Stimulation of 11--hydroxysteroid dehydrogenase type 2 in rat colon but not in k - 《American Journal of Physiology》美国生理学杂志干细胞之家



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作者：Rikke Nørregaard, Torben R. Uhrenholt, Claus Bistrup, Ole Skøtt,  Boye L. Jensen作者单位：Department of Physiology and Pharmacology, University of SouthernDenmark, Odense DK-500 Denmark . W1 A' M/ F0 w) [0 w& u0 f! D
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      【摘要】6 r9 w, n2 r, Z$ e0 `
   Data suggest that mineralocorticoid selectivity is differentially regulatedin epithelial target tissues. We investigated whether the level of dietaryNaCl intake influenced the expression and tissue distribution of11- -hydroxysteroid dehydrogenase type 2 (11 HSD-2), aldosteronereceptor (MR), and glucocorticoid receptor (GR) in rat colon, kidney, andcardiovascular tissue. Rats were fed a diet with 0.01 or 3% NaCl for 10 days.Messenger RNAs were analyzed with ribonuclease protection assay, 11 HSD-2 protein by Western blot analysis, and localization of GR and 11 HSD-2 byimmunohistochemistry. NaCl restriction elevated plasma renin and aldosteroneconcentration, whereas corticosterone was unaltered. In distal colon,11 HSD-2 mRNA and protein were augmented significantly by low-NaCl intakeand 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 HSD-2 mRNAs were not changed in kidney cortex and medulla,left cardiac ventricle, and aorta. Immunofluorescence labeling showed that GRand 11 HSD-2 localization was mutually exclusive in kidney. In colonepithelium, nuclear staining for GR subsided as perinuclear 11 HSD-2immunoreactivity increased with NaCl restriction. As a functional correlate of increased 11 HSD-2 expression in colon, the GR-stimulated sodium-hydrogen exchanger NHE-3 was lowered by NaCl restriction. Inhibition of 11 HSD-2activity by carbenoxolone during NaCl restriction stimulated NHE-3 expressionin colon. Dexamethasone stimulated NHE-3 both in colon and kidney. These dataindicate that mineralocorticoid selectivity is physiologically regulated byNaCl intake at the level of 11 HSD-2 expression and tissue distributionin the distal colon, but not in the kidney. & i2 M3 N, t/ {6 B7 r- F& T9 O% C
      【关键词】 receptor glucocorticoid mineralocorticoid aldosterone corticosterone- J7 ]  S/ \4 g8 Y) G) D  Y
   ALDOSTERONE INITIATES RESPONSES through binding to cytoplasmic mineralocorticoid receptor (MR), which translocates to the cell nucleus andstimulates transcription of responsive genes( 43 ). Receptor activationenhances Na transport in aldosterone-responsive epithelia incolon, kidney, salivary glands, sweat ducts, and skin. In epithelial tissues,glucocorticosteroids bind to and activate MR with the same affinity asmineralocorticoids and their plasma concentration normally exceeds that ofaldosterone by a factor of 10-1,000. Selectivity of aldosterone over glucocorticoids is conferred by 11- -hydroxysteroid dehydrogenase-2 (11 HSD-2), which metabolizes hydroxy-glucocorticoids to inactive ketoglucocorticoids and thereby prevents illicit receptor binding byglucocorticoids ( 13, 18 ). When NaCl balance orextracellular volume is threatened, the normal homeostatic response is an increase in plasma aldosterone concentration that activates MR. Downregulationof 11 HSD-2 activity or large increases of plasma glucocorticoidconcentration lead to glucocorticoid-dependent activation of MR. This takesplace in various settings, such as the syndrome of apparent mineralocorticoidexcess that is caused by loss-of-function mutations in 11 HSD-2, byinhibition of 11 HSD-2 through, e.g., glycyrrhetinic acid contained in licorice, and in Cushing's syndrome with overproduction of glucocorticoid. Thesite(s) of 11 HSD-2 expression and the level of expression are keyfactors that determine aldosterone selectivity, but data on the physiologicalregulation of 11 HSD-2 are sparse. At the functional level, data indicatethat aldosterone-MR selectivity is a dynamic feature of the colon that issubject to physiological regulation by dietary salt intake and/or elevated aldosterone ( 16, 20, 45 ). Both aldosterone andglucocorticoids stimulate electrogenic Na absorption in the colonof rats on a normal sodium intake( 2, 45, 48 ), whereas low-dose dexamethasone and not aldosterone stimulates electroneutral Na absorption ( 2, 3 ). Glucocorticoid sensitivityof electrogenic pathways subsides with sodium restriction in the colon( 16, 20, 45 ), which implies that11 HSD-2 and/or glucocorticoid receptor (GR) is a regulated parameter.Indeed, changes in 11 HSD-2 activity have been demonstrated in colon andkidney ( 30, 36 ), but it is not clearwhether this takes place at the level of mRNA, protein, or tissuedistribution. Moreover, the morphological basis for the sensitivty todexamethasone is not understood. Thus MR and 11 HSD-2 have been mapped in the colon ( 21, 46 ) and in kidney( 7, 12, 21, 24, 26, 28, 34, 44 ), but data on localizationof GR in colon and kidney are sparse and conflicting( 12, 14, 34, 35, 44, 46 ). Thus it is not clearwhether GR is expressed in absorptive surface cells in colon together with11 HSD-2, in secretory crypt cells, or in both. One purpose of thepresent study was to examine whether 11 HSD-2, MR, and GR mRNA expressionand tissue distribution are physiologically regulated in epithelial tissues bychanges in dietary NaCl intake. Moreover, we studied the localization of GRand 11 HSD-2 in kidney and colon and defined cells positive for GR andnegative for 11 HSD-2.

