Department of Physiology and Pharmacology, University of Southern Denmark, Odense DK-5000, Denmark
Submitted 14 February 2003 ; accepted in final form 16 April 2003
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ABSTRACT |
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receptor; glucocorticoid; mineralocorticoid; aldosterone; corticosterone
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MATERIALS AND METHODS |
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All procedures conformed with the Danish national guidelines for the care and handling of animals and with the guidelines from the National Institutes of Health. Male Sprague-Dawley rats (250300 g) had free access to rat chow (2 g/kg Na+ and 5 g/kg Cl; Altromin-1310, Lage, Germany) and tap water. Two series were conducted. In one series, rats were kept on a high-NaCl diet (3% Na+ wt/wt, n = 8), and another group of rats received a low-NaCl diet (Altromin-C1036 with 138 mg Na+/kg and 179 mg Cl/kg) for 10 days (n = 8). In the low-NaCl series, the rats were initially given an intraperitoneal injection of furosemide (2 mg/kg). After 810 days, rats were killed by decapitation, trunk blood was collected in EDTA-coated vials, and organs were rapidly removed, frozen in liquid nitrogen, and stored at 80°C. Kidneys were separated into cortex and medulla and frozen in liquid nitrogen. In a second series, 18 male Sprague-Dawley rats were treated as the low-NaCl diet group above. The rats were divided in three groups of six each; group 1 was treated by subcutaneous injection of dexamethasone ["Decadron"-MSD, University Hospital Pharmacy, Odense, Denmark (50 µg·kg1·day1)], group 2 was given intraperitoneal injections of carbenoxolone [Sigma, Rødovre, Denmark (20 mg·kg1·day1)], and group 3 was injected with vehicle (isotonic glucose). After 10 days, rats were decapitated and blood and organs were collected as described above.
Determination of Plasma Concentrations of Renin, Aldosterone, and Corticosterone
Renin was measured by a microradioimmunoassay for generated ANG I as described (29). Renin concentration is expressed in Goldblatt units compared with renin standards from the National Institutes of Biological Standards and Control, Potters Bar, Hertfordshire, UK. Plasma aldosterone was measured using a commercial kit (COAT-A-COUNT, Diagnostic Products, Los Angeles, CA). The detection limit was 13.0 pg/ml, and the intra-assay coefficient of variation was <4%. Plasma total corticosterone was measured with the radioimmunoassay kit from Amersham's Biotrak series using [125I]corticosterone as a tracer (Hørsholm, Denmark) exactly following the instructions from the manufacturer. The reported range of the assay is 0.78200 ng/ml and with no cross-reactivity for cortisol and aldosterone.
Extraction of RNA
Frozen tissue samples (150200 mg) were homogenized (Polytron PT300, Kinematica, Switzerland) and total RNA was isolated with the RNeasy midi kit (Qiagen, Albertslund, Denmark) according to instructions. RNA was eluated with diethylpyrocarbonate-treated water, and the yield of RNA was quantified by measuring optical density at 260 nm.
RT-PCR, Cloning, and Sequencing
RT-PCR was performed as described
(1) using primer sets below. By
sequence alignment, the primers were designed to anneal to nonhomologous parts
of MR and GR cDNAs. All oligomers were synthesized with restriction sites for
BamHI (sense) and EcoRI (antisense) (Amersham Pharmacia) in
the 5' direction to allow for directional cloning. PCR products were
purified (QiaQuick kit, Qiagen), double digested with BamHI and
EcoRI, then separated on 2% agarose, eluted from the gel (Qiagen),
and ligated to the digested pSP73 vector (Promega) for heat shock uptake in
competent Escherichia coli (DH5). Colonies were confirmed by
PCR and plasmids were isolated by a kit (Qiagen) and digested with
HindIII. Plasmids were sequenced on an ABI Prism genetic analyzer
using the Terminator ready reaction mix (ABI) for the reaction according to
the instructions.
11HSD-2. Sense: 5'-CGC GAA TGT ATG GAG
GTG-'3; antisense: 5'-CAG TTG CTT GCG CTT CTC-'3, covering
bases 682972, 291 bp (acc. no. U22424
[GenBank]
).
