Regulation by glucocorticoids and osmolality of
expression of ROMK (Kir 1.1), the apical K channel of thick
ascending limb
Morgan
Gallazzini1,
Amel
Attmane-Elakeb1,
David B.
Mount2,
Steven C.
Hebert3, and
Maurice
Bichara1
1 Institut National de la Santé et de la Recherche
Médicale U.426, Institut Fédératif Régional
Claude Bernard, Faculté de Médecine Xavier Bichat,
Université Paris 7, 75018 Paris, France; 2 Renal
Division, West Roxbury Veterans Administration Medical Center and
Brigham and Women's Hospital, Harvard Medical School, Boston,
Massachusetts 02115; and 3 Department of Cellular and
Molecular Physiology, Yale University School of Medicine, New Haven,
Connecticut 06520-8025
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ABSTRACT |
Mechanisms
of regulation of ROMK channel mRNA and protein expression in medullary
thick ascending limb (MTAL) were assessed in rat MTAL fragments
incubated for 7 h. ROMK mRNA was quantified by quantitative RT-PCR
and ROMK protein by immunoblotting analysis of crude membranes. Medium
hyperosmolality (450 mosmol/kgH2O; NaCl plus urea added to
isoosmotic medium) increased ROMK mRNA (P < 0.04) and
protein (P < 0.006), and 10 nM dexamethasone also increased ROMK mRNA (P < 0.02). Hyperosmolality and
dexamethasone had no additive effects on ROMK mRNA. NaCl alone, but not
urea or mannitol, reproduced the hyperosmolality effect on ROMK mRNA. 1-Deamino-(8-D-arginine) vasopressin (1 nM) or 0.5 mM
8-bromo-cAMP had no effect per se on ROMK mRNA and protein. However,
8-bromo-cAMP abolished the stimulatory effect of dexamethasone on ROMK
mRNA in the isoosmotic but not in the hyperosmotic medium
(P < 0.004). In in vivo studies, the abundance of ROMK
protein and mRNA increased in adrenalectomized (ADX) rats infused with
dexamethasone compared with ADX rats (P < 0.02). These
results establish glucocorticoids and medium NaCl concentration as
direct regulators of MTAL ROMK mRNA and protein expression, which may
be modulated by cAMP-dependent factors.
regulation of gene expression; ROMK channel; immunoblotting
analysis; quantitative reverse transcriptase-polymerase chain reaction; isolated tubules
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INTRODUCTION |
IN THE THICK ASCENDING
LIMB (TAL) of the nephron, potassium that enters the cell by the
activity of the apical
Na+-K+(NH4+)-2Cl
cotransporter is largely recycled back into the lumen through potassium
channels (5, 17, 32). This K+ recycling has a
major role in NaCl reabsorption by the TAL by providing a potassium
supply to the cotransporter and establishing the lumen-positive
transepithelial potential difference that provides the driving force
for sodium reabsorption through the paracellular pathway. ROMK channels
are believed to constitute the major K+ secretory pathway
in the distal nephron (15, 28). Indeed, the ROMK protein
has been localized by antibodies at the apical membrane of cells of the
part of the renal tubule extending from the beginning of the TAL to the
initial portion of the inner medullary collecting duct (19, 25,
33). The essential role of ROMK channels in TAL transport
functions and thus in the renal regulation of sodium and water balance
is demonstrated by the fact that mutations in the ROMK gene cause
Bartter's syndrome, which is characterized by severe salt wasting and
impaired urinary concentrating ability (14, 18, 30).