MATERIALS AND METHODS. _7 K" Y. c, x- e* n" i' [

In Vivo Protocols9 A5 o0 ]# ]+ N) `. N# ]. _
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All procedures conformed with the Danish national guidelines for the careand handling of animals and with the guidelines from the National Institutesof Health. Male Sprague-Dawley rats (250-300 g) had free access to ratchow (2 g/kg Na and 5 g/kg Cl -; Altromin-1310,Lage, Germany) and tap water. Two series were conducted. In one series, ratswere kept on a high-NaCl diet (3% Na wt/wt, n = 8), andanother group of rats received a low-NaCl diet (Altromin-C1036 with 138 mgNa /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 bydecapitation, trunk blood was collected in EDTA-coated vials, and organs wererapidly 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; group1 was treated by subcutaneous injection of dexamethasone ["Decadron"-MSD, University Hospital Pharmacy, Odense, Denmark (50µg·kg - 1 ·day - 1 )], group 2 was given intraperitoneal injections of carbenoxolone [Sigma, Rødovre, Denmark (20mg·kg - 1 ·day - 1 )], and group 3 was injected with vehicle (isotonic glucose). After 10days, rats were decapitated and blood and organs were collected as describedabove.
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Determination of Plasma Concentrations of Renin, Aldosterone, andCorticosterone

Renin was measured by a microradioimmunoassay for generated ANG I asdescribed ( 29 ). Reninconcentration is expressed in Goldblatt units compared with renin standardsfrom 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 was13.0 pg/ml, and the intra-assay coefficient of variation was with the radioimmunoassay kit fromAmersham's Biotrak series using [ 125 I]corticosterone as a tracer(Hørsholm, Denmark) exactly following the instructions from themanufacturer. The reported range of the assay is 0.78-200 ng/ml and with no cross-reactivity for cortisol and aldosterone.; u4 q# q0 e0 o  ?  V- r3 Z

Extraction of RNA" v8 K" d" h, B: D7 H, l6 Y

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 withdiethylpyrocarbonate-treated water, and the yield of RNA was quantified bymeasuring optical density at 260 nm.: O- b$ A3 w0 k$ o' r" n7 r% U7 a

RT-PCR, Cloning, and Sequencing

RT-PCR was performed as described( 1 ) using primer sets below. Bysequence alignment, the primers were designed to anneal to nonhomologous partsof MR and GR cDNAs. All oligomers were synthesized with restriction sites for Bam HI (sense) and Eco RI (antisense) (Amersham Pharmacia) inthe 5' direction to allow for directional cloning. PCR products werepurified (QiaQuick kit, Qiagen), double digested with Bam HI and Eco RI, 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 Hind III. Plasmids were sequenced on an ABI Prism genetic analyzerusing the Terminator ready reaction mix (ABI) for the reaction according tothe instructions.' F+ @. M, |" D) T

11 HSD-2. Sense: 5'-CGC GAA TGT ATG GAGGTG-'3; antisense: 5'-CAG TTG CTT GCG CTT CTC-'3, coveringbases 682-972, 291 bp (acc. no. U22424  ).! `# p) `( M/ p+ V( @" l, Q$ E7 C$ q

MR. Sense: 5'-GCT TTG ATG GTA GCT GCG-'3; antisense:5'-TGA GCA CCA ATC CGG TAG-'3, covering bases 1912-2065 ofrat MR cDNA, 154 bp (acc. no. M36074  ).
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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  ).% F# \3 j# N; v
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NHE-3. Sense: 5'-ATG GAG AAT CTG GCA CAC-3';antisense: 5'-TGG CAC CCT GGA TAG GAT-3', amplifying bases2183-2395, 213 bp (acc. no. NM012654).
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-Actin. This was as described( 1 ).
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Solution Hybridization and Ribonuclease Protection Assay
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Relative levels of mRNA for 11 HSD-2, MR, GR, and for rat -actin were determined by ribonuclease protection assay( 1 ). The plasmids yieldedradiolabeled antisense RNA transcripts by incubation with SP6 polymerase(Promega) and [ - 32 P]GTP (Amersham Pharmacia Biotech)according to the Promega riboprobe in vitro transcription protocol; 5 x 10 5 cpm of the RNA probes were hybridized with samples of total RNAat 60°C overnight in a final volume of 50 µl. Sequential digestionswere performed with a mixture of RNase A/T1 (Roche, Hvidovre, Denmark) andproteinase K (Roche). The hybrids were separated on 8% denaturingpolyacrylamide gels. Autoradiography was performed at -80°C for 1-3 days (Kodak BioMax film). Radioactivity in the protected probes wasquantitated by excision from the gel and -counting." p0 u+ L' b3 J2 t% R
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Western Blot Analysis