MR. Sense: 5'-GCT TTG ATG GTA GCT GCG-'3; antisense: 5'-TGA GCA CCA ATC CGG TAG-'3, covering bases 19122065 of rat MR cDNA, 154 bp (acc. no. M36074 [GenBank] ).
GR. Sense: 5'-AGG GAT TCA GCA AGC CAC-3'; antisense: 5'-CGC CCA CCT AAC ATG TTG-3', covering bases 16191828, 210 bp (acc. no. M14053 [GenBank] ).
NHE-3. Sense: 5'-ATG GAG AAT CTG GCA CAC-3'; antisense: 5'-TGG CAC CCT GGA TAG GAT-3', amplifying bases 21832395, 213 bp (acc. no. NM012654).
-Actin. This was as described
(1).
Solution Hybridization and Ribonuclease Protection Assay
Relative levels of mRNA for 11HSD-2, MR, GR, and for rat
-actin
were determined by ribonuclease protection assay
(1). The plasmids yielded
radiolabeled antisense RNA transcripts by incubation with SP6 polymerase
(Promega) and [
-32P]GTP (Amersham Pharmacia Biotech)
according to the Promega riboprobe in vitro transcription protocol; 5 x
105 cpm of the RNA probes were hybridized with samples of total RNA
at 60°C overnight in a final volume of 50 µl. Sequential digestions
were performed with a mixture of RNase A/T1 (Roche, Hvidovre, Denmark) and
proteinase K (Roche). The hybrids were separated on 8% denaturing
polyacrylamide gels. Autoradiography was performed at 80°C for
13 days (Kodak BioMax film). Radioactivity in the protected probes was
quantitated by excision from the gel and
-counting.
Western Blot Analysis
Tissues were snap-frozen and homogenized in 1 ml buffer, kept for 10 min on
ice, and subsequently centrifuged at 14,000 g at 4°C for 10 min.
The supernatant was split into 100-µl aliquots and kept at 80°C.
Protein concentrations were determined using the Bio-Rad protein assay, with
BSA as a standard. The samples were mixed with loading buffer, boiled for 2
min, and separated by polyacrylamide gel electrophoresis (10%) at
150200 V for 3040 min. Proteins were electroblotted (Bio-Rad)
onto polyvinylidene difluoride immobilon membranes (Millipore) at 0.8
mA/cm2 for 1 h. After being blotted, the membrane was air dried and
blocked in Tween 20 Tris-base sodium (TTBS; 137 mmol/l NaCl, 20 mmol/l
Tris·HCl, and 0.5% Tween 20) with 5% dry milk for 16 h at 4°C.
Then, the membrane was washed in TTBS and incubated with primary antibody
[polyclonal sheep anti-rat 11HSD-2 (1:3,000); polyclonal rabbit anti-rat
NHE3 (1:2,000)] (both from Chemicon), diluted in TTBS with 2% dry milk for 2 h
at room temperature. Then the membrane was washed and incubated with secondary
antibody [HRP-coupled anti-sheep for anti-11
HSD-2 (1:1,000) and
HRP-coupled anti-rabbit for anti-NHE-3 (1:5,000) (DAKO, Copenhagen, Denmark)]
for 1 h at room temperature. Proteins were detected using Renaissance
Chemiluminescent Reagent Plus kit (Dupont) and exposed to X-ray film (Biomax,
Kodak) for 10 s-5 min.
Immunohistochemical, Immunofluorescence, Double-Immunofluorescence, and Laser Confocal Microscopic Analyses
For immunolabeling, a total of eight rats from the various experimental
groups was anesthetized by intraperitoneal injection of sodium amytal and the
aorta was cannulated below the renal arteries and perfused with 4%
paraformaldehyde in PBS (pH 7.35) for 5 min. The organs were embedded in
paraffin, sections of 15 µm were cut, deparaffinized, and antigen
recovery was routinely performed by microwaving for 20 min in 0.01% sodium
citrate buffer (DAKO) at pH 6. The primary antibodies were as for Western blot
analysis and were diluted as follows: anti-11HSD-2 1:2,000; anti-NHE3
1:250; and anti-rat GR-
1:100 (Santa Cruz). GR-
is the
hormone-binding receptor isoform. Sections were blocked for 45 min in TTBS
with 5% dry milk and then incubated with primary antibody for 16 h at 4°C.
