Stimulation of 11-{beta}-hydroxysteroid dehydrogenase type 2 in rat colon but not in kidney by low dietary NaCl intake

Rikke Nørregaard, Torben R. Uhrenholt, Claus Bistrup, Ole Skøtt, and Boye L. Jensen

Department of Physiology and Pharmacology, University of Southern Denmark, Odense DK-5000, Denmark

Submitted 14 February 2003 ; accepted in final form 16 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Data suggest that mineralocorticoid selectivity is differentially regulated in epithelial target tissues. We investigated whether the level of dietary NaCl intake influenced the expression and tissue distribution of 11-{beta}-hydroxysteroid dehydrogenase type 2 (11{beta}HSD-2), aldosterone receptor (MR), and glucocorticoid receptor (GR) in rat colon, kidney, and cardiovascular tissue. Rats were fed a diet with 0.01 or 3% NaCl for 10 days. Messenger RNAs were analyzed with ribonuclease protection assay, 11{beta}HSD-2 protein by Western blot analysis, and localization of GR and 11{beta}HSD-2 by immunohistochemistry. NaCl restriction elevated plasma renin and aldosterone concentration, whereas corticosterone was unaltered. In distal colon, 11{beta}HSD-2 mRNA and protein were augmented significantly by low-NaCl intake and immunolabeling was widely distributed in crypt and surface epithelium. The MR mRNA level was decreased, whereas GR mRNA was unaltered in distal colon. MR, GR, and 11{beta}HSD-2 mRNAs were not changed in kidney cortex and medulla, left cardiac ventricle, and aorta. Immunofluorescence labeling showed that GR and 11{beta}HSD-2 localization was mutually exclusive in kidney. In colon epithelium, nuclear staining for GR subsided as perinuclear 11{beta}HSD-2 immunoreactivity increased with NaCl restriction. As a functional correlate of increased 11{beta}HSD-2 expression in colon, the GR-stimulated sodium-hydrogen exchanger NHE-3 was lowered by NaCl restriction. Inhibition of 11{beta}HSD-2 activity by carbenoxolone during NaCl restriction stimulated NHE-3 expression in colon. Dexamethasone stimulated NHE-3 both in colon and kidney. These data indicate that mineralocorticoid selectivity is physiologically regulated by NaCl intake at the level of 11{beta}HSD-2 expression and tissue distribution in the distal colon, but not in the kidney.

receptor; glucocorticoid; mineralocorticoid; aldosterone; corticosterone


ALDOSTERONE INITIATES RESPONSES through binding to cytoplasmic mineralocorticoid receptor (MR), which translocates to the cell nucleus and stimulates transcription of responsive genes (43). Receptor activation enhances Na+ transport in aldosterone-responsive epithelia in colon, kidney, salivary glands, sweat ducts, and skin. In epithelial tissues, glucocorticosteroids bind to and activate MR with the same affinity as mineralocorticoids and their plasma concentration normally exceeds that of aldosterone by a factor of 10–1,000. Selectivity of aldosterone over glucocorticoids is conferred by 11-{beta}-hydroxysteroid dehydrogenase-2 (11{beta}HSD-2), which metabolizes hydroxy-glucocorticoids to inactive ketoglucocorticoids and thereby prevents illicit receptor binding by glucocorticoids (13, 18). When NaCl balance or extracellular volume is threatened, the normal homeostatic response is an increase in plasma aldosterone concentration that activates MR. Downregulation of 11{beta}HSD-2 activity or large increases of plasma glucocorticoid concentration lead to glucocorticoid-dependent activation of MR. This takes place in various settings, such as the syndrome of apparent mineralocorticoid excess that is caused by loss-of-function mutations in 11{beta}HSD-2, by inhibition of 11{beta}HSD-2 through, e.g., glycyrrhetinic acid contained in licorice, and in Cushing's syndrome with overproduction of glucocorticoid. The site(s) of 11{beta}HSD-2 expression and the level of expression are key factors that determine aldosterone selectivity, but data on the physiological regulation of 11{beta}HSD-2 are sparse. At the functional level, data indicate that aldosterone-MR selectivity is a dynamic feature of the colon that is subject to physiological regulation by dietary salt intake and/or elevated aldosterone (16, 20, 45). Both aldosterone and glucocorticoids stimulate electrogenic Na+ absorption in the colon of rats on a normal sodium intake (2, 45, 48), whereas low-dose dexamethasone and not aldosterone stimulates electroneutral Na+ absorption (2, 3). Glucocorticoid sensitivity of electrogenic pathways subsides with sodium restriction in the colon (16, 20, 45), which implies that 11{beta}HSD-2 and/or glucocorticoid receptor (GR) is a regulated parameter. Indeed, changes in 11{beta}HSD-2 activity have been demonstrated in colon and kidney (30, 36), but it is not clear whether this takes place at the level of mRNA, protein, or tissue distribution. Moreover, the morphological basis for the sensitivty to dexamethasone is not understood. Thus MR and 11{beta}HSD-2 have been mapped in the colon (21, 46) and in kidney (7, 12, 21, 24, 26, 28, 34, 44), but data on localization of GR in colon and kidney are sparse and conflicting (12, 14, 34, 35, 44, 46). Thus it is not clear whether GR is expressed in absorptive surface cells in colon together with 11{beta}HSD-2, in secretory crypt cells, or in both. One purpose of the present study was to examine whether 11{beta}HSD-2, MR, and GR mRNA expression and tissue distribution are physiologically regulated in epithelial tissues by changes in dietary NaCl intake. Moreover, we studied the localization of GR and 11{beta}HSD-2 in kidney and colon and defined cells positive for GR and negative for 11{beta}HSD-2.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In Vivo Protocols