Tissues were snap-frozen and homogenized in 1 ml buffer, kept for 10 min onice, 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, withBSA as a standard. The samples were mixed with loading buffer, boiled for 2min, and separated by polyacrylamide gel electrophoresis (10%) at150-200 V for 30-40 min. Proteins were electroblotted (Bio-Rad)onto polyvinylidene difluoride immobilon membranes (Millipore) at 0.8mA/cm 2 for 1 h. After being blotted, the membrane was air dried andblocked in Tween 20 Tris-base sodium (TTBS; 137 mmol/l NaCl, 20 mmol/lTris·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 HSD-2 (1:3,000); polyclonal rabbit anti-ratNHE3 (1:2,000)] (both from Chemicon), diluted in TTBS with 2% dry milk for 2 hat room temperature. Then the membrane was washed and incubated with secondaryantibody [HRP-coupled anti-sheep for anti-11 HSD-2 (1:1,000) andHRP-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.3 u1 C  r0 R) y* Q

Immunohistochemical, Immunofluorescence, Double-Immunofluorescence,and Laser Confocal Microscopic Analyses
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For immunolabeling, a total of eight rats from the various experimental groups was anesthetized by intraperitoneal injection of sodium amytal and theaorta was cannulated below the renal arteries and perfused with 4%paraformaldehyde in PBS (pH 7.35) for 5 min. The organs were embedded inparaffin, sections of 1-5 µm were cut, deparaffinized, and antigenrecovery 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 blotanalysis and were diluted as follows: anti-11 HSD-2 1:2,000; anti-NHE31:250; and anti-rat GR- 1:100 (Santa Cruz). GR- is thehormone-binding receptor isoform. Sections were blocked for 45 min in TTBSwith 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 wereincubated for 1 h with HRP-conjugated secondary antibodies (1:500) (DAKO). HRPwas visualized by incubation for 30 s-5 min with 0.01% diaminobenzidine. Forimmunofluorescence microscopy, the primary 11 HSD-2 and GR antibodieswere diluted as above and were added for 2 h at room temperature and thenovernight at 4°C. After being washed with TTBS, the labeling wasvisualized with anti-IgG coupled to Alexa-488 and Alexa-594 (MolecularProbes). For confocal microscopy (Leica DM IRBE), images were obtained using a488-nm excitation wavelength from an air-cooled argon/krypton laser and 510-nmlong-pass filter. The images were produced from an average of four line scansof 1-s duration.

Statistics3 S6 q* r7 C3 g/ w7 n, D) T
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Data are presented as means ± SE. When two sets of data werecompared at the same time, an unpaired Student's t -test was used. P 0.05 was considered significant.
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RESULTS

Plasma Hormone Concentrations# v- J: ^. ~! f5 ^% j
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Plasma concentrations of renin-angiotensin system components and ofcorticosterone were determined in the first experimental series with differentNaCl intakes. Rats on a low dietary NaCl intake ( n = 7) had eightfoldhigher plasma renin concentrations (73.3 ± 12.5 x 10 - 5 vs. 9.4 ± 2.2 x 10 - 5 Goldblatt U/ml) and 300-fold higheraldosterone plasma concentrations (7,100 ± 432.9 vs. 26.7 ± 10.8pg/ml) compared with the high-NaCl intake group ( n = 7). There was atendency that plasma concentration of corticosterone was elevated in responseto 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 asa result of NaCl intake.
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Influence of Dietary NaCl Intake on MR, GR, and 11 HSD-2 mRNAsin Rat Distal Colon, Kidney Regions, and Cardiovascular Tissue
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Sequencing of the cloned MR, GR, and 11 HSD-2 cDNA showed99-100% 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 yieldedsingle hybridization products ( Fig.1 A ). There was a linear relationship between the amountof total RNA in the tested range (5-40 µg) and the amount ofdetectable radioactivity in the protected probes. Probes were completely digested in the absence of template and the probes had the expected relativemolecular size. Pilot experiments showed that there were no significantdifferences in 11 HSD-2, MR, or GR mRNA levels between kidney outer andinner medulla (not shown). In subsequent series, we therefore restricted theanalysis of kidney regions to cortex and whole medulla. Analysis of colon wasrestricted to distal colon. Cardiovascular tissues (left cardiac ventricle andaorta) were analyzed as a nonepithelial control tissue. Different levels ofdietary NaCl intake were not associated with changes in expression of MR, GR,or 11 HSD-2 mRNA in kidney cortex, kidney medulla, left ventricularmyocardium, or aorta ( n = 7 each group)( Fig. 1 B and Table 1 ). -Actin mRNAlevels were not changed by dietary NaCl intake in any organ tested. Thuscorrection with actin for RNA quality and loading did not change theoutcome.# U& J8 Z' g3 e+ B: B3 b* o

