Glucocorticoids have a role in renal cortical ... - Renal Physiology
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<strong>Glucocorticoids</strong> <strong>have</strong> a <strong>role</strong> <strong>in</strong> <strong>renal</strong> <strong>cortical</strong> expression of<br />
the SNAT3 glutam<strong>in</strong>e transporter dur<strong>in</strong>g chronic<br />
metabolic acidosis<br />
Anne M. Kar<strong>in</strong>ch, Cheng-Mao L<strong>in</strong>, Q<strong>in</strong>gHe Meng, M<strong>in</strong>g Pan and Wiley W. Souba<br />
Am J Physiol <strong>Renal</strong> Physiol 292:F448-F455, 2007. First published 5 September 2006;<br />
doi: 10.1152/ajp<strong>renal</strong>.00168.2006<br />
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Am J Physiol <strong>Renal</strong> Physiol 292: F448–F455, 2007.<br />
First published September 5, 2006; doi:10.1152/ajp<strong>renal</strong>.00168.2006.<br />
<strong>Glucocorticoids</strong> <strong>have</strong> a <strong>role</strong> <strong>in</strong> <strong>renal</strong> <strong>cortical</strong> expression of the SNAT3<br />
glutam<strong>in</strong>e transporter dur<strong>in</strong>g chronic metabolic acidosis<br />
Anne M. Kar<strong>in</strong>ch, Cheng-Mao L<strong>in</strong>, Q<strong>in</strong>gHe Meng, M<strong>in</strong>g Pan, and Wiley W. Souba<br />
Department of Surgery, Milton S. Hershey Medical Center, The Pennsylvania<br />
State University College of Medic<strong>in</strong>e, Hershey, Pennsylvania<br />
Submitted 15 May 2006; accepted <strong>in</strong> f<strong>in</strong>al form 27 August 2006<br />
Kar<strong>in</strong>ch AM, L<strong>in</strong> C-M, Meng QH, Pan M, Souba WW. <strong>Glucocorticoids</strong><br />
<strong>have</strong> a <strong>role</strong> <strong>in</strong> <strong>renal</strong> <strong>cortical</strong> expression of the SNAT3<br />
glutam<strong>in</strong>e transporter dur<strong>in</strong>g chronic metabolic acidosis. Am J Physiol<br />
<strong>Renal</strong> Physiol 292: F448–F455, 2007. First published September 5,<br />
2006; doi:10.1152/ajp<strong>renal</strong>.00168.2006.—<strong>Glucocorticoids</strong> are <strong>in</strong>volved<br />
<strong>in</strong> many aspects of regulation of acid-base homeostasis,<br />
<strong>in</strong>clud<strong>in</strong>g the stimulation of <strong>renal</strong> ammoniagenesis dur<strong>in</strong>g chronic<br />
metabolic acidosis. Plasma glutam<strong>in</strong>e is the pr<strong>in</strong>cipal substrate for<br />
ammoniagenesis under these conditions. Expression of the System N<br />
glutam<strong>in</strong>e transporter SNAT3 is <strong>in</strong>creased <strong>in</strong> the <strong>renal</strong> proximal<br />
tubules dur<strong>in</strong>g acidosis. In vivo studies <strong>in</strong> rats us<strong>in</strong>g 1) sham and<br />
ad<strong>renal</strong>ectomized rats, 2) the glucocorticoid receptor antagonist<br />
RU486, and 3) dexamethasone treatment demonstrated <strong>in</strong>volvement<br />
of glucocorticoids <strong>in</strong> regulation of SNAT3 expression. Ad<strong>renal</strong>ectomy<br />
attenuated the acidosis-<strong>in</strong>duced <strong>in</strong>crease <strong>in</strong> <strong>renal</strong> <strong>cortical</strong> SNAT3 mRNA<br />
�40%, and treatment with dexamethasone (1 mg�kg�1�day�1 sc) partially<br />
reversed this effect. RU486 also blunted the acidosis-<strong>in</strong>duced<br />
<strong>in</strong>crease <strong>in</strong> SNAT3 expression �50%. Chronic dexamethasone treatment<br />
(0.1 mg�kg�1�day�1 sc, 6 days) of normal rats slightly <strong>in</strong>creased SNAT3<br />
expression. In all cases, <strong>renal</strong> glutam<strong>in</strong>e arteriovenous difference mirrored<br />
SNAT3 expression and activity <strong>in</strong> the proximal tubules, suggest<strong>in</strong>g<br />
that SNAT3 regulates glutam<strong>in</strong>e uptake dur<strong>in</strong>g acidosis. These studies<br />