It is well established that the activity or the membrane density of
ROMK channels expressed in Xenopus laevis oocytes or in HEK293 cells is acutely regulated by cAMP-dependent protein kinase (20, 23, 34), arachidonic acid and protein kinase C
(21, 22), protein-tyrosine phosphatase and
protein-tyrosine kinase (26), and interactions with
associated proteins (31). In contrast, little is known
about the chronic regulation of ROMK expression in the TAL. Ecelbarger
et al. (13) showed that the abundance of ROMK protein in
the rat outer medulla is augmented by
1-deamino-(8-D-arginine)-vasopressin (dDAVP), restriction
of water intake, and high levels of sodium intake, and decreased by low
levels of sodium intake. However, the direct stimuli and cellular
mechanisms of these chronic regulations of ROMK expression in the TAL
are unknown. Possible candidate mechanisms include direct effects of
cAMP-dependent pathways and the variations in the osmolality of the
surrounding medullary interstitium that accompany the states of water
diuresis and antidiuresis. In addition, beside adenylyl cyclase-coupled
receptors, the TAL possesses specific glucocorticoid receptors (GR),
the activation of which exerts important actions on TAL transport
functions. Indeed, the glucocorticoid dexamethasone has been shown to
stimulate Na+-K+-ATPase activity within a few
hours (12, 29). More recently, we showed that
dexamethasone increases both the expression and activity of the
Na+-K+(NH4+)-2Cl
cotransporter BSC1/NKCC2 of MTALs incubated in vitro (4). Of relevance to these observations, glucocorticoids have long been
known to contribute to the renal urinary concentrating ability, at
least in part, through the maintenance of medullary hyperosmolality (11).
These considerations prompted us to assess directly in vitro the
possible regulation by osmolality, cAMP, and glucocorticoids of the
expression of ROMK mRNA and protein in fragments of medullary TAL
(MTAL) in suspension. To this end, we used the MTAL "shake" suspension previously described (2, 3). The results
establish that ROMK mRNA and protein abundance are regulated directly
by the osmolality of the incubation medium and glucocorticoids not by
dDAVP or cAMP. We also show that in vivo dexamethasone administration increases the abundance of ROMK mRNA and protein in the MTAL.
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MATERIALS AND METHODS |
In Vivo Studies
Male Sprague-Dawley rats (250-300 g) were used and had free
access to standard rat chow and drinking solution until the time of the
experiment. Rats were adrenalectomized (ADX) under light ether
anesthesia and given, as drinking solution, distilled water containing
0.9% NaCl for 6 days before the experiment. A microosmotic pump (Alza,
Palo Alto, CA) was implanted subcutaneously in the nape of some of the
ADX rats, through which we delivered 1.2 µg · 100 g body
wt
1 · day
1 of the glucocorticoid
hormone dexamethasone, a dose that is known to restore normal
glucocorticoid activity, for 6 days (ADX + Dexa). These rats also
had access to 0.9% NaCl in distilled water. Control rats were sham
operated under ether anesthesia and had access to water or to distilled
water containing 0.9% of NaCl. After pentobarbital sodium anesthesia,
the kidneys were rapidly removed and cut into thin slices along the
corticopapillary axis and, under a dissecting microscope, the inner
stripe of outer medulla of each slice was excised and cut into uniform
small pieces that were used for immunoblotting of membrane proteins and
mRNA determinations.
In Vitro Studies
Suspension of rat MTAL tubules.
The method used to isolate MTAL fragments in suspension has been
previously described (1). We established by light and electron microscopy that this suspension was made almost exclusively of
MTALs (
95%), occasional thin limbs, and rare outer medullary collecting tubules, with no isolated cells or proximal tubules (1-3). The MTAL fragments were washed in a 1:1
mixture of Dulbecco's modified Eagle's medium and Ham's nutrient
mixture F-12 supplemented with 5 mM heptanoic acid, 5 mM
L-leucin, 0.1 g/l bovine serum albumin, 200 IU/ml
penicillin G, 250 µg/ml streptomycin, 10 µg/ml minocyclin, 15 mM
HEPES, 10 mM Tris, and 25 mM NaHCO3, pH 7.35, when gassed
with 95% O2-5% CO2 (HDMEM). The MTALs were
then suspended in 60 ml of this medium, placed in a rotary (100 rpm)
shaking water bath at 37°C, and gassed with a humidified 95%
O2-5% CO2 gas mixture. According to results
obtained in a previous study from this laboratory (2), the
MTAL shake suspension was allowed to stabilize during the first 9 h of incubation. Then, samples of MTALs were further incubated for
7 h in the presence or absence of 10 nM dexamethasone, 1 nM dDAVP,
or 0.5 mM 8-bromo-cAMP in is- or hyperosmotic media. We checked in four
preliminary experiments that ROMK mRNA abundance was stable during this
experimental time under control conditions (0.96 ± 0.17 vs.
