Glucocorticoids inhibit transcription and expression of the UT-A urea transporter gene

Tao Peng1, Jeff M. Sands2, and Serena M. Bagnasco1

1 Department of Pathology and 2 Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Dexamethasone treatment increases urea excretion and decreases urea permeability and urea transporter UT-A1 protein abundance in the inner medullary collecting duct (IMCD) of adrenalectomized rats. We examined the effect of dexamethasone treatment for 3 days on the abundance of several UT-A mRNA transcripts in rat renal medulla. By Northern blot analysis, a significant decrease in mRNA expression was observed in the inner medulla of dexamethasone-treated rats compared with controls for UT-A1 (71%), UT-A3 (75%), and UT-A3b (75%), but not for UT-A2. We then tested the effect of 100 nM dexamethasone on the activity of promoter I in the UT-A gene, using LLC-PK1-GR101 cells that express the glucocorticoid receptor. Dexamethasone significantly decreased the activity of rat UT-A promoter I (72%) but did not affect UT-A promoter II. Deletion analysis and site-directed mutagenesis demonstrated that sequences between -423 and -244 are important for this inhibition and that a 10-bp sequence at -363, which binds a nuclear protein in a gel shift assay, is necessary for basal promoter activity. The specific factors involved in repression of UT-A promoter I activity by glucocorticoids remain to be determined.

Slc14a2 gene; kidney


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE EXCRETION OF APPROPRIATELY concentrated urine requires that high urea and NaCl concentrations be maintained in the renal medulla. Urea reabsorption from the inner medullary collecting duct (IMCD) into the renal inner medullary interstitium and urea recycling between vasa recta and thin descending limb of Henle's loop involve facilitated transport by the renal tubular urea transporter UT-A and the erythrocyte urea transporter UT-B. Four major renal isoforms of the UT-A urea transporter have been identified (4, 10, 13) and are encoded by the Slc14a2 gene. We have recently elucidated the organization of the Slc14a2 gene in rats and humans and showed that it includes at least two promoters (1, 7). Water deprivation is characterized by increased inner medullary interstitial osmolality and is associated with increased expression of the UT-A2 and UT-A3 urea transporters (2). Transcription of UT-A1 and UT-A3 isoforms is increased by tonicity, mediated by the tonicity enhancer (TonE) (8), whereas transcription of UT-A2 is increased by vasopressin and cAMP-dependent stimulation (7).

It has been previously demonstrated that glucocorticoids inhibit urea fractional excretion (6) and decrease urea transport and UT-A1 protein abundance in IMCD (9). The effect of glucocorticoids on urea handling by the kidney may affect the ability to adequately concentrate urine when serum glucocorticoids are increased, as, for example, in diabetes mellitus (5). In this study, we examine the mechanisms underlying the effect of glucocorticoids on UT-A urea transporter expression in the renal medulla.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal treatment and tissue collection. Male Sprague-Dawley rats weighing ~260 g were used for these studies. Glucocorticoid-treated animals were injected subcutaneously with dexamethasone (6 µg/100 mg body wt, Sigma, St. Louis, MO) twice a day for 3 days. Dexamethasone-treated animals had unrestricted access to standard rat chow (Prolab Animal Diet RM 3000, PMI Feeds, St. Louis, MO). Control animals did not receive dexamethasone and were pair-fed using the same chow. Immediately after the death of the animals, rat kidneys were dissected to separate the cortex, outer medulla, and inner medulla, which were rapidly frozen in liquid N2 and stored at -80°C.

Northern hybridization. Total RNA was purified by using TriPure Isolation Reagent (Roche, Indianapolis, IN). Northern hybridization, rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and UT-A cDNA probes were as previously described (2). The full UT-A1 probe included nucleotides 493-3262 of UT-A1 cDNA; the UT-A3b probe was obtained by PCR amplification of the alternative 3'-untranslated region sequence of UT-A3, using the sense primer 5'-TGACCAGGCTGGAAGGCTCCTA-3' and antisense primer 5'-GGGCACACAGCTCAACTTTAGAAAC-3' (855 bp). All blots were probed with GAPDH cDNA to ensure uniformity of gel loading. The cDNA probe for tonicity enhancer binding protein (TonEBP) was kindly provided by Dr. H. Moo Kwon (Johns Hopkins University). An aldose reductase (AR) cDNA probe was a generous gift of Drs. Joan Ferraris and Maurice B. Burg (National Institutes of Health). The cDNA templates were labeled with [32P]dCTP (DuPont-NEN, Boston, MA) using a random primed labeling kit (Roche). Autoradiograms of the hybridized membranes were examined by densitometry to quantify the intensity of the signals. To determine the relative abundance, measurements of tested mRNAs were normalized by the GAPDH signal in each lane. Changes in mRNA abundance were compared between dexamethasone-treated rats and control rats, and differences were analyzed by two-tailed, nonpaired t-test, with P < 0.05 indicative of statistical significance.