<strong>in</strong>dicate that glucocorticoids regulate acid-base homeostasis dur<strong>in</strong>g metabolic<br />
acidosis <strong>in</strong> part by regulat<strong>in</strong>g expression of the System N transporter<br />
SNAT3.<br />
dexamethasone; System N expression; ammonium chloride acidosis;<br />
RU486<br />
THE KIDNEY PLAYS A VITAL ROLE <strong>in</strong> ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g acid-base homeostasis<br />
dur<strong>in</strong>g chronic metabolic acidosis. Under acidotic<br />
conditions, a number of adaptive changes occur throughout the<br />
kidney, act<strong>in</strong>g <strong>in</strong> concert to reduce the acid load and restore<br />
acid-base balance. The <strong>renal</strong> proximal convoluted tubule is the<br />
site of remarkable coord<strong>in</strong>ated changes that result <strong>in</strong> <strong>in</strong>creased<br />
ammoniagenesis that is susta<strong>in</strong>ed for the duration of acidosis<br />
(16, 30). These changes <strong>in</strong>clude enhanced uptake and metabolism<br />
of glutam<strong>in</strong>e, the pr<strong>in</strong>cipal substrate for <strong>renal</strong> ammoniagenesis<br />
dur<strong>in</strong>g acidosis (39). Asymmetric secretion from the<br />
proximal tubule of ammonium ions <strong>in</strong>to the ur<strong>in</strong>e and bicarbonate<br />
ions <strong>in</strong>to the blood functions to return acid-base balance<br />
toward normal. Ammonium and bicarbonate ions are both<br />
products of glutam<strong>in</strong>e metabolism, a s<strong>in</strong>gle molecule of glutam<strong>in</strong>e<br />
produc<strong>in</strong>g two ammonium and two bicarbonate ions.<br />
Under normal conditions, the kidney releases low levels of<br />
glutam<strong>in</strong>e <strong>in</strong>to the blood whereas dur<strong>in</strong>g acidosis �40% of the<br />
glutam<strong>in</strong>e presented to the kidney <strong>in</strong> the blood is extracted (36,<br />
Address for repr<strong>in</strong>t requests and other correspondence: W. W. Souba,<br />
College of Medic<strong>in</strong>e, Ohio State University, 254 Meil<strong>in</strong>g Hall, 370 West 9th<br />
Ave., Columbus, OH 43210 (e-mail: chip.souba@osumc.edu).<br />
F448<br />
38), so that the kidney becomes the body’s pr<strong>in</strong>cipal consumer<br />
of glutam<strong>in</strong>e.<br />
Studies us<strong>in</strong>g ad<strong>renal</strong>ectomized (ADX) rats suggest that<br />
glucocorticoids are <strong>in</strong>volved <strong>in</strong> acid-base homeostasis dur<strong>in</strong>g<br />
metabolic acidosis. This observation is consistent with elevated<br />
plasma corticosterone concentration dur<strong>in</strong>g acidosis <strong>in</strong> rats (28,<br />
42). ADX rats that are challenged with an acid load <strong>have</strong> a<br />
reduced ability to <strong>in</strong>crease <strong>renal</strong> ammoniagenesis (33, 46).<br />
Dur<strong>in</strong>g metabolic acidosis, the kidneys of these animals cannot<br />
<strong>in</strong>crease glutam<strong>in</strong>e uptake from the blood or <strong>in</strong>crease glutam<strong>in</strong>e<br />
metabolism to the same degree as ad<strong>renal</strong>-<strong>in</strong>tact rats<br />
(46). Glucocorticoid treatment of ADX rats before <strong>in</strong>duction of<br />
acidosis partially restores their ability to extract glutam<strong>in</strong>e<br />
from the blood (46). <strong>Glucocorticoids</strong> also <strong>in</strong>crease ammonia<br />
excretion <strong>in</strong> the <strong>in</strong>tact animal (19) and ammonia production by<br />
perfused kidneys (44). These studies suggest that glucocorticoids<br />
play a direct or <strong>in</strong>direct <strong>role</strong> <strong>in</strong> regulation of acidosis<strong>in</strong>duced<br />
glutam<strong>in</strong>e metabolism.<br />
We reported that the acidosis-<strong>in</strong>duced <strong>in</strong>crease <strong>in</strong> <strong>renal</strong><br />