0.85 ± 0.16 amol/100 ng RNAtot; not significant; the quantitative
RT-PCR is described below).
Crude Membrane Preparation
Tissues from inner stripe of outer medulla dissection or from
MTAL suspensions were homogenized in a medium composed of 150 mM
sucrose, 12 mM Trizma (Tris base, pH 7.4), 0.1 mM
4-(2-amino-ethyl)-benzenesulfonyl fluoride, and 5 µg/ml leupeptin.
These homogenates were centrifuged at 1,000 g for 5 min in a
Beckman GS-6KR centrifuge with a G-H3.7 rotor, and the supernatants
were further centrifuged at 200,000 g for 60 min in a
Beckman L-70 Ultracentrifuge with a 70 TI rotor. The membrane pellets
were suspended in the above medium and stored at
80°C until use.
Electrophoresis and Immunoblotting of Membrane Proteins
Semiquantification of membrane protein amounts was performed by
immunoblotting after SDS-PAGE. Membranes were solubilized first at
ambient temperature for 20 min in Laemmli medium containing 62.5 mM
Tris · HCl (pH 6.8), 5% SDS, 100 mM dithiothreitol, and 10%
glycerol, then at 65°C for 10 min in the same medium. Samples containing 7 to 20 µg of proteins were loaded into individual lanes
of 10% polyacrylamide minigels (Bio-Rad). Proteins were electrophoretically transferred from the gels to nitrocellulose membranes (Bio-Rad). Equal loading and transfer efficiency were systematically checked by Ponceau red staining of the nitrocellulose membranes. After 1 h of blocking at 37°C with TBS/T containing 5% nonfat milk powder, membranes were exposed overnight at 4°C to an
affinity-purified polyclonal anti-ROMK rabbit antibody (APC001, Alomone
Labs, Jerusalem, Israel) diluted 1:150. This antibody has been
previously documented to reveal both native and heterologously expressed ROMK protein as a ~45-kDa band (24), which
appeared as a 42- to 45-kDa doublet in the present study in MTAL total membranes. The nitrocellulose membranes were then exposed to a horseradish peroxydase-linked anti-rabbit Ig secondary antibody (Bio-Rad) for 1.5 h at ambient temperature. Antibody-antigen
complexes were detected using luminol-based enhanced chemiluminescence
(Amersham-Pharmacia Biotech) before exposure to X-ray film (Fujifilm).
As indicated by the manufacturer, the APC001 anti-ROMK antibody also
reveals a band of unknown identity of 90 kDa. Both the ROMK 42- to
45-kDa doublet and the 90-kDa bands were analyzed by densitometry with use of public domain National Institutes of Health (NIH) Image 1.62 software. The 90-kDa band was used as a control because, as will be
shown below, it does not respond like ROMK to the various experimental conditions.
RNA Extraction, Reverse Transcription, and PCR
Total RNA (RNAtot) was extracted from aliquots of
kidney MTAL with use of the SV Total RNA Isolation System kit
(Promega). To obtain competitor RNAs (RNAc) that differed
from the wild-type ROMK1, 2, 3 mRNAs, deletions of 99 bp, located in
the core exon common to all ROMK isoforms, of the ROMK1, 2, and 3 plasmids were obtained by digestion with Bgl II and Msc I restriction
enzymes (from bp 692 to 790 of ROMK1), followed by blunt-end ligation of the cut plasmids. The deleted ROMK plasmids were subcloned and
linearized with NotI restriction enzyme. In vitro
transcription was then performed with the use of T7 RNA polymerase
(mCAP RNA Capping kit, Stratagene, La Jolla, CA) and
[32P]UTP. The amounts of transcribed RNAc
were determined by the measure of its optical density at 260 nm
corrected for the ratio of TCA-precipitated RNAc to total
RNAc determined by liquid scintillation spectroscopy. We
thus obtained three RNAc that gave identical results in the
RT-PCR described below.
