Increased collecting duct urea transporter expression in Dahl salt-sensitive rat - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:39 |只看该作者 |倒序浏览 |打印

作者：Robert A. Fenton, Chung-Lin Chou, Shana Ageloff, William Brandt, John B. Stokes,  Mark A. Knepper作者单位：1 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and BIood Institute, National Institutes of Health, Bethesda, Maryland 20892; and Department of Internal Medicine, University of Iowa, and Veterans Affairs Medical Center, Iowa City, Iowa 52242


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      【摘要】, ^0 h. a" j( X( B) t+ u3 L! Y
   Because abnormalities of inner medullary function have been proposed in Dahl salt-sensitive (DS) rats vs. salt-resistant (DR) rats, we performed transporter profiling by semiquantitative immunoblotting to determine whether specific solute transporter abundances are altered in inner medullas of DS rats vs. DR rats. Although none of the expressed Na transporters were upregulated in the inner medullas of DS rats compared with DR rats, there were marked increases in the protein abundances of the collecting duct urea transporters UT-A1 (to 212% of DR) and UT-A3 (to 223% of DR). These differences were confirmed by immunocytochemistry. Quantitative real-time RT-PCR showed higher mRNA abundance in DS rats for both UT-A1 (to 256% of DR) and UT-A3 (to 210% of DR). In isolated, perfused inner medullary collecting ducts, urea permeability was significantly greater in DS rats. Because both UT-A1 and UT-A3 are transcriptionally regulated by glucocorticoids, we measured both plasma corticosterone levels and inner medullary 11 -hydroxysteroid dehydrogenase type 2 (11 -HSD2) abundances. Although the plasma corticosterone concentrations were not different between DS and DR rats, immunoblotting and immunocytochemistry revealed a marked elevation of 11 -HSD2 abundance in DS rats. Consistent with the view that an elevated 11 -HSD2 level is responsible for increased urea transporter expression in the inner medullary collecting duct, administration of the 11 -HSD2 inhibitor carbenoxolone to DS rats decreased the abundances of UT-A1 and UT-A3 to levels similar to those seen in DR rats.
      【关键词】 UTA sodiumpotassiumadenosine ‘triphosphatase hydoxysteroid dehydrogenase glucocorticoid
   THE LONG - TERM MAINTENANCE of blood pressure is dependent, in part, on precise regulation of salt and water excretion by the kidney. Several factors are known to regulate renal tubule solute and water transport processes, including vasopressin, angiotensin II, aldosterone, catecholamines, and atrial natriuretic peptide, as well as a variety of autocoids ( 8 ). Quantitatively, the major solute in urine is urea. Although the control of its excretion has not been implicated in blood pressure regulation, it strongly influences both water and salt transport in the nephron and collecting ducts and is likely to play an indirect role in regulation of water and salt balance ( 25 ).6 Q  c; ?3 a9 J$ v; P

The Dahl salt-sensitive (DS) rat is a genetic model of salt-sensitive hypertension, with extracellular fluid (ECF) volume expansion and salt sensitivity ( 41 ). Characteristic of the DS strain, supplemental dietary sodium causes an increase in blood pressure, whereas in the Dahl salt-resistant (DR) strain, supplemental dietary sodium has no effect on blood pressure ( 41 ). Studies in DS rats have reported that they have suppressed plasma and tissue renin activity ( 42 ) and abnormalities in adrenal steroid metabolism ( 39 ) and the sympathetic nervous system ( 37 ). Furthermore, defects in nitric oxide signaling and alterations in the arachidonic acid/cytochrome P -450 pathways in the DS rat are associated with increased salt reabsorption by the thick ascending limb, potentially leading to changes in sodium balance ( 13, 43, 48 )., W$ \; \. n/ O: v- u: x+ ]; U
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In recent years, it has become well established that the renal medulla plays an important role in sodium and water homeostasis and in the long-term control of arterial pressure ( 7 ). Thus abnormal expression of inner medullary solute and water transport proteins may explain the strain differences in blood pressure and ECF volume expansion observed between DS and DR rats. Therefore, the objective of this study was to determine whether there were differences in abundances of several of the renal solute transporter proteins expressed in the kidney inner medulla of the DS compared with the DR rat.8 r0 R4 k, Y3 J. ~
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A major finding in this study was that, with matched intakes of salt, water, protein, and calories, the inner medullary collecting ducts (IMCDs) from DS rats express much greater levels of two urea transporters (UT-A1 and UT-A3) than do DR rats. The increase in urea transporter expression in DS rats was associated with higher urea transport rates in isolated, perfused IMCDs. Because both UT-A1 and UT-A3 urea transporters are negatively regulated by glucocorticoids ( 34, 38 ), we measured the inner medullary levels of 11 -hydroxysteroid dehydrogenase type 2 (11 -HSD2), a corticosterone-inactivating enzyme. The abundance of 11 -HSD2 was markedly increased in the inner medullas of DS rats, pointing to a possible mechanism of urea transporter upregulation.