Cell culture. LLC-PK1 kidney cells (American Type Culture Collection, Rockville, MD) and LLC-PK1-GR101 cells (a generous gift from Dr. S. Russ Price, Emory University) were used in these studies. LLC-PK1-GR101 cells were produced by stable transfection of normal LLC-PK1 cells with an expression plasmid containing the rat glucocorticoid receptor gene ligated to a cytomegalovirus promoter and a gene that confers aminoglycoside resistance (12). They were maintained in DMEM containing 10% FBS, 2 mM glutamine (Mediatech, Herndon, VA), 100 IU/ml of penicillin, 100 IU of streptomycin, and 0.8 mg/ml hygromycin B (Calbiochem, La Jolla, CA).

RU-486 was provided by Dr. S. R. Price, and doses of RU-486 equimolar to dexamethasone were added to cell medium 1-2 h before dexamethasone.

Luciferase reporter plasmid construction. Deletions from the UT-A1 1.3-kb 5'-flanking region containing UT-A promoter I, as previously described (8), were made by PCR, with sense primers corresponding to -1258, -616, -423, and -244 nucelotides of the 1.3-kb sequence and an antisense primer corresponding to 61-85 of UT-A1 cDNA. PCR-amplified products were subcloned into pGL3 Basic firefly luciferase reporter vector (Promega, Madison, WI). A previously described construct for rat UT-A promoter II including a 3.2-kb insert subcloned into pGL3 Basic was used to determine the effect of dexamethasone on promoter II (7). The pGRE-TK glucocorticoid-responsive construct was kindly provided by Dr. Jie Du (University of Kansas).

For site-directed mutagenesis, the glucocorticoid response element (GRE) sequence 5'-AGCAGAACAAGT-3' at -202 was mutated by the Expand High Fidelity PCR System (Roche) using a complementary primer set for the sequence 5'-AGCACTTTAAGTATGCCCAGAGGATGC-3', resulting in the mutated sequence 5'-AGCACTTTAAGT-3'. The TonE sequence 5'-TGGAAAACTCC-3' at -374 in the pGL3 -616 construct was mutated by using a complementary primer set for the sequence 5'-GAATTCCAATGGAGTCCCTCACCCTGAA-3', resulting in the mutated TonE sequence 5'-TGAGGGACTCC-3'. The CCAAT sequence 5'-GAATTCCAATGGAG-3' at -379 in the pGL3 -616 construct was mutated by using a complementary primer set to the sequence 5'-TTCAAGAATTCTCGAGGAGTTTTCC-3', resulting in the mutated CCAAT sequence 5'-GAATTCTCGAGGAG-3'. The sequence 5'-CCCTGAAAGG-3' at -363 was mutated using a complementary primer set for the sequence 5'-GAATTCCAATGGAGTTTTCCACGGTACCAGGCC-3', resulting in the mutated sequence 5'- CGGTACCAGG-3'. These constructs were all sequenced for verification.

Transient transfection and luciferase activity. Cells were seeded 24 h before transfection into 12-well plates (Corning, Marietta, GA). Cells were transfected with the Fugene 6 transfection reagent (Roche), using 0.5-pmol pGL3 constructs as described previously (8). To normalize firefly luciferase activity for differences in transfection efficiencies, cells were cotransfected with 0.01 µg of pRL-TK, a plasmid containing the Renilla luciferase gene under the transcriptional control of the herpes simplex virus thymidine kinase promoter (kindly provided by Dr. S. R. Price). Firefly and Renilla luciferase expression was measured with the Dual Luciferase Assay System (Promega), as described previously (8). To study the effect of glucocorticoids after transfection, cells were incubated in DMEM with 10% charcoal-stripped FBS (to remove endogenous steroids) (12) for 24 h and then treated with dexamethasone (100 nM, 24 h). Luciferase activity was compared between dexamethasone-treated and control cells, and differences were analyzed by two-tailed, nonpaired t-test, with P < 0.05 indicative of statistical significance.