After being washed 2 x 5 and 1 x 15 min in TTBS, the sections were
incubated for 1 h with HRP-conjugated secondary antibodies (1:500) (DAKO). HRP
was visualized by incubation for 30 s-5 min with 0.01% diaminobenzidine. For
immunofluorescence microscopy, the primary 11
HSD-2 and GR antibodies
were diluted as above and were added for 2 h at room temperature and then
overnight at 4°C. After being washed with TTBS, the labeling was
visualized with anti-IgG coupled to Alexa-488 and Alexa-594 (Molecular
Probes). For confocal microscopy (Leica DM IRBE), images were obtained using a
488-nm excitation wavelength from an air-cooled argon/krypton laser and 510-nm
long-pass filter. The images were produced from an average of four line scans
of
1-s duration.
Statistics
Data are presented as means ± SE. When two sets of data were
compared at the same time, an unpaired Student's t-test was used.
P 0.05 was considered significant.
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RESULTS |
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Plasma concentrations of renin-angiotensin system components and of
corticosterone were determined in the first experimental series with different
NaCl intakes. Rats on a low dietary NaCl intake (n = 7) had eightfold
higher plasma renin concentrations (73.3 ± 12.5 x
105 vs. 9.4 ± 2.2 x
105 Goldblatt U/ml) and 300-fold higher
aldosterone plasma concentrations (7,100 ± 432.9 vs. 26.7 ± 10.8
pg/ml) compared with the high-NaCl intake group (n = 7). There was a
tendency that plasma concentration of corticosterone was elevated in response
to a low-NaCl intake (low NaCl 165.3 ± 50 ng/ml vs. high NaCl 62.2
± 18.7 ng/ml). However, this effect was not statistically significant
(P 0.07). There were no differences in average body weight as
a result of NaCl intake.
Influence of Dietary NaCl Intake on MR, GR, and 11HSD-2 mRNAs
in Rat Distal Colon, Kidney Regions, and Cardiovascular Tissue
Sequencing of the cloned MR, GR, and 11HSD-2 cDNA showed
99100% homology with published sequences. Surplus and specificity of
radiolabeled antisense probes for MR, GR, and 11
HSD-2 were tested.
Hybridization of probes to a dilution series of rat kidney total RNA yielded
single hybridization products (Fig.
1A). There was a linear relationship between the amount
of total RNA in the tested range (540 µg) and the amount of
detectable radioactivity in the protected probes. Probes were completely
digested in the absence of template and the probes had the expected relative
molecular size. Pilot experiments showed that there were no significant
differences in 11
HSD-2, MR, or GR mRNA levels between kidney outer and
inner medulla (not shown). In subsequent series, we therefore restricted the
analysis of kidney regions to cortex and whole medulla. Analysis of colon was
restricted to distal colon. Cardiovascular tissues (left cardiac ventricle and
aorta) were analyzed as a nonepithelial control tissue. Different levels of
dietary NaCl intake were not associated with changes in expression of MR, GR,
or 11
HSD-2 mRNA in kidney cortex, kidney medulla, left ventricular
myocardium, or aorta (n = 7 each group)
(Fig. 1B and
Table 1).
-Actin mRNA
levels were not changed by dietary NaCl intake in any organ tested. Thus
correction with actin for RNA quality and loading did not change the
outcome.
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In contrast to kidney and cardiovascular tissue, there was a significant
effect of dietary NaCl intake on mRNA levels for 11HSD-2 and MR in
distal colon (Fig. 2). A low
intake of NaCl increased the
-actin-normalized 11
HSD-2 mRNA level
3.2 times (Fig. 2). Compared
with 11
HSD-2 mRNA, there was an opposite change in MR mRNA abundance in
distal colon, which was significantly lower in rats on low-NaCl diet
(Fig. 2). The level of GR mRNA
in distal colon was not changed by the dietary salt load.