METHODS( J5 `  G/ G! w9 k+ N# V

Standard Animal Protocol1 p  g$ S6 f% w' K6 \

Male SR/JrHsD (DR) and SS/JrHsD (DS) rats (body wt 200-250 g, Harlan Sprague Dawley) were maintained in metabolic cages for the duration of the study (4 days), under controlled temperature and light conditions (12:12-h lightdark cycle). All rats received a gelled diet, providing all nutrients and water and served as a fixed, weighed daily ration, as described elsewhere ( 21 ). Each rat received 40 g gelled diet · 200 g body wt (BW) -1 · day -1. The gelled diet was made up of (per 40 g total) 24.8 ml deionized water, 15 g of special low-NaCl synthetic food (0.0041% Na wt/wt, formula 53140000, Ziegler Bros., Gardner, PA), 2.0 mmol NaCl, and 0.125 g agar. Rats were euthanized or anesthetized after 4 days, and the kidneys of each rat were immediately processed (see Semiquantitative Immunoblotting, Immunocytochemistry, Real-Time RT-PCR, and Isolated, Perfused Tubule).% {* x( M5 |. g1 l8 R. ~

Carbenoxolone Animal Protocol

In one study, after a 2-day equilibration period in metabolic cages, rats (6 DR and 12 DS) were anesthetized with isoflurane for subcutaneous implantation of osmotic minipumps (Alzet, Palo Alto, CA) containing the 11 -HSD2 inhibitor carbenoxolone. Carbenoxolone (Sigma) was solubilized in distilled water. Experimental rats (6 DS) were given carbenoxolone at a dose of 100 mg · kg BW -1 · day -1 for 6 days, whereas control rats (6 DR and 6 DS) received only distilled water vehicle. This dose (when given in drinking water as the natural analog glycyrrhetinic acid) has been shown to be sufficient to effectively block 11 -HSD2 activity ( 55 ).% u* P8 A( e- D* y; P  Q$ r' Y" V6 y

Serum and Urine Collection- e- F+ l( A% T1 E8 ]* W7 B" M
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Twenty-four-hour urine collections were performed throughout the duration of the studies. Urine was preserved with the addition of a small crystal of thymol to the collection tube, and evaporation was prevented with a layer of mineral oil. Serum was collected from trunk blood after decapitation. Sodium, potassium, chloride, creatinine, urea, phosphorus, and bicarbonate levels in serum and urine were determined using an autoanalyzer. Serum corticosterone (Coat-A-Count, Diagnostic Products) and vasopressin (Alpco Diagnostics) concentrations were measured by radioimmunoassay of serum samples.
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Semiquantitative Immunoblotting
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Rats (6 DR and 6 DS) were maintained in metabolic cages (see Standard Animal Protocol) and euthanized by decapitation. The left kidney was immediately removed, and the inner medulla was dissected out. The inner medulla was homogenized in ice-cold isolation solution (250 mM sucrose, 10 mM triethanolamine, pH 7.6, containing 1 mg/ml leupeptin, 0.1 mg/ml phenylmethylsulphonyl fluoride) using a tissue homogenizer (Omni 1000 fitted with a microsawtooth generator) at maximum speed for three 15-s intervals. Total protein concentrations were measured (BCA kit; Pierce Chemical, Rockford, IL), and the samples were solubilized in Laemmli sample buffer at 60°C for 15 min. Semiquantitative immunoblotting was carried out as previously described ( 50 ) to assess the relative abundances of individual proteins in the DR rats compared with DS rats. To ensure equal loading of the gels, preliminary 12% polyacrylamide gels were stained with Coomassie blue and densitometry of major bands was performed. For immunoblotting, SDS-PAGE was performed on 7.5, 10, or 12% polyacrylamide gels (Ready Gels, Bio-Rad, Hercules, CA). The proteins were transferred electrophoretically (Mini Trans-Blot Cell, Bio-Rad) to nitrocellulose membranes (Bio-Rad). Membranes were blocked for 1 h at room temperature with 5% nonfat dry milk and probed overnight at 4°C with the appropriate affinity-purified antibodies. Membranes were washed and exposed to one of the following horseradish peroxidase-labeled secondary antibodies for 1 h at room temperature: rabbit anti-sheep IgG (no. 81-8620, diluted 1:5,000, Zymed Laboratories); goat antirabbit IgG (no. 31463, diluted 1:5,000, Pierce Chemical); or rabbit anti-mouse IgG (no. 31450, diluted 1:5,000, Pierce Chemical). After being washed, sites of antibody-antigen reaction were visualized using a luminol-based enhanced chemiluminescence substrate (LumiGLO, Kirkegaard and Perry Laboratories, Gaithersburg, MD) before exposure to light-sensitive film (no.165-1579, Kodak). The band densities were quantitated by laser densitometry (model PDS1-P90, Molecular Dynamics, San Jose, CA). All band density values were normalized to the mean of the DR control group to facilitate comparisons.  h+ l& p; q! K7 N! [# f5 r; L5 k