Fig. 1. A : hybridization of radiolabeled antisense probes for11- -hydroxysteroid dehydrogenase type 2 (11 HSD-2) ( top ),glucocorticoid receptor (GR; middle ), and mineralocorticoid receptor(MR; bottom ) mRNAs with whole kidney total RNA. Autoradiographsdisplay the results of ribonuclease protection assays in which increasingamounts of total kidney RNA (5-40 µg) were hybridized with theappropriate antisense probe. Radioactivity in the protected probes wasasssayed by cutting the hybrids out of the gel and counting them in a -counter. Assays were linear in the tested range, indicating surplus ofprobe. B : effect of dietary NaCl intake on levels of mRNA for11 HSD-2, MR, GR, and -actin in rat kidney cortex. Autoradiographsdisplay the result of ribonuclease protection assays for 11 HSD-2, MR,GR, and -actin mRNAs using RNA from rat kidney cortices from rats onhigh- and low-NaCl intake. Twenty micrograms of total RNA were used forsolution hybridization with the antisense probes. C : quantitativeevaluation of the ribonuclease protection assays for 11 HSD-2( top ), MR ( middle ), and GR ( bottom ) mRNAs in kidneycortex. Radioactivity in the protected probes was assayed by cutting thehybrids out of the gel and counting them in a -counter. The results are -actin-normalized mean values ± SE of 7 separatedeterminations.+ I2 ?6 H# k6 l: ~- i
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Table 1. Effect of NaCl intake on mRNA levels for 11 HSD-2, MR, and GR incardiovascular tissue and kidney medulla% b  T' s! J8 R2 M: V# F& u

In contrast to kidney and cardiovascular tissue, there was a significanteffect of dietary NaCl intake on mRNA levels for 11 HSD-2 and MR indistal colon ( Fig. 2 ). A lowintake of NaCl increased the -actin-normalized 11 HSD-2 mRNA level3.2 times ( Fig. 2 ). Comparedwith 11 HSD-2 mRNA, there was an opposite change in MR mRNA abundance indistal 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.' {/ \' r- W4 R6 Z7 T# }9 ]
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Fig. 2. Effect of dietary NaCl intake on mRNA levels for 11 HSD-2, MR, GR, and -actin in rat distal colon. A : autoradiographs depicting theresult of ribonuclease protection assays for 11 HSD-2, MR, GR, and -actin mRNAs in rat distal colon. Thirty micrograms of total RNA wereused for solution hybridization with the antisense probes, except for -actin where 5 µg were applied. B : quantitative evaluationof the ribonuclease protection assays for 11 HSD-2 ( top ), MR( middle ), and GR ( bottom ) mRNAs in rat distal colon.Radioactivity in the protected probes was asssayed by cutting the hybrids outof the gel and counting them in a -counter. The results shown are -actin-normalized mean values ± SE of 7 separate determinations.* P& F' |/ M# D5 T

Effect of Dietary NaCl Intake on 11 HSD-2 Protein in Rat DistalColon and Kidney Cortex% Q6 \, C& v2 E6 x7 F" h" b" Q
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11 HSD-2 protein expression was examined in distal colon and kidneycortex by immunoblotting. In serially diluted rat kidney protein extracts, thepolyclonal 11 HSD-2 antibody (sheep anti-rat) yielded a single band withexpected size (42-44 kDa; not shown) at a dilution of 1:3,000. When 25µg 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, aninverse relationship between dietary NaCl load and 11 HSD-2 protein levelwas evident ( Fig. 3 ). Thus11 HSD-2 mRNA and protein were concordantly stimulated by low dietaryNaCl intake in rat distal colon. In contrast, there was no detectable difference in the 11 HSD-2 protein level in kidney cortices from the twodiet regimens ( Fig. 3 ).Negative controls with an identical amount of protein loaded followed byblotting and incubation without primary antibody were always run in parallel. In the absence of primary antibody, no signals were detected with colon orkidney cortex extracts.
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Fig. 3. Effect of dietary NaCl intake on 11 HSD-2 protein level in rat kidneycortex and distal colon. Immunoblotting was followed by chemiluminescencedetection of 11 HSD-2 protein by labeling with the specific sheepanti-rat 11 HSD-2 antibody. Twenty-five micrograms of protein aliquotsfrom kidney cortex and distal colon samples from the 2 NaCl regimens werecompared. The antibody labeled a distinct protein in both colon and kidneywith a molecular mass of 42-45 kDa.
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Effect of NaCl Intake on Cellular and Subcellular Localization of11 HSD-2 and GR in Rat Distal Colon