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Effect of Dietary NaCl Intake on 11HSD-2 Protein in Rat Distal
Colon and Kidney Cortex
11HSD-2 protein expression was examined in distal colon and kidney
cortex by immunoblotting. In serially diluted rat kidney protein extracts, the
polyclonal 11
HSD-2 antibody (sheep anti-rat) yielded a single band with
expected size (4244 kDa; not shown) at a dilution of 1:3,000. When 25
µg protein from distal colon were loaded and immunoblotted from four rats
in the low-NaCl group and compared with four rats in the high-NaCl group, an
inverse relationship between dietary NaCl load and 11
HSD-2 protein level
was evident (Fig. 3). Thus
11
HSD-2 mRNA and protein were concordantly stimulated by low dietary
NaCl intake in rat distal colon. In contrast, there was no detectable
difference in the 11
HSD-2 protein level in kidney cortices from the two
diet regimens (Fig. 3).
Negative controls with an identical amount of protein loaded followed by
blotting and incubation without primary antibody were always run in parallel.
In the absence of primary antibody, no signals were detected with colon or
kidney cortex extracts.
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Effect of NaCl Intake on Cellular and Subcellular Localization of
11HSD-2 and GR in Rat Distal Colon
11HSD-2. Immunoperoxidase microscopy on semithin
paraffin-embedded sections from the low-NaCl intake group showed marked
labeling associated with the epithelium both in the crypts and at the luminal
surface (Fig. 4A). In
contrast, 11
HSD-2 immunolabeling was faint and hardly detectable in few
epithelial cells in the high-NaCl group
(Fig. 4B). Similar
results were obtained with immunofluorescence confocal microscopy, but the
larger resolution provided showed that fluorescence was particularly strong in
the perinuclear area of midcrypt and basal crypt cells in the low-NaCl group,
whereas in apical crypt and surface cells, the signal was more diffusely
associated with cytoplasm (Fig.
4C). A confocal image from a basal crypt at larger
magnification confirmed the notion that 11
HSD-2 was strictly perinuclear
(Fig. 4C,
inset) and thus not associated with cell nuclei. A colon section from
a high-NaCl rat displayed fluorescence signals from few cells in a minority of
crypts and no surface cells were labeled
(Fig. 4D).
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GR-. In colon epithelial cells from a low-NaCl rat,
GR-
immunoreactivity was associated with cell nuclei. In the basal
third of the crypts, few scattered epithelial cells were positive, whereas in
the midthird a more continous labeling associated with nuclei was observed. In
the superficial third and in surface epithelial cells, labeling was scarce
(Fig. 5A). Strong
labeling was associated with nuclei of smooth muscle cells of the circular
layer and resident cells in the lamina propria. Controls without primary
antibody or preabsorbed with immunizing peptide were negative, as shown in an
adjacent tissue section in Fig.
5B.
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Next, we performed double-immunofluorescence labeling of colon sections for
GR- and 11
HSD-2. Labeling confirmed the separate subcellular
localization of GR-
and 11
HSD-2 seen above; in immunopositive
cells, GR-
was associated with nuclei (red fluorescence signal),
whereas 11
HSD-2 was strictly perinuclear
(Fig. 5C). Moreover,
the double-staining procedure revealed that there was an inverse relationship
between strong perinuclear fluorescence for 11
HSD-2 and low, or absent,
nuclear signals for GR-
. Thus the basal crypt cells with particularly
marked 11
HSD-2 signals displayed no nuclear labeling for GR-
,
whereas cells negative for 11
HSD-2, e.g., smooth muscle seen below the
mucosa, showed strong nuclear signals for GR-
(Fig. 5C).
Effect of Dietary NaCl on Cellular and Subcellular Distribution of
11HSD-2 and GR in Rat Kidney
Labeling of a kidney section for 11HSD-2 and GR-
with the
double-immunofluorescence technique showed at low-power magnification that
immunofluorescence signals for 11
HSD-2 (green) were associated with
distinct tubules both in the cortical labyrinth and in the medullary rays,
whereas fluorescence signals for GR-
were associated primarily with
glomeruli and proximal tubules (Fig.