Immunocytochemistry) k- P/ Q4 r- y+ O& `

Three DR and three DS rats were maintained in metabolic cages (see Standard Animal Protocol ) and anesthetized with isoflurane administered via a nose cone. The kidneys were fixed by perfusion via the abdominal aorta with cold PBS (pH 7.4) for 60 s, followed by cold 4% paraformaldehyde in PBS (pH 7.4) for 3 min. The kidneys were removed, and the midregion was sectioned into 5-mm transverse sections and postfixed for 1 h in cold 4% paraformaldehyde/PBS, followed by 3 x 10-min washes with PBS. The tissue was dehydrated in a series of ethanol concentrations and left overnight in xylene. The tissue was embedded in paraffin, and 5-µm sections were cut (Histo-Path of America) and mounted on Superfrost Plus slides (VWR International). Staining was performed as described previously ( 11 ) using the following horseradish peroxidase-conjugated secondary antibodies diluted 1:200: goat anti-rabbit IgG (P448, DAKO); rabbit antisheep IgG (no. 81-8620, Zymed Laboratories); and rabbit anti-mouse IgG (no. 31450, Pierce Chemical). Light microscopy was performed with a Nikon E800 upright microscope.
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Antibodies5 l$ y3 [- k& ?, {2 D

Rabbit polyclonal antibodies produced in our laboratory were utilized to label three splice variants of the UT-A urea transporter family: UT-A1, UT-A2 ( 35 ), and UT-A3 ( 52 ). In addition, we used previously characterized rabbit polyclonal antibodies to aquaporin-1 ( 50 ), aquaporin-2 ( 9 ), aquaporin-3 ( 10 ), aquaporin-4 ( 49 ), and all three subunits (,, ) of the epithelial Na channel (ENaC) of the collecting duct ( 30 ). The antisera were affinity purified against the immunizing peptides as previously described ( 21 ). Specificity of the antibodies has been demonstrated by showing unique peptide-ablatable bands on immunoblots and a unique distribution of labeling by immunocytochemistry. For immunoblotting, antibodies were used at a final IgG concentration of between 0.1 and 0.2 µg/ml. For immunocytochemistry, antibodies were used at a final IgG concentration of between 0.4 and 0.5 µg/ml. Commercial antibodies used in the study were: a mouse monoclonal antibody to the Na-K-ATPase 1 -subunit (no. 05-369, diluted 1:1,000 for immunoblotting and 1:300 for immunocytochemistry, Upstate Biotechnology, Lake Placid, NY) and a sheep polyclonal antibody to 11 -HSD2 (no. AB1296, diluted 1:1,500 for immunoblotting and 1:300 for immunocytochemistry, Chemicon, Temecula, CA).
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Real-Time RT-PCR
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Rats (6 DR and 6 DS) were maintained in metabolic cages (see Standard Animal Protocol ) and euthanized by decapitation. Total RNA was isolated from the left kidney inner medulla of DR or DS rats as described previously ( 11 ). Potential contaminating genomic DNA was removed from the RNA preparations by a 30-min incubation with DNase I (DNA-free, Ambion). Total RNA (1 µg) was reverse transcribed using oligo(dT) and Superscript II RT (Invitrogen) following the manufacturer's recommended protocol. RT-negative controls were performed to assess the presence of possible genomic contamination of RNA samples. PCR primers were designed to be specific for the rat cDNAs: UT-A1 (GenBank no. U77971  ), UT-A2 (GenBank no. U09957  ), UT-A3 (GenBank no. AF041788  ), aquaporin-2 (GenBank no. D13906  ), aquaporin-3 (GenBank no. NM_031703  ), aquaporin-4 (GenBank no. AF144082  ), Na-K-ATPase 1 -subunit (GenBank no. M14512  ), -ENaC (GenBank no. X70497  ), -ENaC (GenBank no. X77932  ), -ENaC (GenBank no. X77933  ), and 11 -HSD2 (GenBank no. NM_017081  ). These primers were designed to amplify targets between 80 and 150 bp in length, with minimal secondary structure. Real-time PCR was performed on an ABI Prism 7900HT system, using 10 nl of cDNA, 18 pmol (each) of gene-specific primers, and the Quantitect SYBR green PCR kit (Qiagen) according to the manufacturer's protocol. Typical threshold cycle (C T ) values for these experiments were in the range of 20-24. Specificity of the amplified product was determined using melting curve-analysis software, gel electrophoresis, and sequencing. Relative quantitation of gene expression was determined using the comparative C T method ( 27 ) with validation experiments performed to determine that amplification efficiencies were equal between control and experimental groups ( 27 ) as outlined at http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf. All experiments were performed at least twice (on separate days) to demonstrate reproducibility. All values were normalized to the mean of the DR control group to facilitate comparisons.% ~5 g. k, E! r/ E  {