The primers (GIBCO BRL) for cDNA synthesis and PCR amplification were
chosen from the published ROMK1, 2, and 3 sequences with the help of
Oligo 4.04 Primer Analysis software (National Biosciences, Plymouth,
MN). The sequences of the primers were 5'-GAC CTC CCA GAG TTC TAC-3'
(sense) and 5'-AGG GCT GTT GTG GTC AAT AA-3' (antisense). The sense
primer was directed against a segment common to ROMK1, 2, 3, and 6 but
located within the intron retained in the ROMK core exon. This retained
intron is subject to low-frequency alternative splicing that generates
a set of truncated hydrophilic ROMK isoforms of unknown function, which thus were not amplified by our method. The primers used in the present
study yielded, as expected, only one PCR product common to ROMK1, 2, and 3 of 510 bp from MTAL RNAtot and the ROMK cDNA plasmids
and of 411 bp from the RNAc and the deleted ROMK plasmids, respectively. After 30 PCR cycles, PCR products resulting from nonspecific hybridization were never observed. The identities of the
PCR products were confirmed by digestion with SalI, which generated two bands of 74 and 436 bp from the wild DNA and of 74 and
337 bp from the competitive DNA as expected.
cDNAs were synthesized from MTAL RNAtot and
RNAc by reverse transcription at 37°C for 60 min with 200 U Moloney murine leukemia virus reverse transcriptase (Life
Technologies), 30 pmol of antisense primer, 4 µg of yeast transfer
RNA, 1 mM of each deoxyribonucleotide (dNTP), 10 mM DTT, 2 U of
ribonuclease inhibitor (GIBCO BRL), and RT buffer in a final volume of
22 µl. Reverse transcriptase was then inactivated at 95°C for 5 min. Each reaction was performed in parallel with an otherwise
identical one that contained no reverse transcriptase.
For PCR, 10 µl of the cDNA solution were supplemented with PCR
buffer, 5.4 mM MgCl2, 30 pmol of sense and antisense
primers, 1 mM of each dNTP, and 5 U of Taq DNA polymerase
(Life Technologies) in a final volume of 50 µl. Samples were
denatured at 94°C for 5 min, which were followed by cycles consisting
of denaturation at 94°C (1 min), annealing at 50°C (1 min), and
extension at 72°C (1.5 min). PCR was completed by a final extension
step at 72°C for 10 min. Quantitative PCR was performed using 27 cycles of amplification of cDNAs simultaneously obtained from a fixed
amount of MTAL RNAtot (25 to 100 ng, as appropriate) and
0.27 to 2 amol of RNAc. Under these PCR conditions,
heteroduplexes of PCR amplicons were never observed. The PCR amplicons
were resolved by 1.8% agarose gel electrophoresis and stained with
ethidium bromide. The bands were digitized, and quantification was
performed by densitometry with use of NIH Image 1.62 software. To
correct for differences in molecular weight, the densitometry values of
the competitive DNA bands were multiplied by the 510/411-bp ratio. We
checked that the amplification efficiencies of the wild and competitive DNAs were identical with up to 28 PCR cycles (0.41 ± 0.04 vs. 0.42 ± 0.04 per cycle, n = 4 for both) and that
the amounts of amplicons obtained after 27 PCR cycles were well within
the exponential phase of amplification. The ROMK mRNA abundance was
calculated from the linear log-log scale plot of the ratio of the
fluorescence intensities of RNAc to RNAtot PCR
products against the known amount of RNAc added in each
reaction tube; r values for these linear plots were all
>0.99. Results are expressed in attomoles of ROMK mRNA per 100 ng
RNAtot.
Statistics
Results are expressed as means ± SE. Statistical
significance between experimental groups was assessed by Student's
paired or unpaired t-test or by one-way ANOVA completed by a
t-test using the within-groups residual variance of ANOVA,
as appropriate.
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RESULTS |
In Vitro Studies
For the present experiments, tubule fragments were incubated in
experimental media for 7 h. The MTAL is surrounded in vivo by the
interstitial medium of variable osmolality of the inner stripe of the
outer medulla of the kidney depending on the state of water diuresis or
antidiuresis and is subjected to tonic influences by cAMP-generating
peptide hormones such as AVP, glucagon, and calcitonin
(27). Thus, we first assessed possible effects on ROMK
expression by hyperosmolality and dDAVP/8-bromo-cAMP. The hyperosmotic
HDMEM was obtained by adding 50 mM NaCl and 50 mM urea to normal HDMEM
as physiologically occurs during antidiuresis. As shown in Fig.