Electrophoretic mobility shift assay. Nuclear extracts were harvested from confluent LLC-PK1-GR101 cells after incubation with 100 nM dexamethasone for 24 h; control cells were treated with vehicle only. Nuclear protein extracts were prepared as previously described (8). For the electrophoretic mobility shift assay (EMSA), each 5 µg of nuclear extract were incubated for 30 min at room temperature with 32P-radiolabeled probe for the sequence at -363 5'-GAATTCCAATGGAGTTTTCCACCCTGAAAGGCC-3' in the reaction buffer containing 12 mM HEPES, 4 mM Tris · HCl, 1 mM EDTA, 1 mM dithiothreitol, 60 mM KCl, 12% glycerol (vol/vol), 1 mM phenylmethylsulfonyl fluoride, and 2 µg poly(dIdC). The unlabeled oligonucleotide probe was used in 100× concentration to inhibit specific binding. Electrophoresis was performed with a 5% polyacrylamide gel followed by autoradiography. A supershift assay was performed as previously described (8) using an antibody (1 µl/reaction) to the 65-kDa subunit of the nuclear factor (NF)-kappa B protein (Santa Cruz Biotechnology, Santa Cruz, CA).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Dexamethasone treatment inhibits UT-A expression in rat kidney. The dose of dexamethasone given to rats in this study was intended to artificially raise glucocorticoids to stress levels for 3 days (9). The dexamethasone-treated animals showed decreased body weight compared with controls (dexamethasone-treated, -20 ± 7 g; controls, -1 ± 15 g, n = 5, P < 0.05). Urine osmolality was lower in dexamethasone-treated animals (756 ± 440 mosmol/kgH2O) but not significantly different from controls (1,397 ± 332 mosmol/kgH2O, n = 5, P < 0.7). By Northern blot analysis, we detected a significant decrease in the mRNA abundance of UT-A1, UT-A3, and UT-A3b in the inner medulla of dexamethasone-treated rats (Fig. 1). Quantitative analysis of the expression of UT-A1, UT-A3, and UT-A3b mRNA in dexamethasone-treated rats showed average decreases of 70-75% compared with pair-fed control animals, whereas no significant changes were observed for UT-A2 (Fig. 1B).


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Fig. 1.   Glucocorticoid treatment inhibits urea transporter UT-A mRNA expression in renal inner medulla. A: representative Northern blot analysis of UT-A mRNA expression in the inner medulla of dexamethasone-treated (Dex) and untreated (Control) rats. A full-size UT-A1 cDNA probe was used to detect UT-A1, UT-A2, and UT-A3 on the basis of different transcript sizes. A probe specific for the unique 3'-untranslated region of UT-A3b was used for identification of this isoform (see MATERIALS AND METHODS). B: quantitative analysis of UT-A mRNA abundance. Densitometric measurements of expression in untreated (open bars) and dexamethasone-treated rat kidney (filled bars) are shown. Values are means ± SD of UT-A/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA; n = 4. The experiment was repeated twice using different sets of animals with similar results. *Significantly different from control (P < 0.05).

Dexamethasone-treated rats had somewhat lower urine osmolality, and we previously demonstrated that expression of the renal UT-A mRNA transcripts varies with changes in hydration in vivo, due to tonicity-responsive transcriptional control (2, 8). Therefore, we tested the expression of AR, another tonicity-responsive gene in the renal inner medulla, and of the transcription factor TonEBP/NFAT5, to see whether dexamethasone may affect tonicity-responsive regulation of gene expression. Dexamethasone failed to produce any significant difference in the expression of AR or TonEBP mRNA in the kidney of dexamethasone-treated animals compared with untreated controls (Fig. 2), suggesting that glucocorticoids do not affect the tonicity-responsive regulatory pathway controlling expression of AR, TonEBP, and UT-A.


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Fig. 2.   Lack of effect of glucocorticoid treatment on aldose reductase (AR) and tonicity enhancer binding protein (TonEBP) mRNA expression in renal inner medulla. A: representative Northern blot analysis of AR and TonEBP mRNA expression in the inner medulla (IM) of dexamethasone-treated (Dex) and untreated (Control) rats. No signal was detected in the cortex (C) of either group. B: quantitative analysis of mRNA abundance. Densitometric measurements of expression in untreated (open bars) and dexamethasone-treated rat inner medulla (filled bars) are shown. Values are means ± SD of the AR/GAPDH ratio and TonEBP/GAPDH mRNA, n = 3.