Isolated, Perfused Tubules

Urea permeability was measured in IMCDs using the isolated tubule microperfusion technique ( 6 ). IMCD segments were microdissected from the midregion of the inner medulla (40-70% of the distance from the inner-outer medullary junction to the papillary tip of the rat kidney). The dissection solution contained (in mM) 125 NaCl, 25 NaHCO 3, 2K 2 HPO 4, 2 CaCl 2, 1.2 MgSO 4, 5.5 glucose, and 5 creatinine. The tubules were transferred to a perfusion chamber, mounted on an inverted microscope, cannulated by concentric pipettes, and perfused in vitro. The perfusate and the pertitubular bath solutions were identical to the dissection solution except that in the bath solution 5 mM creatinine was replaced by 5 mM urea. Therefore, the tubules were perfused with solutions of equal osmolality, but with a 5 mM bath-to-lumen urea gradient. The urea permeability was determined by measuring the urea flux resulting from the transepithelial urea gradient as described previously ( 6 ). The urea concentrations in the perfusate, bath, and collected fluid were measured fluorometrically using a continuous-flow ultramicrofluorometer and an enzymatic assay (BUN Reagent Kit 64-20, Sigma).. P: b9 R* J& Z9 o

Presentation of Data and Statistical Analyses) T7 S( @; P" g: B9 V$ U

Quantification of the band densities from immunoblots was carried out by laser densitometry (Molecular Dynamics). To facilitate comparisons, we normalized the densitometry values such that the mean for the DR rats is defined as 100 (arbitrary units). Quantitative data are presented as means ± SE. Statistical comparisons were accomplished by unpaired t -test. P values7 p  \3 b; n; D% ^. K% N
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RESULTS
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Semiquantitative Immunoblotting

Sodium transporter protein abundances. Despite a propensity for increased total renal NaCl reabsorption in DS rats, there was a marked decrease in the inner medullary abundance of the 1 -subunit of the Na-K-ATPase in DS rats compared with DR rats ( Fig. 1 ). The mean Na-K-ATPase 1 -subunit abundance was 23% of the mean for DR rats. The band density corresponding to the -subunit of ENaC was also significantly decreased in DS rats ( Fig. 1 ). Band densities corresponding to - and -subunits of ENaC were not significantly changed.
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Fig. 1. Immunoblots assessing sodium transporter and epithelial sodium channel (ENaC) abundances in inner medulla homogenates from Dahl salt-resistant and salt-sensitive rats. Each lane was loaded with a sample from a different rat. Values are mean band densities normalized so that the mean for the salt-resistant rats is defined as 100. * Significant change in mean band densities among groups.

UT-A urea transporters. Figure 2 shows immunoblots for three urea transporter proteins in the inner medulla: UT-A1 and UT-A3 (expressed in the IMCD) ( 51, 52 ) and UT-A2 [expressed in type 1 (short-loop) and type 3 (long-loop) thin descending limbs of the loop of Henle] ( 53 ). As can be seen, the two collecting duct urea transporters were markedly increased in abundance, whereas the loop of Henle isoform was unchanged. Band densities for UT-A1 and UT-A3 were increased to 212 and 223% of DR values, respectively ( Fig. 2 ). The abundance of UT-A2 was also unchanged in the outer medulla (not shown).  e% K2 C2 r* p; z# i

Fig. 2. Immunoblots assessing urea transporter (UT-A) abundances in inner medulla homogenates from Dahl salt-resistant and saltsensitive rats. Each lane was loaded with a sample from a different rat. Values are mean band densities normalized such that the mean for the salt-resistant rats is defined as 100. * Significant change in mean band densities among groups.