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 (250–300 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 8–10 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 0.78–200 ng/ml and with no cross-reactivity for cortisol and aldosterone.

Extraction of RNA

Frozen tissue samples (150–200 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{alpha}). 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.

11{beta}HSD-2. Sense: 5'-CGC GAA TGT ATG GAG GTG-'3; antisense: 5'-CAG TTG CTT GCG CTT CTC-'3, covering bases 682–972, 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 1912–2065 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 1619–1828, 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 2183–2395, 213 bp (acc. no. NM012654).

{beta}-Actin. This was as described (1).

Solution Hybridization and Ribonuclease Protection Assay

Relative levels of mRNA for 11{beta}HSD-2, MR, GR, and for rat {beta}-actin were determined by ribonuclease protection assay (1). The plasmids yielded radiolabeled antisense RNA transcripts by incubation with SP6 polymerase (Promega) and [{alpha}-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 1–3 days (Kodak BioMax film). Radioactivity in the protected probes was quantitated by excision from the gel and {beta}-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 150–200 V for 30–40 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 11{beta}HSD-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{beta}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 1–5 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-11{beta}HSD-2 1:2,000; anti-NHE3 1:250; and anti-rat GR-{alpha} 1:100 (Santa Cruz). GR-{alpha} 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{beta}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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasma Hormone Concentrations

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 {cong} 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 11{beta}HSD-2 mRNAs in Rat Distal Colon, Kidney Regions, and Cardiovascular Tissue

Sequencing of the cloned MR, GR, and 11{beta}HSD-2 cDNA showed 99–100% homology with published sequences. Surplus and specificity of radiolabeled antisense probes for MR, GR, and 11{beta}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 (5–40 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{beta}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{beta}HSD-2 mRNA in kidney cortex, kidney medulla, left ventricular myocardium, or aorta (n = 7 each group) (Fig. 1B and Table 1). {beta}-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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Fig. 1. A: hybridization of radiolabeled antisense probes for 11-{beta}-hydroxysteroid dehydrogenase type 2 (11{beta}HSD-2) (top), glucocorticoid receptor (GR; middle), and mineralocorticoid receptor (MR; bottom) mRNAs with whole kidney total RNA. Autoradiographs display the results of ribonuclease protection assays in which increasing amounts of total kidney RNA (5–40 µg) were hybridized with the appropriate antisense probe. Radioactivity in the protected probes was asssayed by cutting the hybrids out of the gel and counting them in a {beta}-counter. Assays were linear in the tested range, indicating surplus of probe. B: effect of dietary NaCl intake on levels of mRNA for 11{beta}HSD-2, MR, GR, and {beta}-actin in rat kidney cortex. Autoradiographs display the result of ribonuclease protection assays for 11{beta}HSD-2, MR, GR, and {beta}-actin mRNAs using RNA from rat kidney cortices from rats on high- and low-NaCl intake. Twenty micrograms of total RNA were used for solution hybridization with the antisense probes. C: quantitative evaluation of the ribonuclease protection assays for 11{beta}HSD-2 (top), MR (middle), and GR (bottom) mRNAs in kidney cortex. Radioactivity in the protected probes was assayed by cutting the hybrids out of the gel and counting them in a {beta}-counter. The results are {beta}-actin-normalized mean values ± SE of 7 separate determinations.