1, hyperosmolality increased ROMK mRNA
abundance from 0.31 ± 0.03 amol/100 ng RNAtot in control
isosmotic medium to 0.55 ± 0.12 (P < 0.04). ROMK
protein abundance was also increased moderately by ~28% but very
significantly (P < 0.006) by hyperosmolality, whereas
the 90-kDa band was not affected (Fig.
2). By contrast, the V2
receptor-specific analog dDAVP (10
9 M) or 0.5 mM
8-bromo-cAMP had no effect on ROMK mRNA or protein abundance in both
the isosmotic and hyperosmotic medium (Table 1). To gain some insight into the
hyperosmolality effect, the following experiments were performed. As
shown in Fig. 3, a 75 mM increase in
medium NaCl concentration augmented ROMK mRNA abundance from 0.40 ± 0.04 amol/100 ng RNAtot in control isoosmotic medium to
0.65 ± 0.03 (P < 0.006). Such an effect was not
seen with 150 mM urea or mannitol (Fig. 3). The dose-response curve
depicted in Fig. 4 shows that ROMK mRNA
abundance was regulated between 170 and 205 mM medium NaCl
concentration. Thus these results establish that the hyperosmolality
effect was mediated via the increase in the NaCl concentration
specifically.

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Fig. 1.
Effect of medium hyperosmolality (450 mosmol/kgH2O) on ROMK mRNA abundance after 7 h of
incubation of medullary thick ascending limb (MTAL) fragments. Lines
connect results obtained in the same experiment.
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Fig. 2.
Effect of medium hyperosmolality (Hyperosmo.; 450 mosmol/kgH2O) on ROMK protein (45 kDa) and on 90-kDa band
abundance after 7 h of incubation of MTAL fragments. A:
representative immunoblot in crude membranes from MTAL fragments.
B: band densities (arbitrary units) of immunoblots made in
duplicate in 6 independent experiments. The augmentation of ROMK bands
(+28 ± 12%) is significantly higher than that of the 90-kDa band
(+9 ± 9%; P < 0.04). Bars represent means ± SE. Numbers within bars are the numbers of measurements. NS, not
significant.
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Fig. 3.
Effect of hyperosmolality caused by 150 mM urea and
mannitol and by 75 mM NaCl on ROMK mRNA abundance. Each bar represents
means ± SE of 3 measurements.
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Fig. 4.
Dose-response curve of the effect of medium NaCl concentration on
ROMK mRNA abundance. Each point represents means ± SE of at least
3 measurements.
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We previously showed that glucocorticoids regulate the expression of
the
Na+-K+(NH4+)-2Cl
cotransporter BSC1/NKCC2 in the MTAL through interactions with cAMP-dependent factors (22). Accordingly, we assessed
possible effects of glucocorticoids on ROMK expression. Dexamethasone
(10 nM) increased the abundance of ROMK mRNA from 0.36 ± 0.04 in
the control isosmotic medium to 0.67 ± 0.11 amol/100 ng
RNAtot (P < 0.02; Fig.
5). This dexamethasone-induced increase
in ROMK mRNA was abolished in the additional presence of
8-bromo-cAMP (0.47 ± 0.09 amol/100 ng RNAtot with
dexamethasone plus 8-bromo-cAMP; not significant compared with
control and P < 0.004 compared with dexamethasone
alone; Fig. 5). The abundance of ROMK protein or the 90-kDa band was
not significantly affected under any of these experimental conditions
(Fig. 6).

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Fig. 5.
Effect of 10 nM dexamathasone (DEX) and DEX + 0.5 mM
8-bromo-cAMP (cAMP) on ROMK mRNA abundance after 7 h of incubation
of MTAL fragments in isosmotic medium. Lines connect results obtained
in the same experiment.
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Fig. 6.