glutam<strong>in</strong>e uptake is accomplished by <strong>in</strong>creased expression of<br />
the System N transporter <strong>in</strong> the proximal convoluted tubule of<br />
acidotic rats (23). This observation was recently confirmed by<br />
others (37). The System N transporter, previously known as<br />
SN1, has been renamed SNAT3 <strong>in</strong> recognition of its identity as<br />
a member of the SCL38 gene family of sodium-coupled neutral<br />
am<strong>in</strong>o acid transporters (25). SNAT3 is located <strong>in</strong> the basolateral<br />
membrane of proximal tubule epithelial cells (37) and<br />
transports one glutam<strong>in</strong>e molecule and one sodium ion <strong>in</strong><br />
exchange for one proton (7, 10). The transporter mediates<br />
glutam<strong>in</strong>e flux <strong>in</strong> both directions, depend<strong>in</strong>g on substrate<br />
gradients (7) and is associated with an uncoupled proton<br />
conductance that can serve to m<strong>in</strong>imize the proton flux produced<br />
by a transport-coupled proton antiport (7, 10).<br />
We hypothesized that glucocorticoids enhance ammoniagenesis<br />
dur<strong>in</strong>g metabolic acidosis <strong>in</strong> part by regulation of<br />
expression of the SNAT3 transporter. We studied regulation of<br />
<strong>renal</strong> <strong>cortical</strong> SNAT3 expression by glucocorticoids dur<strong>in</strong>g<br />
chronic metabolic acidosis <strong>in</strong> vivo us<strong>in</strong>g normal and ADX rats<br />
and the glucocorticoid receptor antagonist RU486. Data from<br />
our studies suggest that glucocorticoids ma<strong>in</strong>ta<strong>in</strong> acid-base<br />
homeostasis, <strong>in</strong> part, by alter<strong>in</strong>g <strong>renal</strong> glutam<strong>in</strong>e uptake via<br />
regulation of expression of the SNAT3 transporter.<br />
MATERIALS AND METHODS<br />
Animals. Animals were treated <strong>in</strong> accordance with regulations of<br />
the Institutional Animal Use and Care Committee. Male Sprague-<br />
The costs of publication of this article were defrayed <strong>in</strong> part by the payment<br />
of page charges. The article must therefore be hereby marked “advertisement”<br />
<strong>in</strong> accordance with 18 U.S.C. Section 1734 solely to <strong>in</strong>dicate this fact.<br />
0363-6127/07 $8.00 Copyright © 2007 the American Physiological Society http://www.ajp<strong>renal</strong>.org<br />
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GLUCOCORTICOIDS AND RENAL SNAT3 EXPRESSION<br />
Dawley rats (Charles River, Wilm<strong>in</strong>gton, MA), weigh<strong>in</strong>g 250–300 g,<br />
were ma<strong>in</strong>ta<strong>in</strong>ed <strong>in</strong> a 12:12-h light-dark cycle with unrestricted access<br />
to rat chow and water or water conta<strong>in</strong><strong>in</strong>g 1.5% NH4Cl. Metabolic<br />
acidosis was ma<strong>in</strong>ta<strong>in</strong>ed for vary<strong>in</strong>g lengths of time, up to 6 days.<br />
ADX (bilateral) rats and sham-operated controls purchased from<br />
Charles River were used for some experiments. Both sham and ADX<br />
rats were ma<strong>in</strong>ta<strong>in</strong>ed on 0.9% sal<strong>in</strong>e substituted for their dr<strong>in</strong>k<strong>in</strong>g<br />
water before experimentation. We followed a modification of the<br />
protocol of Welbourne and colleagues (46) that adjusts the NH4Cl<br />
concentration because ADX rats dr<strong>in</strong>k approximately twice the volume<br />
that <strong>in</strong>tact rats dr<strong>in</strong>k. ADX rats were given 0.6% NH4Cl <strong>in</strong> sal<strong>in</strong>e,<br />
and sham acidotic rats were given 1.2% NH4Cl <strong>in</strong> sal<strong>in</strong>e so that they<br />
consumed approximately equal acid loads. ADX rats were exposed to<br />