6A). At higher magnification, 11
HSD-2 was observed
in distal convoluted tubules, connecting tubules, and cortical and outer
medullary collecting ducts (Fig. 6,
B and C). Immunofluorescence for 11
HSD-2
was associated with the majority of collecting duct cells, but single,
scattered cells were not labeled (Fig. 6,
B and C, inset). These
11
HSD-2-negative cells are most likely intercalated cells as previously
reported (7). 11
HSD-2
labeling gradually waned in the outer medulla and was absent in the innermost
portion (papilla) of the collecting ducts (not shown). In
11
HSD-2-positive cells, immunoreactivity was seen particularly in the
perinuclear region and not in cell nuclei
(Fig. 6B,
inset). Different NaCl intakes had no effect on distribution or
subcellular localization of 11
HSD-2 in kidney. In the absence of primary
11
HSD-2 antibody or secondary antibody, there was no labeling of the
tissue (not shown).
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At high-power magnification, GR- labeling (red fluorescence signal)
was detected in glomeruli and proximal convoluted tubules, where signals were
associated primarily with the cytoplasm, whereas all other
GR-
-immunopositive structures were labeled in the nucleus
(Fig. 6, B and
C). In the medullary rays, loop of Henle nuclei were
GR-
immunopositive (Fig.
6C), and distinct labeling was associated with
11
HSD-2-negative, intercalated cell nuclei of the collecting ducts
(Fig. 6B,
inset). 11
HSD-2-positive principal cells in collecting ducts
were GR-
negative (Fig. 6,
B and C, inset). Moreover, nuclei in
vascular smooth muscle cells and endothelial cells were labeled for
GR-
. In the inner medulla, GR-
immunoreactivity was associated
with interstitial cells and not found in collecting ducts (not shown).
Controls without GR-
antibody or with preabsorption of the primary
antibody by the peptide used for immunization were negative, as for colon (not
shown).
Physiological Significance of Enhanced 11HSD-2 Activity for
Glucocorticoid Sensitivity in Distal Colon Epithelial Cells
On the basis of the above results, we hypothesized that an increase of
11HSD-2 expression in colon is responsible for downregulation of
glucocorticoid-stimulated pathways, e.g., the sodium-hydrogen exchanger NHE-3,
which is observed with chronic NaCl restriction or aldosterone infusion
(16,
20,
23,
45). To explore this
hypothesis, rats on a low-NaCl intake were subcutaneously injected with
dexamethasone (50
µg·kg1·day1)
that is resistant against metabolism by 11
HSD-2 and selectively
activates GR. A second group was treated with carbenoxolone (20
mg·kg1·day1
by intraperitoneal injection) that directly inhibits 11
HSD-2
activity.
We first examined whether level and distribution of NHE-3 changed with the applied NaCl regimens. Immunoblotting of colon protein from the high- and low-NaCl intake groups for NHE-3 confirmed that a low-NaCl intake reduced NHE-3 protein levels in distal colon (Fig. 7C) (23). Next, immunohistochemistry was used to examine the cellular localization of NHE-3 in colon from rats on high- or low-NaCl intake. NHE-3 was localized in the apical membranes of surface cells in the distal colon in rats on a high-NaCl intake (Fig. 7A), whereas labeling was hardly detectable in colon after a low-NaCl intake (Fig. 7B). We did not detect any differences in NHE-3 mRNA or protein levels in kidney cortex subsequent to changes in NaCl intake (not shown). Thus the actin-normalized NHE-3 mRNA level in the low-NaCl group was 87.7 ± 4.8 arbitrary units (n = 7) compared with 90.9 ± 6.1 arbitrary units (n = 7) in the high-NaCl group.