Aquaporin water channels. In the inner medulla, there were no significant differences in the band densities corresponding to aquaporin-1, -2, -3 and -4 (not shown).
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Immunocytochemistry

To confirm the large increases observed in UT-A1 and UT-A3 protein and the decreased abundance of the Na-K-ATPase 1 -subunit, immunocytochemistry was performed using immunoperoxidase techniques. Figure 3 shows representative high-power images of IMCDs demonstrating marked increases in UT-A1 and UT-A3 labeling in principal cells of DS rats. Labeling conditions and exposure settings on the microscope were identical for both pairs of images. There was no obvious cellular redistribution of UT-A1 or UT-A3 labeling in collecting duct cells, and low-power magnification (not shown) revealed that the tubular distribution of urea transporter expression along the IMCD was similar in both DR and DS rats. Similar observations were made in two additional pairs of rats.  @: z- y# q9 Y% p0 N

Fig. 3. Immunoperoxidase labeling of UT-A1 and UT-A3 in rat inner medulla. High-power images demonstrate a marked increase in labeling of collecting duct cells in the Dahl salt-sensitive rats ( right ) compared with salt-resistant rats ( left ).
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Figure 4 shows high-power images demonstrating a marked decrease in Na-K-ATPase 1 -subunit labeling in the DS rats compared with DR rats. However, there was no obvious redistribution of labeling in collecting duct cells. Similar observations were made in two additional pairs of rats. Immunoblots for UT-A1, UT-A3, and Na-K-ATPase 1 -subunit (not shown) from the unfixed, contralateral kidney revealed similar results to those observed in Figs. 1 and 2.

Fig. 4. Immunoperoxidase labeling of Na-K-ATPase 1 -subunit in rat inner medulla. High-power images demonstrate a marked decrease in Na-K-ATPase 1 -subunit labeling in the Dahl salt-sensitive rats ( right ) compared with salt-resistant rats ( left ).

Transporter mRNA Abundances% O% R7 g* e$ u$ l, G6 Z9 D& n" G) [
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To address the possibility that the observed differences in protein abundance observed between DS and DR rats are due to altered levels of the corresponding mRNA transcripts, quantitative real-time RT-PCR was used to assess mRNA transcript levels in the renal inner medulla. A summary of the relative abundances of mRNA transcripts in DS compared with DR rats is shown in Table 1. The normalized inner medullary mRNA abundances of both UT-A1 and UT-A3 were significantly greater in DS compared with DR rats. Similar to protein abundance, a significant decrease in -ENaC mRNA transcript abundance was observed for DS rats compared with DR rats. However, although the normalized inner medullary mRNA level for the Na-K-ATPase 1 -subunit was lower in DS rats compared with DR rats, this was not significant.

Table 1. Comparison of mRNA abundances in inner medulla of Dahl salt-sensitive and salt-resistant rats) G5 p* O6 n4 h2 m& b' a& @7 q
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Urea Permeability in Isolated, Perfused IMCDs

To determine whether the increase in abundance of UT-A1 and UT-A3 protein in DS rats contributed to greater urea permeability in the IMCD, urea flux measurements were performed using isolated, perfused tubules ( 6 ). In the absence of vasopressin, urea permeability of terminal IMCD segments was significantly greater ( P x 10 -5 cm/s) compared with DR (28.9 ± 1.9 x 10 -5 cm/s) rats ( Fig. 5 ). In the presence of vasopressin (0.1 nM), terminal IMCD urea permeability was increased in both DR and DS rats. A significantly ( P 0.001) greater urea permeability was observed in DS rats (122.4 ± 6.5 x 10 -5 cm/s) compared with DR rats (89.4 ± 4.4 x 10 -5 cm/s) ( Fig. 5 ). Immunoblots (not shown) performed on the contralateral kidney inner medulla confirmed the large differences in UT-A1 (normalized band densities: DS, 251 ± 38; DR, 100 ± 21, P band densities: DS, 244 ± 40; DR, 100 ± 33, P, B* r0 {% u( W) ~5 g# M; w/ O

