1 Department of Pathology and 2 Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
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)-
B
protein (Santa Cruz Biotechnology, Santa Cruz, CA).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
|
|
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.
|
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).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
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
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
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].
6.
Knepper, MA,
Danielson RA,
Saidel GM,
and
Johnston KH.
Effects of dietary protein restriction and glucocorticoid administration on urea excretion in rats.
Kidney Int
8:
303-315,
1975[ISI][Medline].
7.
Nakayama, Y,
Naruse M,
Karakashian A,
Peng T,
Sands JM,
and
Bagnasco SM.
Cloning of the rat Slc14a2 gene and genomic organization of the UT-A urea transporter.
Biochim Biophys Acta Gene Struct Expression
1518:
19-26,
2001[ISI][Medline].
8.
Nakayama, Y,
Peng T,
Sands JM,
and
Bagnasco S.
The TonE/TonEBP pathway mediates tonicity-responsive regulation of UT-A urea transporter expression.
J Biol Chem
275:
38275-38280,
2000
9.
Naruse, M,
Klein JD,
Ashkar ZM,
Jacobs JD,
and
Sands JM.
Glucocorticoids downregulate the rat vasopressin-regulated urea transporter in rat terminal inner medullary collecting ducts.
J Am Soc Nephrol
8:
517-523,
1997[Abstract].
10.
Shayakul, C,
Steel A,
and
Hediger MA.
Molecular cloning and characterization of the vasopressin-regulated urea transporter of rat kidney collecting ducts.
J Clin Invest
98:
2580-2587,
1996
11.
Smotherman, WP,
Hennessy JW,
and
Levine S.
Plasma corticosterone levels during recovery from LiCl produced taste aversion.
Behav Biol
16:
401-412,
1976[ISI][Medline].
12.
Wang, X,
Jurkovitz C,
and
Price SR.
Regulation of branched-chain ketoacid dehydrogenase flux by extracellular pH and glucocorticoids.
Am J Physiol Cell Physiol
272:
C2031-C2036,
1997
13.
You, G,
Smith CP,
Kanai Y,
Lee WS,
Stelzner M,
and
Hediger MA.
Cloning and characterization of the vasopressin-regulated urea transporter.
Nature
365:
844-847,
1993[ISI][Medline].