 

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Table 1. Effect of NaCl intake on mRNA levels for 11{beta}HSD-2, MR, and GR in cardiovascular tissue and kidney medulla

 

In contrast to kidney and cardiovascular tissue, there was a significant effect of dietary NaCl intake on mRNA levels for 11{beta}HSD-2 and MR in distal colon (Fig. 2). A low intake of NaCl increased the {beta}-actin-normalized 11{beta}HSD-2 mRNA level 3.2 times (Fig. 2). Compared with 11{beta}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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Fig. 2. Effect of dietary NaCl intake on mRNA levels for 11{beta}HSD-2, MR, GR, and {beta}-actin in rat distal colon. A: autoradiographs depicting the result of ribonuclease protection assays for 11{beta}HSD-2, MR, GR, and {beta}-actin mRNAs in rat distal colon. Thirty micrograms of total RNA were used for solution hybridization with the antisense probes, except for {beta}-actin where 5 µg were applied. B: quantitative evaluation of the ribonuclease protection assays for 11{beta}HSD-2 (top), MR (middle), and GR (bottom) mRNAs in rat distal colon. Radioactivity in the protected probes was asssayed by cutting the hybrids out of the gel and counting them in a {beta}-counter. The results shown are {beta}-actin-normalized mean values ± SE of 7 separate determinations. *P < 0.05.

 

Effect of Dietary NaCl Intake on 11{beta}HSD-2 Protein in Rat Distal Colon and Kidney Cortex

11{beta}HSD-2 protein expression was examined in distal colon and kidney cortex by immunoblotting. In serially diluted rat kidney protein extracts, the polyclonal 11{beta}HSD-2 antibody (sheep anti-rat) yielded a single band with expected size (42–44 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{beta}HSD-2 protein level was evident (Fig. 3). Thus 11{beta}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{beta}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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Fig. 3. Effect of dietary NaCl intake on 11{beta}HSD-2 protein level in rat kidney cortex and distal colon. Immunoblotting was followed by chemiluminescence detection of 11{beta}HSD-2 protein by labeling with the specific sheep anti-rat 11{beta}HSD-2 antibody. Twenty-five micrograms of protein aliquots from kidney cortex and distal colon samples from the 2 NaCl regimens were compared. The antibody labeled a distinct protein in both colon and kidney with a molecular mass of ~42–45 kDa.

 

Effect of NaCl Intake on Cellular and Subcellular Localization of 11{beta}HSD-2 and GR in Rat Distal Colon

11{beta}HSD-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{beta}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{beta}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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Fig. 4. Effect of dietary NaCl intake on cellular and subcellular distribution of 11{beta}HSD-2 in rat distal colon analyzed by immunoperoxidase and immunofluorescence confocal microscopy. Immunoperoxidase microscopy showed that 11{beta}HSD-2 was widely distributed and associated with epithelial cells along colon crypts and luminal surface in rats fed a diet low in NaCl (A), whereas immunoreactivity was faint and restricted in rats given a diet rich in NaCl (B). Bars = 50 µm. C and D: confocal immunofluorescence microscopy of colon sections from rats given a low-NaCl diet (C) and a high-NaCl diet (D). The sections were labeled with primary anti-11{beta}HSD-2 antibody and secondary antibody was coupled to Alexa-488 fluorophore. 11{beta}HSD-2 (green fluorescence) was strongly associated with the perinuclear area in the basal and midcrypt epithelial cells after a low-NaCl ingestion. Basal colon crypt cells from low-salt rats at high magnification displayed perinuclear localization of 11{beta}HSD-2 (green fluorescence). 11{beta}HSD-2 fluorescence was detectable in few crypts per section after ingestion of a high-salt intake (D).

 

GR-{alpha}. In colon epithelial cells from a low-NaCl rat, GR-{alpha} 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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Fig. 5. Single- and double-immunofluorescence and immunohistochemical labeling of distal colon for GR-{alpha} and 11{beta}HSD-2 using paraffin-embedded sections from rats on a low-NaCl intake. A: GR-{alpha}-immunopositive labeling was restricted to cell nuclei in the basal to midcrypt epithelial cells and was not observed in luminal colonocytes. B: in an adjacent tissue section, immunoreactivity was absent when the primary antibody was preincubated with the antigen used for immunization. Bars = 50 µm. C: double-immunofluorescence labeling of a rat distal colon section from a low-salt animal for GR-{alpha} (red fluorescence) and 11{beta}HSD-2 (green fluorescence). GR-{alpha} immunofluorescence was nuclear. The intensity of nuclear GR-{alpha} fluorescence in the epithelium was inversely related to the presence of 11{beta}HSD-2 in the perinuclear area. Elongated GR-{alpha}-positive cell nuclei were associated with 11{beta}HSD-2-negative smooth muscle cells in the lamina muscularis below the mucosa.