Fig. 5. Summary of urea permeability ( P urea ) in the inner medullary collecting duct using isolated, perfused tubules in the presence or absence of AVP. For basal urea permeability measurements, 6 tubules were perfused for each group; for vasopressin-stimulated urea permeability measurements, 10 tubules were perfused for each group. * Significant change in urea permeability between groups.
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Role of Renal 11 -HSD2
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The major findings in this study were the large increases in abundances of the collecting duct urea transporters, UT-A1 and UT-A3. Urea transporter proteins and transcripts are regulated by a number of factors ( 44 ). Among these factors are glucocorticoids, which increase the fractional excretion of urea and decrease urea permeability ( 24 ). This decrease in urea permeability is associated with a decrease in UT-A1 protein abundance in the IMCD ( 34 ). Subsequent studies have shown that glucocorticoid administration causes a decrease in the expression of UT-A1 and UT-A3 mRNA transcripts, with no apparent change in UT-A2 mRNA abundance ( 38 ). Therefore, we hypothesized that the large increase in UT-A1 and UT-A3 protein abundance observed in the DS rats, with no change in UT-A2 abundance, may be due to a decrease in glucocorticoid levels, either systemically or in the collecting duct cells. Similar to observations by other groups ( 42 ), in our study serum corticosterone concentrations did not show any significant differences between the strains ( Table 2 ). We subsequently assessed the relative abundance of renal 11 -HSD2, the enzyme responsible for glucocorticoid degradation in the kidney, in the inner medullas of DS and DR rats. We hypothesized that an increase in the activity or abundance of 11 -HSD2 in IMCD of DS rats may be responsible for a decrease in local glucocorticoid levels and thus lead to an increase in the urea transporters UT-A1 and UT-A3. In DS rat inner medullary homogenates, immunoblots showed a significant increase in 11 -HSD2 protein abundance ( Fig. 6 A ). Immunocytochemistry confirmed this result; representative highpower images of IMCDs demonstrated a marked increase in 11 -HSD2 labeling in IMCD cells ( Fig. 6 B ). Labeling conditions and exposure settings on the microscope were identical for both images. Similar observations were made in two additional pairs of rats. Interestingly, the mRNA abundance of 11 -HSD2 was not significantly greater in DS (1.33 ± 0.14) compared with DR (1.00 ± 0.06) rats.
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Table 2. Urinary excretion and serum analysis. o" g; k! V7 A3 }$ e" M# i6 F! b* M
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Fig. 6. A : immunoblot assessing the abundance of renal 11 -hydroxysteroid dehydrogenase type 2 (11 -HSD2) in the inner medullary collecting duct of Dahl salt-resistant and salt-sensitive rats. Each lane was loaded with a sample from a different rat. Values are mean band densities normalized so that the mean for the salt-resistant rats is defined as 100. * Significant change in mean band densities between groups. B : immunoperoxidase labeling of 11 -HSD2 in rat inner medulla. High-power images demonstrate a marked increase in labeling of collecting duct cells in the Dahl salt-sensitive rats ( right ) compared with salt-resistant rats ( left ).4 P- a0 v6 o4 k8 V4 ~2 v
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To address whether 11 -HSD2 enzyme activity affects urea transporter abundance, we administered carbenoxolone, an inhibitor of 11 -HSD2 activity to DS rats. As shown in Fig. 7, administering 100 mg · kg BW -1 · day -1 carbenoxolone by osmotic minipump to DS rats for 6 days resulted in a decreased abundance of UT-A1 (to 75% of control, P of control, P This decrease in IMCD urea transporter abundance was also associated with an increase in the fractional excretion of urea ( Table 2 ). Inhibition of 11 -HSD2 activity caused a significant increase in -ENaC abundance (to 315% of control, P densities corresponding to the Na-K-ATPase 1 -subunit and - and -subunits of ENaC were not significantly changed.
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Fig. 7. Immunoblots assessing the effects of administering carbenoxolone (CBX; 100 mg · kg body wt -1 · day -1 ) to Dahl salt-sensitive rats. Relative transporter abundances for Dahl salt-resistant rats were also studied to confirm previous findings. Each lane was loaded with a sample from a different rat. Values are mean band densities normalized so that the mean for the salt-sensitive rats is defined as 100. * Significant change in mean band densities between groups.
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DISCUSSION