11 HSD-2. Immunoperoxidase microscopy on semithinparaffin-embedded sections from the low-NaCl intake group showed markedlabeling associated with the epithelium both in the crypts and at the luminalsurface ( Fig. 4 A ). Incontrast, 11 HSD-2 immunolabeling was faint and hardly detectable in fewepithelial cells in the high-NaCl group( Fig. 4 B ). Similarresults were obtained with immunofluorescence confocal microscopy, but thelarger resolution provided showed that fluorescence was particularly strong inthe perinuclear area of midcrypt and basal crypt cells in the low-NaCl group,whereas in apical crypt and surface cells, the signal was more diffuselyassociated with cytoplasm ( Fig.4 C ). A confocal image from a basal crypt at largermagnification confirmed the notion that 11 HSD-2 was strictly perinuclear ( Fig. 4 C, inset ) and thus not associated with cell nuclei. A colon section froma high-NaCl rat displayed fluorescence signals from few cells in a minority ofcrypts and no surface cells were labeled( Fig. 4 D ).5 E4 z3 v+ C# v1 {8 K% J0 M1 u

Fig. 4. Effect of dietary NaCl intake on cellular and subcellular distribution of11 HSD-2 in rat distal colon analyzed by immunoperoxidase andimmunofluorescence confocal microscopy. Immunoperoxidase microscopy showedthat 11 HSD-2 was widely distributed and associated with epithelial cellsalong colon crypts and luminal surface in rats fed a diet low in NaCl( A ), whereas immunoreactivity was faint and restricted in rats givena diet rich in NaCl ( B ). Bars = 50 µm. C and D :confocal immunofluorescence microscopy of colon sections from rats given alow-NaCl diet ( C ) and a high-NaCl diet ( D ). The sectionswere labeled with primary anti-11 HSD-2 antibody and secondary antibodywas coupled to Alexa-488 fluorophore. 11 HSD-2 (green fluorescence) wasstrongly associated with the perinuclear area in the basal and midcryptepithelial cells after a low-NaCl ingestion. Basal colon crypt cells fromlow-salt rats at high magnification displayed perinuclear localization of11 HSD-2 (green fluorescence). 11 HSD-2 fluorescence was detectablein few crypts per section after ingestion of a high-salt intake( D ).* S+ S2 D9 q7 V) ~0 V
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GR-. In colon epithelial cells from a low-NaCl rat,GR- immunoreactivity was associated with cell nuclei. In the basalthird of the crypts, few scattered epithelial cells were positive, whereas inthe midthird a more continous labeling associated with nuclei was observed. Inthe superficial third and in surface epithelial cells, labeling was scarce( Fig. 5 A ). Stronglabeling was associated with nuclei of smooth muscle cells of the circular layer and resident cells in the lamina propria. Controls without primaryantibody or preabsorbed with immunizing peptide were negative, as shown in anadjacent tissue section in Fig.5 B.

Fig. 5. Single- and double-immunofluorescence and immunohistochemical labeling ofdistal colon for GR- and 11 HSD-2 using paraffin-embedded sectionsfrom rats on a low-NaCl intake. A : GR- -immunopositive labelingwas restricted to cell nuclei in the basal to midcrypt epithelial cells andwas not observed in luminal colonocytes. B : in an adjacent tissuesection, immunoreactivity was absent when the primary antibody waspreincubated with the antigen used for immunization. Bars = 50 µm. C : double-immunofluorescence labeling of a rat distal colon sectionfrom a low-salt animal for GR- (red fluorescence) and 11 HSD-2(green fluorescence). GR- immunofluorescence was nuclear. The intensityof nuclear GR- fluorescence in the epithelium was inversely related tothe presence of 11 HSD-2 in the perinuclear area. ElongatedGR- -positive cell nuclei were associated with 11 HSD-2-negativesmooth muscle cells in the lamina muscularis below the mucosa.
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Next, we performed double-immunofluorescence labeling of colon sections forGR- and 11 HSD-2. Labeling confirmed the separate subcellularlocalization of GR- and 11 HSD-2 seen above; in immunopositivecells, GR- was associated with nuclei (red fluorescence signal),whereas 11 HSD-2 was strictly perinuclear( Fig. 5 C ). Moreover,the double-staining procedure revealed that there was an inverse relationshipbetween strong perinuclear fluorescence for 11 HSD-2 and low, or absent,nuclear signals for GR-. Thus the basal crypt cells with particularlymarked 11 HSD-2 signals displayed no nuclear labeling for GR-,whereas cells negative for 11 HSD-2, e.g., smooth muscle seen below themucosa, showed strong nuclear signals for GR- ( Fig. 5 C ).