 

Next, we performed double-immunofluorescence labeling of colon sections for GR-{alpha} and 11{beta}HSD-2. Labeling confirmed the separate subcellular localization of GR-{alpha} and 11{beta}HSD-2 seen above; in immunopositive cells, GR-{alpha} was associated with nuclei (red fluorescence signal), whereas 11{beta}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{beta}HSD-2 and low, or absent, nuclear signals for GR-{alpha}. Thus the basal crypt cells with particularly marked 11{beta}HSD-2 signals displayed no nuclear labeling for GR-{alpha}, whereas cells negative for 11{beta}HSD-2, e.g., smooth muscle seen below the mucosa, showed strong nuclear signals for GR-{alpha} (Fig. 5C).

Effect of Dietary NaCl on Cellular and Subcellular Distribution of 11{beta}HSD-2 and GR in Rat Kidney

Labeling of a kidney section for 11{beta}HSD-2 and GR-{alpha} with the double-immunofluorescence technique showed at low-power magnification that immunofluorescence signals for 11{beta}HSD-2 (green) were associated with distinct tubules both in the cortical labyrinth and in the medullary rays, whereas fluorescence signals for GR-{alpha} were associated primarily with glomeruli and proximal tubules (Fig. 6A). At higher magnification, 11{beta}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{beta}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{beta}HSD-2-negative cells are most likely intercalated cells as previously reported (7). 11{beta}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{beta}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{beta}HSD-2 in kidney. In the absence of primary 11{beta}HSD-2 antibody or secondary antibody, there was no labeling of the tissue (not shown).



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Fig. 6. Double-immunofluorescence labeling of rat kidney from a rat fed a low-salt diet for GR-{alpha} and 11{beta}HSD-2 using paraffin-embedded 1-µm sections. A: at low magnification, kidney cortex displayed GR-{alpha} signal (red fluorescence signal) associated with proximal convoluted tubules and glomeruli, whereas 11{beta}HSD-2 (green fluorescence signal) was seen in distal convoluted tubule, connecting tubule, and collecting duct. There was no colocalization between GR-{alpha} and 11{beta}HSD-2. Bar = 200 µm. B: in 2 merging cortical collecting ducts, GR-{alpha} (red fluorescence) was present in 11{beta}HSD-2-negative intercalated cell nuclei (inset) and was absent from 11{beta}HSD-2-positive principal cell nuclei and cytoplasm. Bar = 50 µm. C: in a medullary ray of the deep renal cortex, GR-{alpha} (red fluorescence) was present in loops of Henle, predominantly in nuclei, whereas proximal tubules were labeled both in cytoplasm and nuclei. 11{beta}HSD-2 (green fluorescence) was localized in a collecting duct with a course parallel to the loops of Henle. Bar = 50 µm.

 

At high-power magnification, GR-{alpha} labeling (red fluorescence signal) was detected in glomeruli and proximal convoluted tubules, where signals were associated primarily with the cytoplasm, whereas all other GR-{alpha}-immunopositive structures were labeled in the nucleus (Fig. 6, B and C). In the medullary rays, loop of Henle nuclei were GR-{alpha} immunopositive (Fig. 6C), and distinct labeling was associated with 11{beta}HSD-2-negative, intercalated cell nuclei of the collecting ducts (Fig. 6B, inset). 11{beta}HSD-2-positive principal cells in collecting ducts were GR-{alpha} negative (Fig. 6, B and C, inset). Moreover, nuclei in vascular smooth muscle cells and endothelial cells were labeled for GR-{alpha}. In the inner medulla, GR-{alpha} immunoreactivity was associated with interstitial cells and not found in collecting ducts (not shown). Controls without GR-{alpha} 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 11{beta}HSD-2 Activity for Glucocorticoid Sensitivity in Distal Colon Epithelial Cells

On the basis of the above results, we hypothesized that an increase of 11{beta}HSD-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{beta}HSD-2 and selectively activates GR. A second group was treated with carbenoxolone (20 mg·kg1·day1 by intraperitoneal injection) that directly inhibits 11{beta}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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Fig. 7. Effect of dexamethasone (D) and carbenoxolone (C) on NHE-3 expression in colon during a low-NaCl intake. Immunoperoxidase microscopy of NHE-3 in rat distal colon using paraffin-embedded sections is shown. NHE-3 immunoreactivity was associated with the apical membrane of epithelial cells lining the upper part of the crypts and the luminal surface in colon from animals on a high-NaCl intake (A), whereas labeling was scarce and hard to detect in low-NaCl intake rats (B). Bars = 50 µm. C: immunoblotting of NHE-3 in rat distal colon showing the effect of NaCl intake on NHE-3 protein level. The antibody labeled a distinct band at 85 kDa that was not seen in the absence of primary antibody and after preabsorption of the primary antibody with peptide used for immunization. D: auto-radiograph (top) showing ribonuclease protection assay for NHE-3 mRNA with rat distal colon total RNA. Bottom: quantitative evaluation of the ribonuclease protection assay for NHE-3 mRNA in rat distal colon from rats treated with dexametheasone (50 µg·kg1·day1) and carbenoxolone (20 mg·kg1·day1). Radioactivity in the protected NHE-3 probe was asssayed by cutting the hybrids out of the gel and counting them in a {beta}-counter. The results shown are {beta}-actin-normalized mean values ± SE of 5 separate determinations. LS, low salt. *P < 0.05.