UT-A Urea Transporters in DS vs. DR Rats: v3 s9 S4 Y; r, h* h* |* V

Urea transporters play an important role in the maintenance of water balance and can have an indirect influence on NaCl balance as discussed below. Two urea transporter genes are expressed in human, rat, and mouse genomes: UT-A ( 2, 12, 33 ) and UT-B ( 28 ). In the mouse and rat, UT-A encodes for a number of splicing variants that are expressed chiefly in epithelia ( 44 ). In the rat, UT-B encodes for a single protein that is primarily expressed in red blood cells and renal vasculature ( 44 ). Within the kidney, three UT-A isoforms are expressed at a protein level: UT-A1 and UT-A3 are expressed solely in the IMCD ( 35, 52 ), whereas UT-A2 is expressed in the thin descending limbs of the loop of Henle ( 53 ). In the present study, UT-A1 and UT-A3, both believed to contribute to the very high urea permeability of the IMCD, were found to be markedly upregulated in DS compared with DR rats, whereas the loop of Henle isoform was not.# l/ N2 T* ~# I( O
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The basic model of urea handling in the renal medulla was elucidated by Berliner and colleagues 45 years ago ( 3 ) and has been confirmed and refined by subsequent studies. Early portions of the collecting duct, i.e., the cortical and outer medullary collecting ducts, do not express urea transporters and have very low urea permeability ( 23, 45 ). Hence vasopressin-dependent water absorption in the cortical collecting duct and outer medullary collecting duct concentrates the urea in the lumen. Once the tubule fluid reaches the IMCD, which expresses urea transporters and has high urea permeability ( 45 ), the urea exits the IMCD into the inner medullary interstitium. This urea is trapped by countercurrent exchange processes and, in the steady state, inner medullary interstitial urea rises to nearly the same level as the IMCD luminal urea. By this mechanism, the interstitial urea can osmotically balance the very high urea concentration in the lumen, thereby nullifying the tendency for luminal urea to obligate water for its excretion. In contrast, in the absence of urea transporters in the IMCD, urea would behave as an osmotic diuretic, increasing the excretion of water and nonurea solutes." h1 U7 |/ a2 M% _: c0 [
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Accordingly, modulation of urea transporter activity is a potential means by which water and salt excretion could be regulated. Thus the higher UT-A1 and UT-A3 urea transporter expression (Figs. 2 and 3 ) and the associated increase in urea permeability ( Fig. 5 ) in DS rats relative to DR rats could have implications with regard to regulation of ECF volume and, in part, the long-term maintenance of blood pressure in these two rat strains.

Previous studies in Dahl rats have uncovered roles for a variety of factors in differential regulation of renal function in DS vs. DR rats, including nitric oxide ( 5, 13, 19 ), prostaglandins ( 41 ), cytochrome P -450 metabolites of arachidonic acid ( 29, 43 ), and endothelin ( 17, 18 ). To date, none of these factors has been studied with regard to a possible role in renal urea transporter regulation.
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Urea transporters are regulated by both long-term and short-term mechanisms. Short-term regulation of urea transporters in the IMCD is mediated by vasopressin ( 46 ) and appears to involve transporter protein phosphorylation ( 56 ). Furthermore, angiotensin II significantly increases vasopressin-stimulated urea permeability in terminal IMCDs and may augment the maximal urea permeability response to vasopressin ( 20 ). Long-term mechanisms involve changes in urea transporter protein abundance seen in response to changes in dietary protein intake ( 44 ), glucocorticoids ( 34, 38 ), and lithium ( 22 ). However, vasopressin has alternate long-term effects on multiple UT-A isoforms. For example, vasopressin does not alter the abundance of the collecting duct isoform UT-A1 ( 4, 51 ), but it does increase the abundance of the loop of Henle isoform UT-A2 ( 53 ). In our study, although circulating vasopressin levels were relatively high in both DR and DS rats compared with normal rats, no differences in vasopressin levels were observed between the DR and DS strains. This indicates that vasopressin is unlikely to be responsible for the differences in UT-A1 and UT-A3 abundances observed. Among the most potent regulators of urea transporters in the IMCD are glucocorticoids, which downregulate transporter activity and abundance by transcriptional mechanisms ( 34, 38 ). The pattern of UT-A expression observed in the present study is consistent with regulation by glucocorticoids. The increased UT-A1 and UT-A3 urea transporter protein abundances in DS rats were associated with increases in the corresponding mRNAs, consistent with differences in UT-A gene transcription (or altered mRNA stability) ( 38 ). Although the circulating levels of corticosterone, the major glucocorticoid hormone in rats, were not elevated in DS vs. DR rats, we found a marked increase in the abundance of 11 -HSD2 protein in the IMCDs of DS rats. Conceivably, this could have been causative in the increases in UT-A1 and UT-A3 expression in the IMCD. Higher 11 -HSD2 could result in a decrease in intracellular levels of corticosterone, which in turn may cause corticosterone-mediated downregulation of urea transporter gene expression to be reduced. Previous studies have demonstrated that 11 -HSD2 protein levels ( 4 ) or activity ( 1 ) can be regulated in the collecting duct as part of the overall regulation of collecting duct function.3 q8 `  f8 T" L/ E- ~6 E