Dexamethasone inhibits the activity of UT-A promoter I. We hypothesized that the decreased renal expression of UT-A1, UT-A3, and UT-A3b mRNA may be due to an inhibitory effect on UT-A promoter I activity, which controls their transcription. We examined the luciferase activity of various UT-A promoter I constructs in LLC-PK1-GR101 cells, a strain of LLC-PK1 cells permanently transfected to express the glucocorticoid receptor (12), with and without dexamethasone treatment. Dexamethasone inhibited UT-A promoter I activity in LLC-PK1-GR101 cells and stimulated the activity of the glucocorticoid-sensitive promoter pGRE-TK, which was included as a positive control (Fig. 3). Dexamethasone did not affect UT-A promoter I activity in LLC-PK1 cells that do not express the glucocorticoid receptor (40.74 ± 9.30 in treated cell vs. 40.8 ± 5.10 in untreated controls). The steroid-receptor antagonist RU-486 blocked the difference between UT-A promoter I activity in dexamethasone-treated (20.13 ± 1.86) and untreated LLC-PK1-GR101 cells (18.86 ± 0.65). However, it also blunted the UT-A promoter activity in control cells not treated with dexamethasone from 41.24 ± 0.42 to 24.7 ± 0.79, raising questions about the specificity of the RU-486 effect. Although we did not observe any effect of dexamethasone on the expression of the UT-A2 transcript, we tested the effect of dexamethasone on the activity of UT-A promoter II, which controls transcription of UT-A2. Dexamethasone did not depress the activity of UT-A promoter II (0.29 ± 0.029 in dexamethasone-treated cells vs. 0.18 ± 0.01 in untreated controls), suggesting that glucocorticoids do not inhibit transcription of UT-A2.


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Fig. 3.   Dexamethasone inhibits UT-A promoter I activity. Effect of dexamethasone on the luciferase activity of LLC- PK1-GR101 cells transfected with the glucocorticoid-responsive plasmid pGRE-TK and with the 1.3-kb UT-A promoter I construct (pUT-A1 1.3). Dexamethasone (100 nM) significantly stimulates the activity of pGRE-TK promoter and significantly inhibits the activity of UT-A promoter I. Values are means ± SD of firefly/Renilla luciferase, n = 4. Experiment was repeated twice using different plasmid preparations with similar results. *Significantly different from control (P < 0.05).

Dexamethasone inhibition is mediated by specific UT-A promoter sequences. To localize the promoter elements necessary for the inhibitory effect of dexamethasone on UT-A promoter I, we tested progressive deletions of the UT-A promoter I construct. Analysis of the luciferase activity of cells transfected with partially deleted constructs shows preservation of the inhibitory effect with the 1.3-, 0.7-, and 0.5-kb, but not with the 0.3-kb, construct (Fig. 4A). This finding suggested that the inhibition may be mediated by sequences upstream of -244 and that the GRE element at -202 is not involved. Indeed, mutation of the GRE within the 0.7-kb construct did not prevent dexamethasone inhibition of promoter activity (77% dexamethasone-induced decrease for mutated GRE compared with 70% dexamethasone-induced decrease in the wild-type 0.7-kb construct).


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Fig. 4.   Promoter sequences mediating dexamethasone repression. A: luciferase activity of cells transfected with progressive deletions of the UT-A promoter I with (open bars) and without (filled bars) dexamethasone treatment. The pattern of dexamethasone inhibition of promoter activity suggests that sequences between -423 and -244 mediate this effect. B: effect of site-directed mutagenesis of individual cis-elements in the UT-A promoter I sequence between -616 and +85 on promoter activity with (open bars) and without (filled bars) dexamethasone treatment. Control, wild-type 0.7-kb (-616, +85) promoter construct; m, site-specific mutation within the 0.7-kb promoter construct; TonE, tonicity enhancer. Values are means ± SD of firefly/Renilla (F/R) luciferase; n = 4. Experiments were repeated at least twice using different preparations of each plasmid, with similar results. *Significantly different from control (P < 0.05).