Effect of 10 nM DEX (D) and DEX + cAMP on ROMK
protein (45 kDa) and on 90-kDa band abundance after 7 h of
incubation of MTAL fragments in isosmotic medium. A:
representative immunoblot in crude membranes from MTAL fragments.
B: band densities (arbitrary units) of immunoblots made in
duplicate in 3 independent experiments. Bars represent means ± SE. Numbers within bars are the numbers of measurements. C, control.
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Thus both dexamethasone and hyperosmolality increased ROMK mRNA
compared with the control isosmotic experimental condition. We thus
assessed whether the effects of hyperosmolality and glucocorticoids were additive. As shown in Fig. 7, the
effects on ROMK mRNA abundance of hyperosmolality and dexamethasone
were not additive. However, in contrast to what was observed in the
isosmotic medium, the presence of 8-bromo-cAMP in addition to
dexamethasone in the hyperosmotic medium did not alter the stimulating
effect of these latter agents on ROMK mRNA abundance (Fig. 7). ROMK
protein abundance was augmented ~33% by hyperosmolality but not
significantly in this experimental series compared by ANOVA with the
level seen in the isosmotic medium (Fig.
8). However, hyperosmolality plus
dexamethasone and hyperosmolality plus dexamethasone plus cAMP
significantly augmented by ~45 (P < 0.04) and
~64% (P < 0.01), respectively, the ROMK protein
abundance. The 90-kDa band abundance was affected by none of these
experimental conditions (Fig. 8).

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Fig. 7.
Effect of hyperosmotic medium (450 mosmol/kgH2O; H), hyperosmotic medium + 10 nM DEX
(H + D), and hyperosmotic medium + 10 nM DEX + cAMP
(H + D + cAMP) on ROMK mRNA abundance after 7 h of
incubation of MTAL fragments.
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Fig. 8.
Effect of H (450 mosmol/kgH2O), H + D,
and H + D + cAMP on ROMK protein (45 kDa) and on 90-kDa band
abundance after 7 h of incubation of MTAL fragments. A:
representative immunoblot in crude membranes from MTAL fragments.
B: band densities (arbitrary units) of immunoblots made in
duplicate in 4 independent experiments. C, control isoosmotic medium.
Bars represent means ± SE. Numbers within bars are the numbers of
measurements.
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In Vivo Studies
To assess the physiological significance of the effects of
dexamethasone observed in vitro, we performed the following in vivo
studies. As shown in Table 2, there was
no significant difference in the abundance of ROMK protein and mRNA
between ADX and control rats that drank normal water or 0.9% NaCl.
However, adrenalectomy is a complex condition in which several factors
may have had opposing effects on ROMK expression, as discussed below.
Thus, in another experimental series, results obtained from five ADX
rats were compared with those obtained from five ADX + Dexa rats.
As shown in Fig. 9, dexamethasone
administration increased ROMK protein abundance in crude membranes of
the inner stripe of the outer medulla by ~61% (161 ± 16 arbitrary units in ADX + Dexa vs. 100 ± 8 in ADX;
P < 0.004). It may be noted that the 90-kDa band was decreased by dexamethasone administration (Fig. 9) and increased by
adrenalectomy when control rats drank 0.9% NaCl (Table 2), as opposed
to what was observed for ROMK. The dexamethasone-induced increase in
ROMK protein abundance was accompanied by a ~67% increase in ROMK
mRNA abundance (9.2 ± 0.5 amol/100 ng RNAtot in ADX + Dexa
vs. 5.5 ± 0.5 in ADX; P < 0.002; Fig.
10). These results establish that
glucocorticoid administration enhances ROMK mRNA and protein expression
in the MTAL.
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Table 2.
Quantification of ROMK protein and mRNA abundance in inner stripe of
outer medulla of control and ADX rats
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Fig. 9.
A: immunoblot of ROMK protein (45 kDa) and 90-kDa band
in crude membranes from inner stripe of outer medulla in 5 adrenalectomized (ADX) and 5 ADX + DEX (Dexa) rats. B:
band densities (arbitrary units) of immunoblots made in duplicate in
ADX and ADX + Dexa rats. Bars represent means ± SE.