To further assess the potential link among glucocorticoids, 11 -HSD2, and the increased inner medullary urea transporters, we administered carbenoxolone, an inhibitor of 11 -HSD2 activity, for 6 days via an osmotic minipump. DS rats administered carbenoxolone had a significantly higher serum corticosterone concentration compared with control DS rats ( Table 2 ). In the inner medullas of DS rats, carbenoxolone caused both UT-A1 and UT-A3 protein abundance to decrease significantly compared with control DS rats. This result indicates, for the first time, that 11 -HSD2 activity is an important factor in urea transporter regulation and supports the view that increased 11 -HSD2 activity may be involved in the increase in urea transporter gene expression in the IMCDs of DS rats. However, it must be noted that the effects of carbenoxolone on urea transporter expression would be expected in any rat strain and are not likely to be specific for the Dahl rat model.
8 }2 K$ ?/ b6 w1 {' a
Although, as reviewed above, changes in urea transporter activity in the IMCD could theoretically affect water and salt transport, a functional linkage between the increase in IMCD urea transporter expression and the higher blood pressure in DS vs. DR rats has not been established. However, recent analysis of the human UT-A gene revealed seven single nucleotide polymorphisms, two of which were associated with altered blood pressure in males ( 40 ). It is unclear whether the single nucleotide polymorphisms observed in this study result in differences in IMCD urea transport that could potentially explain the hypertension observed.( S" ~( Y# ?% T
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IMCD Sodium Transporters in DS vs. DR Rats( u7 d8 N* I  V2 \/ i4 m4 v

One of the objectives of the present study was to identify Na transporters that might be upregulated in DS vs. DR rats, thus contributing to the higher systemic blood pressure seen in the former. Although many different apical transporters are expressed along the renal tubule, only ENaC is expressed in the apical plasma membrane of the IMCD ( 15 ). This channel is made up of three different subunits,,, and, coded for by different genes ( 14 ). The -subunit appears to be the chief target for regulation by adrenal corticosteroids ( 30, 31, 47 ). In the present study, none of the subunits were increased in abundance in the inner medullas of DS compared with DR rats. In fact, the -subunit was strongly downregulated, perhaps as a result of the marked increase in 11 -HSD2 expression in the same cells. Consistent with this view, the protein abundance of -ENaC was markedly elevated when 11 -HSD2 activity was inhibited with carbenoxolone. This is an important observation, because there is no prior evidence in the mammalian kidney that 11 -HSD2 inhibition mimics the action of aldosterone, as seen here. Prior studies by Stokes and colleagues ( 32, 37 ) have provided evidence for an important role for glucocorticoids in the regulation of ENaC abundance and activity ( 16, 26 ) in the IMCD, supporting the view that baseline 11 -HSD2 levels in these cells are not so high as to constitutively consume all glucocorticoid presented to them. Thus the results from our study show that 11 -HSD2 abundance has the potential to modulate glucocorticoid action in IMCD cells.4 \) w" d: M% q- j

Summary

In this study, we have provided novel evidence that the abundances of two of the collecting duct urea transporters, namely, UT-A1 and UT-A3, are increased in the DS rat compared with the DR rat. Furthermore, an increase in 11 -HSD2 abundance observed in the DS rat may contribute to this increased UT-A expression in the IMCD. Further studies are required to determine whether urea transporter regulation can play a direct role in the hypertension observed in these rats.% Z1 A) W" @* a5 e3 @

NOTE ADDED IN PROOF

Subsequent measurements of creatinine concentrations using HPLC analysis have been performed. The creatinine clearance values (ml/h) resulting from these measurements are as follows: DR rats, 67.2 ± 3.6; DS rats, 68.4 ± 1.8; and DS rats CBX, 69.1 ± 5.7. These results indicate that creatinine clearances are not significantly different among the groups.

ACKNOWLEDGMENTS
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The authors are grateful for the assistance of Christian A. Combs, manager of the National Heart, Lung, and Blood Institute (NHLBI) Light Microscopy Imaging Facility at the National Institutes of Health (NIH). The authors also thank David Caden at the Laboratory of Animal Medicine and Surgery, NHLBI, for carrying out careful analysis of the serum and urine samples.

This study was funded by the Intramural Budget of the NHLBI (Z01-HL-01282-KE to M. A. Knepper). Further funding was provided by NIH Grants DK-52617 and HL-55006 (to J. B. Stokes) and by a grant from the Department of Veterans Affairs (to J. B. Stokes).
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