increase in NHE3 activity. There are two possible explanations. First, the
T3-induced increase in NHE3
protein is much larger in the intracellular than the plasma membrane
pool. Second, the increase in NHE3 protein is likely not linearly
reflected by the
dpHi/dt
functional assay. The increased NHE3 protein is preceded by increased
NHE3 transcript. Significant increases in NHE3 transcript can be
detected as early as 4 h after T3
treatment. We demonstrate that the increase in NHE3 transcript is not
due to increased transcript stability but rather to increased
transcription of the NHE3 gene. This is in contrast to other
thyroid-responsive genes such as the Na-K-ATPase
- and
-subunits
and malic enzyme, where large increments in transcript levels were
associated with little or no changes in transcription rate (3, 9, 12, 15). However, the main discrepancy between our study and that of Azuma
and co-workers (2) is that we found an increase in NHE3 protein in
adult rat cortex caused by T3
using three independent antisera. In a recent study, we showed in
neonatal rats that hyperthyroidism is associated with increased,
whereas hypothyroidism is associated with decreased, NHE3 transcript,
protein, and activity in renal cortex (5). These findings in whole
animals strongly support the physiological relevance of the current
cell culture model.
To investigate the activation of NHE3 gene transcription, we examined
NHE3 genomic sequences with a CAT reporter in OK cells. The
5'-flanking sequence of the rat NHE3 gene has been identified and
shown to be active in the cis
configuration in reporter constructs in renal epithelial
cells (8, 16). In addition,
trans-activation of the
5'-flanking sequences by glucocorticoids has been demonstrated (8, 16). In the present study, we show that both the 2.2- and 0.82-kb
promoter fragments are activated by
T3. Thus far we have not detected
differences of sufficient magnitude and consistency between the 2.2- and 0.82-kb constructs to draw conclusions on relative
T3 sensitivity. We previously
demonstrated low baseline expression of both of these constructs in
fibroblasts. Addition of T3 to 3T3
fibroblasts transiently transfected with the 2.2-kb promoter construct,
along with a plasmid expressing the thyroid hormone receptor-
(THR-
; gift from Dr. Kevin Petty, University of Southwestern Medical
Center, Dallas, TX) failed to elicit measurable CAT activity (data not
shown), indicating that T3/THR-
per se is insufficient to activate the NHE3 promoter in a nonepithelial cellular context. The 2.2-kb nucleotide sequence upstream to the transcriptional start site harbors multiple consensus hexamers that
conform partially to classical thyroid-responsive elements (TREs) (7,
8, 14, 16, 26). Relative to glucocorticoid-responsive elements (GREs),
TREs in general tolerate a greater flexibility in
half-site primary sequence variability and orientation (7). However,
two half-sites in tandem are usually required for
cis-activation (7, 13). The closest
TRE half-sites in tandem in the NHE3 promoter are separated by 10 nucleotides. The ability of the 0.82-kb fragment that contains only
four TRE half-sites to respond to T3 suggests that the
T3 response may involve multiple
transcription factors other than the THRs and their cognate
dimerization partners.
In addition to gene transcription and message stability, it has been
proposed, although not proven, that
T3 can increase specific protein
expression via either stabilization of protein or direct stimulation of
translation (9, 27). Even if T3
directly activates NHE3 translation, it is extremely difficult to
interpret an increase in NHE3 translation rate in the presence of an
expanded pool of translatable NHE3 transcripts. We elected to create a
situation in vivo where mRNA was clamped constant by expressing the rat NHE3 gene using a constitutive promoter. When rat NHE3 mRNA levels were
kept constant, T3 had no effect on
rat NHE3 abundance, even though the cells preserve an intact response
to T3 as manifested by the rise in
native OK NHE3 transcript and protein levels. This suggests that
T3 exerts a minimal effect on NHE3
protein translation and stability. We recognize that the present data
do not exclude the alternative explanation that the required regulatory
sequences for translational regulation may reside in untranslated
regions that are missing in the rat NHE3 cDNA construct.
In summary, we demonstrate that T3
increases proximal tubule apical membrane Na/H exchange by directly
activating NHE3 gene transcription, and at least part of the regulatory
sequence for this activation resides in the 5'-flanking region of
the NHE3 gene. This may be an important mechanism in the determination of proximal tubule NaCl and NaHCO3
absorption in hyperthyroid and hypothyroid states and during the
maturation of the proximal nephron.
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ACKNOWLEDGEMENTS |
We acknowledge the technical assistance of Sherry Scobee, Daniel
Harris, Vangipuram Dwarakanath, and Ladonna Crowder. We thank Dr. David
Russell and Dr. Robert Alpern for their helpful discussions and Dr.
Alpern for his careful reading of the manuscript.
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FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grants DK-41612 (M. Baum) and DK-48482 (O. W. Moe)
and by the Department of Veterans Affairs Research Service (O. W. Moe).
A. Cano was a recipient of an American Heart Association Clinician
Scientist Award.
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.
Address for reprint requests: O. W. Moe, Dept. of Internal Medicine,
5323 Harry Hines Blvd., Dallas, TX 75235-8856.
Received 20 April 1998; accepted in final form 8 September 1998.
 |
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