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Fig. 10.
Determination of ROMK mRNA abundance in inner stripe of
outer medulla of 5 ADX and 5 ADX + Dexa rats.
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DISCUSSION |
This study is the first, to our knowledge, to assess the direct in
vitro effects of glucocorticoids, dDAVP and cAMP, and hyperosmolality on ROMK expression in rat MTAL fragments. The main observations were
that 1) ROMK mRNA abundance was increased when MTALs were incubated in a hyperosmotic medium or in the presence of 10 nM dexamethasone; 2) the effects of hyperosmolality and
glucocorticoids on mRNA abundance were not additive; 3) an
increase in ROMK protein abundance was seen after 7 h of
incubation in a hyperosmotic medium; 4) the hyperosmolality
effect was mediated by the increase in medium NaCl concentration, but
not by urea or mannitol, and ROMK mRNA abundance was regulated by NaCl
concentrations between 170 and 205 mM; 5) in vivo
administration of dexamethasone over 6 days increased ROMK mRNA and
protein abundance in the MTAL; and 6) finally, dDAVP or
8-bromo-cAMP alone had no significant effect per se on ROMK mRNA or
protein abundance but may modulate the effects of hyperosmolality and glucocorticoids.
The effects on ROMK protein expression in the MTAL of changes in sodium
chloride intake, water restriction, and dDAVP administration were
previously assessed in vivo (13) but not those of
glucocorticoid administration. We thus performed in vivo studies using
ADX rats and ADX rats supplemented with dexamethasone. Comparing the
level of ROMK expression in ADX rats to that in normal rats to estimate the effects of glucocorticoid deficiency can hardly be achieved satisfactorily because adrenalectomy is a complex condition with respect to ROMK expression. For example, adrenalectomy is associated with increased circulating AVP concentrations probably due to impaired
cardiac function (8), and dDAVP administration and water
restriction that stimulates the endogenous AVP secretion have been
shown to strongly stimulate ROMK expression in the MTAL (13). In addition, NaCl administration, which was used in
ADX rats to minimize urinary NaCl losses, has also been shown to
stimulate ROMK expression (13). With these issues in mind,
we observed that the abundance of ROMK mRNA or protein in the inner
stripe of outer medulla was not significantly different after
adrenalectomy from that in control rats drinking normal water or 0.9%
NaCl. On the other hand, supplementing ADX rats with dexamethasone
appears as a better means of assessing the effects of glucocorticoids on ROMK expression. Glucocorticoid administration to ADX rats strongly
stimulated ROMK mRNA and protein expression in the inner stripe of
outer medulla compared with ADX rats. These results must reflect
changes of ROMK expression in the MTAL because the level of ROMK
expression in this segment is much higher than in the outer medullary
collecting duct (OMCD) (7, 25, 33) and because the MTAL
mass of tissue is approximately sixfold that of the OMCD. Thus these
results establish that glucocorticoids enhance ROMK mRNA and protein
expression in the MTAL when administered in vivo. Furthermore, when 10 nM dexamethasone was directly applied to MTALs incubated in an
isosmotic medium, ROMK mRNA expression was stimulated. Thus
glucocorticoids directly regulate ROMK mRNA expression in the MTAL in
vitro. The present finding that glucocorticoids physiologically
stimulate ROMK expression would be consistent with the observations
that dexamethasone also stimulates BSC1/NKCC2 expression and activity
(4) and Na+-K+-ATPase activity in
the MTAL (12, 29). Thus glucocorticoids coordinately
stimulate basolateral Na+-K+-ATPase and apical
BSC1/NKCC2 and ROMK to increase NaCl absorption by the MTAL, which
explains at least in part the role of glucocorticoids in the ability of
the kidney to maximally concentrate or dilute the urine.