Effect of Dietary NaCl on Cellular and Subcellular Distribution of11 HSD-2 and GR in Rat Kidney) Y' S/ r9 K' C8 R  D
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Labeling of a kidney section for 11 HSD-2 and GR- with thedouble-immunofluorescence technique showed at low-power magnification thatimmunofluorescence signals for 11 HSD-2 (green) were associated withdistinct 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.6 A ). At higher magnification, 11 HSD-2 was observedin distal convoluted tubules, connecting tubules, and cortical and outermedullary collecting ducts ( Fig. 6, B and C ). Immunofluorescence for 11 HSD-2was associated with the majority of collecting duct cells, but single,scattered cells were not labeled ( Fig. 6, B and C, inset ). These11 HSD-2-negative cells are most likely intercalated cells as previouslyreported ( 7 ). 11 HSD-2labeling gradually waned in the outer medulla and was absent in the innermostportion (papilla) of the collecting ducts (not shown). In11 HSD-2-positive cells, immunoreactivity was seen particularly in theperinuclear region and not in cell nuclei( Fig. 6 B, inset ). Different NaCl intakes had no effect on distribution orsubcellular localization of 11 HSD-2 in kidney. In the absence of primary11 HSD-2 antibody or secondary antibody, there was no labeling of thetissue (not shown).) e/ J8 Z) o1 x( q, Y) z

Fig. 6. Double-immunofluorescence labeling of rat kidney from a rat fed a low-saltdiet for GR- and 11 HSD-2 using paraffin-embedded 1-µmsections. A : at low magnification, kidney cortex displayed GR- signal (red fluorescence signal) associated with proximal convoluted tubulesand glomeruli, whereas 11 HSD-2 (green fluorescence signal) was seen indistal convoluted tubule, connecting tubule, and collecting duct. There was nocolocalization between GR- and 11 HSD-2. Bar = 200 µm. B : in 2 merging cortical collecting ducts, GR- (redfluorescence) was present in 11 HSD-2-negative intercalated cell nuclei( inset ) and was absent from 11 HSD-2-positive principal cellnuclei and cytoplasm. Bar = 50 µm. C : in a medullary ray of thedeep renal cortex, GR- (red fluorescence) was present in loops ofHenle, predominantly in nuclei, whereas proximal tubules were labeled both incytoplasm and nuclei. 11 HSD-2 (green fluorescence) was localized in acollecting duct with a course parallel to the loops of Henle. Bar = 50µm.

At high-power magnification, GR- labeling (red fluorescence signal)was detected in glomeruli and proximal convoluted tubules, where signals wereassociated primarily with the cytoplasm, whereas all otherGR- -immunopositive structures were labeled in the nucleus( Fig. 6, B and C ). In the medullary rays, loop of Henle nuclei wereGR- immunopositive ( Fig.6 C ), and distinct labeling was associated with11 HSD-2-negative, intercalated cell nuclei of the collecting ducts( Fig. 6 B, inset ). 11 HSD-2-positive principal cells in collecting ductswere GR- negative ( Fig. 6, B and C, inset ). Moreover, nuclei invascular smooth muscle cells and endothelial cells were labeled forGR-. In the inner medulla, GR- immunoreactivity was associatedwith interstitial cells and not found in collecting ducts (not shown).Controls without GR- antibody or with preabsorption of the primaryantibody by the peptide used for immunization were negative, as for colon (notshown).) v1 e# R. ?1 k0 Q
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Physiological Significance of Enhanced 11 HSD-2 Activity forGlucocorticoid Sensitivity in Distal Colon Epithelial Cells" A. ~* w2 Y+ V2 j  \4 e/ `

On the basis of the above results, we hypothesized that an increase of11 HSD-2 expression in colon is responsible for downregulation ofglucocorticoid-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 withdexamethasone (50µg·kg - 1 ·day - 1 ) that is resistant against metabolism by 11 HSD-2 and selectively activates GR. A second group was treated with carbenoxolone (20mg·kg - 1 ·day - 1 by intraperitoneal injection) that directly inhibits 11 HSD-2activity.
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We first examined whether level and distribution of NHE-3 changed with theapplied NaCl regimens. Immunoblotting of colon protein from the high- andlow-NaCl intake groups for NHE-3 confirmed that a low-NaCl intake reducedNHE-3 protein levels in distal colon ( Fig.7 C ) ( 23 ).Next, immunohistochemistry was used to examine the cellular localization ofNHE-3 in colon from rats on high- or low-NaCl intake. NHE-3 was localized inthe apical membranes of surface cells in the distal colon in rats on ahigh-NaCl intake ( Fig.7 A ), whereas labeling was hardly detectable in colonafter a low-NaCl intake ( Fig.7 B ). We did not detect any differences in NHE-3 mRNA orprotein 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 was87.7 ± 4.8 arbitrary units ( n = 7) compared with 90.9 ±6.1 arbitrary units ( n = 7) in the high-NaCl group.3 C7 t# w! v' I7 M. X
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Fig. 7. Effect of dexamethasone (D) and carbenoxolone (C) on NHE-3 expression incolon during a low-NaCl intake. Immunoperoxidase microscopy of NHE-3 in ratdistal colon using paraffin-embedded sections is shown. NHE-3 immunoreactivitywas associated with the apical membrane of epithelial cells lining the upperpart of the crypts and the luminal surface in colon from animals on ahigh-NaCl intake ( A ), whereas labeling was scarce and hard to detectin low-NaCl intake rats ( B ). Bars = 50 µm. C :immunoblotting of NHE-3 in rat distal colon showing the effect of NaCl intakeon NHE-3 protein level. The antibody labeled a distinct band at 85 kDa thatwas not seen in the absence of primary antibody and after preabsorption of theprimary antibody with peptide used for immunization. D :auto-radiograph ( top ) showing ribonuclease protection assay for NHE-3mRNA with rat distal colon total RNA. Bottom : quantitative evaluationof the ribonuclease protection assay for NHE-3 mRNA in rat distal colon fromrats treated with dexametheasone (50µg·kg - 1 ·day - 1 )and carbenoxolone (20mg·kg - 1 ·day - 1 ).Radioactivity in the protected NHE-3 probe was asssayed by cutting the hybridsout of the gel and counting them in a -counter. The results shown are -actin-normalized mean values ± SE of 5 separate determinations.LS, low salt. * P; \! V& I- ^4 Z