We focused our attention on the promoter region between -423 (0.5-kb construct) and -244 (0.3-kb construct), seeking to identify sequences that may mediate glucocorticoid repression of the UT-A promoter I. This region includes several consensus sequences for known cis-elements: CCAAT at -379, TonE at -374, and a sequence at -363 that resembles, but does not completely fit, the consensus for the binding site of NF-kappa B. Mutation of CCAAT at -379 did not produce any effect (Fig. 4B). Mutation of the contiguous TonE at -374 was also ineffective, further indicating that the glucocorticoid inhibition was not acting through the TonE/TonEBP pathway, which mediates tonicity-responsive transactivation of UT-A gene expression. However, when we tested a construct with a mutation of the -363 sequence, UT-A promoter I activity in both dexamethasone-treated (0.027 ± 0.005) and untreated cells (0.034 ± 0.013) was lower than the least active 0.3-kb promoter deletion construct (Fig. 4, A and B). This indicates that this sequence may be important for the basal activity of UT-A promoter I. By EMSA we detected evidence of specific protein binding to this motif, which is slightly decreased in the nuclei of dexamethasone-treated cells (Fig. 5). Given the similarity with the NF-kappa B consensus, we performed a supershift assay with an antibody for the p65 subunit of NF-kappa B. The result of this assay does not show evidence of a supershift in the size of the DNA-protein complex and does not support the possibility that the DNA-protein complex may include NF-kappa B (Fig. 5B).


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Fig. 5.   DNA-protein complex at the -363 binding site in the UT-A promoter I sequence. A: electrophoretic mobility shift assay with nuclear extracts of dexamethasone-treated (Dex) and untreated (C) LLC-PK1-GR101, using oligonucleotide probe for the -363 sequence. Lane 1, free radiolabeled oligonucleotide probe without nuclear extract. DNA-protein complex (lane 2, arrow) is less intense in Dex-treated cells (lane 3) in the absence of competing oligonucleotide (-). The 2 lanes on the right show effective competition of specific binding by 100× unlabeled oligonucleotide. B: supershift assay using an antibody (ab) to the p65 subunit of nuclear factor-kappa B. Lane 1, free probe; lane 2, control cells without antibody; lane 3, Dex-treated cells without antibody; lane 4, control cells with antibody; lane 5, Dex-treated cells with antibody. The arrow indicates the DNA-protein complex, which does not show supershift in the presence of the p65 antibody. The experiments were repeated twice with similar results.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we demonstrate that dexamethasone treatment for 3 days inhibits expression of the UT-A1, UT-A3, and UT-A3b urea transporter transcripts in the inner medulla of rat kidney. We show that this effect is due, at least in part, to dexamethasone inhibition of the activity of UT-A promoter I, which controls the transcription of these UT-A transporters. Our findings indicate that glucocorticoid repression of UT-A transcription is working through a mechanism that is not GRE or TonE mediated.

Our in vivo observation of decreased UT-A expression in the kidney inner medulla of dexamethasone-treated rats is consistent with earlier data by Knepper et al. (6), who reported that urea fractional excretion increases in adrenalectomized rats treated with dexamethasone for 3 days. The decrease in UT-A1, UT-A3, and UT-A3b mRNA abundance in inner medulla in our study fits nicely with the observation by Naruse et al. (9) that the urea permeability of perfused IMCD segments and UT-A1 protein abundance in inner medullary tip decrease in adrenalectomized rats treated with dexamethasone for 7 days.

Although the difference did not reach statistical significance, the dexamethasone-treated rats showed on average lower urine osmolality than untreated controls, which may result from decreased urea reabsorption into the medullary interstitium, lower interstitial urea levels, and reduced interstitial osmolality, with consequent excretion of less concentrated urine. The role of glucocorticoid in the regulation of renal urea transport may be significant in the setting of diabetes mellitus, contributing to the urine-concentration deficit observed in diabetic patients, and may occur during treatment with anti-inflammatory steroids in a variety of diseases. Studies of rats with streptozocin-induced diabetes seem to support this possibility (5). Glucocorticoids are elevated in the serum of lithium-treated rats (11), which have reduced interstitial urea and NaCl concentration (3). It is possible that increased glucocorticoids may affect urea transport and urine concentration, contributing to the diabetes insipidus that develops as a side effect of lithium therapy in patients affected by bipolar disorders.

In this study, we investigated the mechanism by which dexamethasone decreases renal UT-A expression by examining the transcriptional regulation of the two UT-A promoters. Previously, we characterized the rat Slc14a2 gene that encodes the known rat UT-A isoforms, and we identified two promoters (7). Promoter I controls transcription of UT-A1, UT-A3, and UT-A3b and is located at the 5'-end of the gene. Promoter II controls transcription of UT-A2 and is located between exons 12 and 13 (7).