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During 10 days of NaCl restriction, control rats gained weight from 190
± 9to224 ± 11g(n = 6), as did the carbenoxolone-treated
rats (186 ± 14 to 210 ± 15 g) (n = 6), whereas
dexamethasone administration basically stopped growth (177 ± 14 vs. 174
± 12 g) (n = 6). Dexamethasone and carbenoxolone significantly
increased NHE-3 mRNA abundance in distal colon
(Fig. 7D). In kidney
cortex, the NHE-3 mRNA level was significantly increased by dexamethasone (not
shown, control 644.9 ± 40 cpm vs. dexamethasone 866.3 ± 56 cpm;
P < 0.05), whereas the effect of carbonoxolone was not
statistically significant. Thus using NHE-3 as a marker for the
glucocorticoid-GR pathway, a significant 11HSD-2 activity reduces
glucocorticoid-GR-mediated responses during NaCl restriction in colon.
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DISCUSSION |
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The present data on localization of 11HSD-2 are essentially in
accordance with previous studies
(7,
21,
24,
26,
34,
44,
46) and add new information by
showing 1) that 11
HSD-2 is strictly localized in the
perinuclear area in both colon and kidney, 2) that its abundance and
distribution along colon crypts are regulated by NaCl intake, and 3)
that there is an axial heterogeneity in the subcellular distribution of
11
HSD-2 in colon crypts. We found expression of 11
HSD-2 only in
principal cells of collecting ducts, in accordance with previous
immunohistochemical (6,
7) and activity data
(34) and in agreement with
selective expression of MR and MR target proteins, e.g., ENaC subunits, in
principal cells (7,
19,
28,
38,
44). The present finding of a
predominantly perinuclear localization of 11
HSD-2 is consistent with
most, but not all, in vitro studies
(32,
35,
41). The perinuclear
localization was particularly prominent in colonic crypt cells during low-NaCl
intake and in kidney cortical collecting duct principal cells. The strong
perinuclear labeling of the basal stem cells in the colon indicates an
enhanced translational activity in the endoplasmatic reticulum during cell
maturation. A secretory phenotype is associated with low differentiation and
dominates in the crypts (25).
The present data are compatible with the view that less differentiated cells
are recruited for electrogenic ENaC-mediated sodium absorption during sodium
restriction.
In the kidney, previous data suggest that glucocorticoid receptor
activation is functionally important in vessels and glomeruli
(4) and in proximal tubules
where gluconeogenesis, ammoniogenesis and phosphate, sodium and acid transport
are influenced (5). This is in
agreement with the present localization of the hormone-binding GR isoform
GR- and with data showing GR mRNA expression and autoradiographic
binding of dexamethasone in this nephron segment
(14,
27,
44). We found GR-
immunoreactivity in the loop of Henle, which has previously been demonstrated
at functional mRNA and protein levels
(12,
14,
44). In the collecting ducts,
we observed GR-
immunoreactivity only in intercalated cells, consistent
with a much lower expression of GR mRNA in collecting ducts compared with
proximal tubules and loop of Henle
(44). Data showed significanly
lower activity of 11
HSD-2 in intercalated cells compared with principal
cells (34), which allows for
effects of glucocorticoid on distal tubular acidification
(10). Together, these
observations point to a functional and morphological segregation of steroid
action in renal collecting ducts. However, in vitro data showed that GR can
stimulate electrogenic sodium transport
(22), which agrees with GR
immunolabeling in one previous report
(12). The applied antibody,
raised and used by Farman et al.
(12), did not label proximal
tubules, glomeruli, or intercalated cells. We have no obvious explanation for
this discrepancy, but as mentioned above, we believe that our localization
data are in good agreement with the majority of molecular and functional
studies. Some discrepancies might have arisen from studies that show specific
binding of 3H-corticosterone to principal cells because this
binding is predominantly mediated by interaction with 11
HSD-2 and not GR
(33).
The lack of change in 11HSD-2 mRNA and protein expression in the
kidney after ingestion of a low-salt diet is consistent with mRNA data from
rats and activity data from humans
(15,
30). The absence of changes in
GR mRNA or distribution is also in accordance with previous reports
(11). In contrast, MR mRNA
decreased in the colon in response to a low-NaCl intake. We did not further
address the mechanism responsible for this effect, but infusion of aldosterone
and dexamethasone did not change MR mRNA expression in either kidney or colon
(11,
31).