 

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 11{beta}HSD-2 activity reduces glucocorticoid-GR-mediated responses during NaCl restriction in colon.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present report, we show in rats that a NaCl-restricted diet leads to parallel increases in renin-angiotensin-aldosterone system components in plasma and 11{beta}HSD-2 mRNA, protein, and immunolabeling in colon but not in kidney or cardiovascular tissue. Nuclear labeling for GR-{alpha} and perinuclear staining for 11{beta}HSD-2 were inversely related in both colon and kidney. In the kidney, there was a mutually exclusive segmental localization of GR and 11{beta}HSD-2. Inhibition of 11{beta}HSD-2 during sodium restriction enhanced the abundance of a GR-stimulated transporter, NHE-3, in the colon. On the basis of the data, we suggest that in distal colon, glucocorticoid signaling via its cognate receptor and via MR is impeded during sodium restriction and selectivity for the aldosterone-MR pathway is favored. Our results suggest that in the kidney there is a division between glucocorticoid-sensitive sites, which are localized in proximal nephron segments and loops of Henle, and aldosterone-sensitive sites in principal cells of the distal nephron and collecting duct system.

The present data on localization of 11{beta}HSD-2 are essentially in accordance with previous studies (7, 21, 24, 26, 34, 44, 46) and add new information by showing 1) that 11{beta}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{beta}HSD-2 in colon crypts. We found expression of 11{beta}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{beta}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-{alpha} and with data showing GR mRNA expression and autoradiographic binding of dexamethasone in this nephron segment (14, 27, 44). We found GR-{alpha} 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-{alpha} 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{beta}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{beta}HSD-2 and not GR (33).

The lack of change in 11{beta}HSD-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-{alpha} was more strongly associated with crypt cells than with surface cells in the colon (39). The overlap of GR and 11{beta}HSD-2 in crypts during sodium restriction is in agreement with data showing that inhibition of 11{beta}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-{alpha} 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{beta}HSD-2 and nuclear GR-{alpha} is compatible with 11{beta}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 11{beta}HSD-2 activity (40). Functional data support the present finding of physiological regulation of 11{beta}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{beta}HSD-2 activity. In accordance, we found that the 11{beta}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{beta}HSD-2 in separate cells. NHE-3 is expressed with GR predominantly in proximal tubules where 11{beta}HSD-2 is absent (42). Whereas 11{beta}HSD-2 has a low Km for glucocorticoids and is essentially irreversible in the dehydrogenase direction, the enzyme isoform 11{beta}HSD-1 can act in a reversible fashion and has been suggested to modulate glucocorticoid responses by either decreasing or increasing local concentrations. 11{beta}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{beta}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{beta}HSD-1 activity correlated directly with NaCl intake in canine kidney proximal tubules (8), and it is assumed that 11{beta}HSD-1 is a predominant reductase in vivo, an increase of 11{beta}HSD-1 activity could have contributed to this response. Unspecific inhibition of 11{beta}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{beta}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{beta}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{beta}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.


    DISCLOSURES
 
This work was supported by grants from the Danish Medical Research Council (22010159), the Novo Nordisk Foundation, The Danish Heart Foundation (99223622743, 01123022896, 021233A2982, 012161A22939), the Danish Medical Association Research Fund, A. J. Andersens Foundation, and Ms. Ruth T. E. Koenig-Petersens Foundation for kidney diseases.


    ACKNOWLEDGMENTS
 
The technical assistance of M. Fredenslund, K. Kejling, and I. Andersen is gratefully acknowledged. The authors thank A. M. Carter for linguistic revision.


    FOOTNOTES
 

Address for reprint requests and other correspondence: B. L. Jensen, Dept. of Physiology and Pharmacology, Univ. of Southern Denmark, Winsloewparken 21, 3 DK-5000 Odense C, Denmark (E-mail: bljensen{at}health.sdu.dk).

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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