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 significantlyincreased NHE-3 mRNA abundance in distal colon ( Fig. 7 D ). In kidneycortex, the NHE-3 mRNA level was significantly increased by dexamethasone (notshown, control 644.9 ± 40 cpm vs. dexamethasone 866.3 ± 56 cpm; P whereas the effect of carbonoxolone was notstatistically significant. Thus using NHE-3 as a marker for theglucocorticoid-GR pathway, a significant 11 HSD-2 activity reducesglucocorticoid-GR-mediated responses during NaCl restriction in colon.% B( b8 N& d4 C, t$ o

DISCUSSION
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In the present report, we show in rats that a NaCl-restricted diet leads toparallel increases in renin-angiotensin-aldosterone system components inplasma and 11 HSD-2 mRNA, protein, and immunolabeling in colon but not inkidney or cardiovascular tissue. Nuclear labeling for GR- andperinuclear staining for 11 HSD-2 were inversely related in both colonand kidney. In the kidney, there was a mutually exclusive segmentallocalization of GR and 11 HSD-2. Inhibition of 11 HSD-2 duringsodium 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 impededduring sodium restriction and selectivity for the aldosterone-MR pathway isfavored. Our results suggest that in the kidney there is a division betweenglucocorticoid-sensitive sites, which are localized in proximal nephronsegments and loops of Henle, and aldosterone-sensitive sites in principal cells of the distal nephron and collecting duct system.
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The present data on localization of 11 HSD-2 are essentially inaccordance with previous studies( 7, 21, 24, 26, 34, 44, 46 ) and add new information byshowing 1 ) that 11 HSD-2 is strictly localized in theperinuclear area in both colon and kidney, 2 ) that its abundance anddistribution along colon crypts are regulated by NaCl intake, and 3 )that there is an axial heterogeneity in the subcellular distribution of11 HSD-2 in colon crypts. We found expression of 11 HSD-2 only inprincipal cells of collecting ducts, in accordance with previousimmunohistochemical ( 6, 7 ) and activity data( 34 ) and in agreement withselective expression of MR and MR target proteins, e.g., ENaC subunits, inprincipal cells ( 7, 19, 28, 38, 44 ). The present finding of apredominantly perinuclear localization of 11 HSD-2 is consistent withmost, but not all, in vitro studies( 32, 35, 41 ). The perinuclearlocalization was particularly prominent in colonic crypt cells during low-NaClintake and in kidney cortical collecting duct principal cells. The strongperinuclear labeling of the basal stem cells in the colon indicates anenhanced translational activity in the endoplasmatic reticulum during cellmaturation. A secretory phenotype is associated with low differentiation anddominates in the crypts ( 25 ).The present data are compatible with the view that less differentiated cellsare recruited for electrogenic ENaC-mediated sodium absorption during sodium restriction.& d) i2 b- T' P" M! r9 i' c

In the kidney, previous data suggest that glucocorticoid receptor activation is functionally important in vessels and glomeruli ( 4 ) and in proximal tubuleswhere gluconeogenesis, ammoniogenesis and phosphate, sodium and acid transportare influenced ( 5 ). This is inagreement with the present localization of the hormone-binding GR isoformGR- and with data showing GR mRNA expression and autoradiographicbinding of dexamethasone in this nephron segment ( 14, 27, 44 ). We found GR- immunoreactivity in the loop of Henle, which has previously been demonstratedat functional mRNA and protein levels( 12, 14, 44 ). In the collecting ducts, we observed GR- immunoreactivity only in intercalated cells, consistentwith a much lower expression of GR mRNA in collecting ducts compared withproximal tubules and loop of Henle( 44 ). Data showed significanlylower activity of 11 HSD-2 in intercalated cells compared with principalcells ( 34 ), which allows for effects of glucocorticoid on distal tubular acidification( 10 ). Together, theseobservations point to a functional and morphological segregation of steroidaction in renal collecting ducts. However, in vitro data showed that GR canstimulate electrogenic sodium transport( 22 ), which agrees with GRimmunolabeling in one previous report( 12 ). The applied antibody,raised and used by Farman et al.( 12 ), did not label proximaltubules, glomeruli, or intercalated cells. We have no obvious explanation forthis discrepancy, but as mentioned above, we believe that our localization data are in good agreement with the majority of molecular and functionalstudies. Some discrepancies might have arisen from studies that show specificbinding of 3 H-corticosterone to principal cells because thisbinding is predominantly mediated by interaction with 11 HSD-2 and not GR( 33 ).