Consistent with previous observations, immunoreactivity for hormone-binding
GR- was more strongly associated with crypt cells than with surface
cells in the colon (39). The
overlap of GR and 11
HSD-2 in crypts during sodium restriction is in
agreement with data showing that inhibition of 11
HSD-2 in isolated
colonic cells leads to a marked increase in corticosterone binding to both GR
and MR (40). Binding of
glucocorticoid to GR leads to dimerization and translocation to the nucleus.
We observed a predominantly nuclear localization of GR-
immunoreactivity in epithelial cells and smooth muscle cells in colon and in
most GR-positive cells in kidney, suggesting activation of GR. The inverse
relationship between perinuclear 11
HSD-2 and nuclear GR-
is
compatible with 11
HSD-2-mediated metabolization of glucocorticoid
leading to decreased GR binding, activation, and translocation in vivo.
In contrast to more proximal intestinal segments, colonic epithelial cells
contain a significant basal 11HSD-2 activity
(40). Functional data support
the present finding of physiological regulation of 11
HSD-2 in distal
colon. Thus chronic elevation of plasma aldosterone stimulates electrogenic
sodium absorption, through ENaC, and decreases the contribution from
electroneutral Na+ absorption
(20), which disappears after 1
wk (45). In the colon,
combined Na+/H+ and
Cl/HCO3 exchange is the
predominant mechanism of electroneutral Na+ absorption
(2,
3,
16,
17,
25), which is stimulated by
glucocorticoids (2,
3). The dominant
Na+/H+ exchange isoforms in colon, NHE-2 and -3, are
downregulated by 7 days of aldosterone treatment or low-NaCl intake
(23), whereas after 3 days, no
such effect is seen (9).
Together, the available functional data indicate a progressive suppression of
glucocorticoid-regulated transport proteins in colon during Na+
restriction and/or elevated aldosterone. We suggest this effect is
attributable to enhanced 11
HSD-2 activity. In accordance, we found that
the 11
HSD-2 blocker carbenoxolone stimulated NHE-3 expression in colon
during sodium restriction. The lack of effect of carbenoxolone on NHE-3
expression in kidney corroborates the molecular data showing expression of GR
and 11
HSD-2 in separate cells. NHE-3 is expressed with GR predominantly
in proximal tubules where 11
HSD-2 is absent
(42). Whereas 11
HSD-2
has a low Km for glucocorticoids and is essentially
irreversible in the dehydrogenase direction, the enzyme isoform 11
HSD-1
can act in a reversible fashion and has been suggested to modulate
glucocorticoid responses by either decreasing or increasing local
concentrations. 11
HSD-1 is present in rat kidney proximal tubules and
thus colocalizes with GR (8,
37), but it is absent in rat
colon epithelium (40,
47). Although we did not
measure 11
HSD-1 activity, it is less likely to have contributed to the
the present results in rat colon. We found that NHE-3 was stimulated in kidney
following a high-NaCl intake with no concomitant change in GR or circulating
corticosterone. Because 11
HSD-1 activity correlated directly with NaCl
intake in canine kidney proximal tubules
(8), and it is assumed that
11
HSD-1 is a predominant reductase in vivo, an increase of 11
HSD-1
activity could have contributed to this response. Unspecific inhibition of
11
HSD-1 activity by carbenoxolone could potentially have decreased or
increased GR activation in proximal tubules in the present study, depending on
the diretion of 11
HSD-1 activity. However, we observed no significant
change in renal NHE-3 abundance in carbenoxolone-treated animals compared with
controls. The described time course of changes in functional behavior and
transport protein expression in distal colon after a change in NaCl intake
closely matches the present change in 11
HSD-2 expression. Because of the
slow time course of these changes, it is tempting to suggest that the decrease
in electroneutral Na+ transport includes cell turnover, where crypt
cells are "committed" to electrogenic transport at a rate that
matches apical shedding of cells. Together, the data indicate a key role for
11
HSD-2 in distal colon epithelium as a switch between states of mixed
steroid sensitivity and selective mineralocorticoid sensitivity dictated by
NaCl intake. In contrast, the kidney maintains a segment-specific
mineralocorticoid sensitivity and glucocorticoid sensitivity that are
independent of dietary salt intake.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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