In previous in vivo studies, rats that were water restricted for 7 days, which stimulates the endogenous AVP secretion, or that were
administered dDAVP for 7 days exhibited an enhanced abundance of ROMK
protein in the MTAL compared with control rats, as assessed by
immunolocalization and immunoblotting analysis (13). The
mechanisms of the latter regulations were not investigated in this
previous work (13) but results obtained in vitro in the
present study indicate that AVP and cAMP-dependent pathways per se are
not directly responsible for the increased ROMK expression. Indeed,
both dDAVP and 8-bromo-cAMP had no effect on ROMK mRNA and protein
abundance after 7 h of incubation. However, increases in medium
NaCl concentration did increase ROMK mRNA and protein expression in the
present study. These observations taken together thus suggest that
chronic water restriction and dDAVP administration stimulate ROMK
expression indirectly, at least in part, through the increase in the
medullary NaCl concentration that occurs under these conditions. Note
that a high-sodium diet increased and a low-sodium diet decreased ROMK
protein expression in a previous study (13), which also
may have been due to the changes in the medullary NaCl concentration
that follow these high- and low-sodium intakes. Thus the medullary NaCl
concentration directly regulates ROMK expression in the MTAL. Note that
the NaCl effect seems very specific since ROMK expression was not
enhanced by urea or mannitol, which can affect gene expression by
various mechanisms (9, 10, 16).
The intracellular mechanisms by which hyperosmolality caused by NaCl
and glucocorticoids enhanced ROMK mRNA and protein expression in the
MTAL were not investigated in the present study. However, it must be
emphasized that the promoter region 5' of exon 1 in the human ROMK
gene, KCNJ1, contains both a glucocorticoid response element and a
sequence 91% identical to the tonicity-responsive enhancer
(TonE)/osmotic response element consensus [TGGAAANNYNY (9, 10,
16)] that are located 27 and 358 bp, respectively, 5' of the
transcription start point of exon 1 (10). This suggests that high medium NaCl concentration and glucocorticoids may stimulate the ROMK gene transcription rate through activation of TonE binding protein (9) and GR, respectively. However, it may be noted that TonE is also usually activated by mannitol in cultured cells, whereas mannitol had no effect on ROMK mRNA expression in freshly harvested MTALs in the present study, which might have been due to
differences between cultured and fresh cells. Otherwise, other intracellular events, such as altered ROMK mRNA decay and ROMK protein
synthesis and/or degradation, may have combined to explain our results.
Regulation of intermediate protein(s) expression may also have
occurred. Further work is needed to address these issues.
In summary, ROMK expression in the MTAL is regulated in vivo by changes
in sodium and water intake (13) and by glucocorticoid administration (present study). Results obtained in vitro in the present work establish medium NaCl concentration and glucocorticoids as
direct regulators of ROMK expression. As stated above, luminal K+ recycling through ROMK channels has a major role in NaCl
reabsorption by the TAL by providing a potassium supply to the
cotransporter BSC1/NKCC2 and establishing the lumen-positive
transepithelial potential difference that provides the driving force
for sodium reabsorption through the paracellular pathway. Present and
previous (4) findings show that BSC1/NKCC2 and ROMK mRNA
and protein expressions in the MTAL are coordinately regulated by
various mechanisms including glucocorticoids, cAMP-dependent factors, and medium NaCl concentration to set MTAL NaCl absorption at a level
appropriate to the renal regulation of sodium and water balance.
Furthermore, the effects of glucocorticoids on MTAL ROMK and BSC1/NKCC2
described in the present and previous (4) studies may
explain, at least in part, the well-known inability of the kidney to
maximally concentrate or dilute the urine during adrenal insufficiency.
 |
ACKNOWLEDGEMENTS |
This study was supported by grants from the Institut National de la
Recherche Médicale and the Université Paris 7. Amel Attmane-Elakeb is supported by a grant from La Fondation pour la
Recherche Médicale; David B. Mount is supported by National Institutes of Health (NIH) Grant RO1-DK-57708 and by an Advanced Career
Development Award from the Veterans Administration; and Steven C. Hebert is supported by NIH Grant DK-54999.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: M. Bichara, INSERM U.426/IFR 02 Claude Bernard, B.P. 416-16, rue Henri Huchard, 75870 Paris Cedex 18, France (E-mail:
bichara{at}bichat.inserm.fr).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 21, 2003;10.1152/ajprenal.00255.2002
Received 16 July 2002; accepted in final form 16 January 2003.
 |
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