The lack of change in 11 HSD-2 mRNA and protein expression in thekidney after ingestion of a low-salt diet is consistent with mRNA data fromrats and activity data from humans( 15, 30 ). The absence of changes inGR 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 furtheraddress the mechanism responsible for this effect, but infusion of aldosteroneand dexamethasone did not change MR mRNA expression in either kidney or colon( 11, 31 ).: s9 m5 y' V9 _

Consistent with previous observations, immunoreactivity for hormone-bindingGR- was more strongly associated with crypt cells than with surfacecells in the colon ( 39 ). Theoverlap 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 GRand MR ( 40 ). Binding ofglucocorticoid 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 inverserelationship between perinuclear 11 HSD-2 and nuclear GR- iscompatible with 11 HSD-2-mediated metabolization of glucocorticoid leading to decreased GR binding, activation, and translocation in vivo.4 `4 d# h8 D/ B8 {7 w- ~
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In contrast to more proximal intestinal segments, colonic epithelial cellscontain a significant basal 11 HSD-2 activity( 40 ). Functional data supportthe present finding of physiological regulation of 11 HSD-2 in distalcolon. Thus chronic elevation of plasma aldosterone stimulates electrogenicsodium absorption, through ENaC, and decreases the contribution fromelectroneutral Na absorption( 20 ), which disappears after 1wk ( 45 ). In the colon,combined Na /H andCl - /HCO 3 - exchange is thepredominant mechanism of electroneutral Na absorption ( 2, 3, 16, 17, 25 ), which is stimulated byglucocorticoids ( 2, 3 ). The dominantNa /H exchange isoforms in colon, NHE-2 and -3, aredownregulated by 7 days of aldosterone treatment or low-NaCl intake( 23 ), whereas after 3 days, nosuch effect is seen ( 9 ).Together, the available functional data indicate a progressive suppression ofglucocorticoid-regulated transport proteins in colon during Na restriction and/or elevated aldosterone. We suggest this effect isattributable to enhanced 11 HSD-2 activity. In accordance, we found thatthe 11 HSD-2 blocker carbenoxolone stimulated NHE-3 expression in colonduring sodium restriction. The lack of effect of carbenoxolone on NHE-3expression in kidney corroborates the molecular data showing expression of GRand 11 HSD-2 in separate cells. NHE-3 is expressed with GR predominantlyin proximal tubules where 11 HSD-2 is absent ( 42 ). Whereas 11 HSD-2has a low K m for glucocorticoids and is essentiallyirreversible in the dehydrogenase direction, the enzyme isoform 11 HSD-1can act in a reversible fashion and has been suggested to modulateglucocorticoid responses by either decreasing or increasing localconcentrations. 11 HSD-1 is present in rat kidney proximal tubules andthus colocalizes with GR ( 8, 37 ), but it is absent in ratcolon epithelium ( 40, 47 ). Although we did notmeasure 11 HSD-1 activity, it is less likely to have contributed to thethe 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 circulatingcorticosterone. Because 11 HSD-1 activity correlated directly with NaClintake in canine kidney proximal tubules ( 8 ), and it is assumed that11 HSD-1 is a predominant reductase in vivo, an increase of 11 HSD-1activity could have contributed to this response. Unspecific inhibition of11 HSD-1 activity by carbenoxolone could potentially have decreased orincreased GR activation in proximal tubules in the present study, depending onthe diretion of 11 HSD-1 activity. However, we observed no significantchange in renal NHE-3 abundance in carbenoxolone-treated animals compared withcontrols. The described time course of changes in functional behavior andtransport protein expression in distal colon after a change in NaCl intakeclosely matches the present change in 11 HSD-2 expression. Because of theslow time course of these changes, it is tempting to suggest that the decreasein electroneutral Na transport includes cell turnover, where cryptcells are "committed" to electrogenic transport at a rate thatmatches apical shedding of cells. Together, the data indicate a key role for11 HSD-2 in distal colon epithelium as a switch between states of mixedsteroid sensitivity and selective mineralocorticoid sensitivity dictated byNaCl intake. In contrast, the kidney maintains a segment-specific mineralocorticoid sensitivity and glucocorticoid sensitivity that areindependent of dietary salt intake.

DISCLOSURES/ Z6 U: \) K1 V. {/ s- G
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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 MedicalAssociation Research Fund, A. J. Andersens Foundation, and Ms. Ruth T. E.Koenig-Petersens Foundation for kidney diseases.
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ACKNOWLEDGMENTS

The technical assistance of M. Fredenslund, K. Kejling, and I. Andersen isgratefully acknowledged. The authors thank A. M. Carter for linguisticrevision.
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