We demonstrated that the abundance of UT-A2, UT-A3, and UT-A3b transcripts is increased in the inner medulla of water-deprived rats (2). The increased expression of UT-A3 and UT-A3b in dehydrated animals seems to be due to tonicity-responsive stimulation of their transcription by activation of the UT-A promoter I through the TonE/TonEBP pathway (8). Increased transcription of UT-A2 in water-deprived rats results from activation of UT-A promoter II by cAMP-dependent pathways (7).

In the present study, the reduced expression of UT-A1, UT-A3, and UT-A3b in dexamethasone-treated rats results from transcriptional repression of UT-A promoter I. The mechanism for the glucocorticoid-induced transrepression of promoter I is distinct from the transactivation mechanisms involving the TonE/TonEBP pathway, because the expression of the tonicity-regulated genes AR and TonEBP were unchanged in dexamethasone-treated rats. Furthermore, mutation of the TonE site in promoter I did not prevent transrepression by dexamethasone. Involvement of cAMP pathways also seems unlikely, because there are no cAMP responsive elements in UT-A promoter I. Our deletion and site-directed mutagenesis studies rule out involvement of the GRE consensus sequence present in promoter I in the inhibitory response to dexamethasone. Interestingly, mutation of the sequence at -363 induced a dramatic ablation of promoter I activity, suggesting that this may be an important site for basal activation of promoter I. By EMSA assay, we show that this sequence forms a DNA-protein complex. The nature of the binding protein remains to be determined. Dexamethasone treatment mildly decreases the intensity of this DNA-protein binding, but it is unclear whether changes in this interaction mediate dexamethasone repression of promoter I activity.

In summary, our study shows that glucocorticoids repress the activity of UT-A promoter I, leading to decreased transcription of UT-A1, UT-A3, and UT-A3b from the UT-A gene and resulting in decreased abundance of UT-A mRNA and UT-A protein in the renal medulla. Further investigation is necessary to identify the factor(s) responsible for the transcriptional repression of the UT-A urea transporter gene expression by glucocorticoids in the kidney.


    ACKNOWLEDGEMENTS

We are grateful to Dr. S. Russ Price for providing crucial reagents, helpful suggestions, and a critical review of the manuscript.


    FOOTNOTES

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-53917, R01-DK-41707, and P01-DK-50268. Part of this work was presented in abstract form at the 37th annual meeting of the American Society of Nephrology, San Francisco, CA, October 2001, and has been published (J Am Soc Nephrol 13: 13A, 2001).

Address for reprint requests and other correspondence: S. M. Bagnasco, Dept. of Pathology, Emory Univ. School of Medicine, WMB Rm. 7105 A, 1639 Pierce Dr., NE, Atlanta GA 30322 (E-mail: sbagnas{at}emory.edu).

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.

10.1152/ajprenal.00262.2001

Received 20 August 2001; accepted in final form 26 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bagnasco, SM, Peng T, Janech MG, Karakashian A, and Sands JM. Cloning and characterization of the human urea transporter UT-A1 and mapping of the human Slc14a2 gene. Am J Physiol Renal Physiol 281: F400-F406, 2001[Abstract/Free Full Text].

2.   Bagnasco, SM, Peng T, Nakayama Y, and Sands JM. Differential expression of individual UT-A urea transporter isoforms in rat kidney. J Am Soc Nephrol 11: 1980-1986, 2000[Abstract/Free Full Text].

3.   Christensen, S, Kusano E, Yususfi ANK, Murayama N, and Dousa PT. Pathogenesis of nephrogenic diabetes insipidus due to chronic administration of lithium in rats. J Clin Invest 75: 1869-1879, 1985[ISI][Medline].

4.   Karakashian, A, Timmer RT, Klein DJ, Gunn RB, Sands JM, and Bagnasco SM. Cloning and characterization of two new isoforms of the rat kidney urea transporter: UT-A3 and UT-A4. J Am Soc Nephrol 10: 230-237, 1999[Abstract/Free Full Text].

5.   Klein, DJ, Price SR, Bailey JL, Jacobs JD, and Sands JM. Glucocorticoids mediate a decrease in AVP-regulated urea transporter in diabetic rat inner medulla. Am J Physiol Renal Physiol 273: F949-F953, 1997[ISI][Medline].

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Am J Physiol Renal Fluid Electrolyte Physiol 282(5):F853-F858
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