acidosis for only 2 days because of significant mortality <strong>in</strong> acidotic<br />
ADX rats after 3 days. For some experiments, normal rats were<br />
treated acutely (5 mg/kg sc, once) or chronically (100 �g/rat sc, once<br />
a day for up to 6 days) with dexamethasone. The body weight of<br />
control rats <strong>in</strong>creased steadily dur<strong>in</strong>g the experimental period (259 �<br />
4gatday 0, 295 � 4gatday 6), whereas the body weight of<br />
chronically treated rats decreased for the first 3 days and then<br />
stabilized (255 � 3atday 0, 240 � 4gatday 6).<br />
Rats were killed after anesthesia with ketam<strong>in</strong>e/acepromaz<strong>in</strong>e/<br />
xylaz<strong>in</strong>e (80:1:12 mg/kg body wt). The kidneys were removed, and<br />
the <strong>renal</strong> cortex was immediately dissected out and processed for<br />
RNA isolation or membrane preparation. At the time of death, blood<br />
was drawn from the descend<strong>in</strong>g aorta and the <strong>renal</strong> ve<strong>in</strong> for measurement<br />
of blood gases, pH, and plasma glutam<strong>in</strong>e concentration.<br />
Measurement of blood gases, pH, and glutam<strong>in</strong>e concentration.<br />
Arterial blood pH and PCO2 were measured us<strong>in</strong>g an IL BG3 bloodgas<br />
mach<strong>in</strong>e (model 1420, Instrumentation Laboratory, Lex<strong>in</strong>gton,<br />
�<br />
MA). Blood HCO3 concentration was automatically calculated from<br />
the measured pH and PCO2. Plasma glutam<strong>in</strong>e levels were measured <strong>in</strong><br />
triplicate by a modified spectrophotometric assay us<strong>in</strong>g a colorimetric<br />
assay kit (Boer<strong>in</strong>ger Mannheim, Mannheim, Germany) adapted to a<br />
96-well plate format with a microplate spectrophotometer. Glutam<strong>in</strong>e<br />
arteriovenous (AV) difference was calculated as (arterial � <strong>renal</strong><br />
ve<strong>in</strong>) plasma glutam<strong>in</strong>e concentration.<br />
Kidney dissection. Kidney dissection was carried out under approximately<br />
threefold magnification. The decapsulated kidney was first cut<br />
lengthwise <strong>in</strong>to two symmetrical halves. For dissection of the cortex,<br />
a th<strong>in</strong> slice (1.5–2 mm thick) was first cut from the curved outer<br />
surface. Additional cortex was dissected us<strong>in</strong>g f<strong>in</strong>e scissors by trimm<strong>in</strong>g<br />
away the outer 1.5–2 mm, us<strong>in</strong>g the visible arcuate arteries as a<br />
guide to the junction of the cortex and medulla.<br />
Northern blot analysis. Total RNA was isolated from the <strong>renal</strong><br />
cortex us<strong>in</strong>g the Totally RNA system (Ambion, Aust<strong>in</strong>, TX) or from<br />
cultured cells us<strong>in</strong>g an RNeasy M<strong>in</strong>i kit (Qiagen, Valencia, CA). RNA<br />
was separated on a 1% formaldehyde gel, transferred to nylon membrane<br />
(Genescreen, New England Nuclear), and hybridized with a<br />
SNAT3-specific oligonucleotide probe. The SNAT3 oligonucleotide<br />
sequence was 5�-GTGCAGAAGGCTTCAGCAGTGTCAGGTTGG-<br />
3�. The oligonucleotide was radioactively 3�-end labeled us<strong>in</strong>g term<strong>in</strong>al<br />
transferase and �-[ 32P]dATP. For quantification of SNAT3<br />
mRNA, autoradiographs were scanned us<strong>in</strong>g a laser densitometer<br />
(Dynamic Biosystems). Glyceraldehyde-3 phosphate dehydrogenase<br />
(GAPDH) and �-act<strong>in</strong> were both found to be unsuitable for RNA<br />
load<strong>in</strong>g normalization because acidosis <strong>in</strong>creased mRNA levels of<br />
both. The 18S ribosomal RNA subunit was therefore used for normalization.<br />
Isolation of <strong>cortical</strong> basolateral membrane vesicles. Basolateral<br />
membrane vesicles (BLMV) were isolated from <strong>renal</strong> cortex by<br />
Percoll density gradient centrifugation as previously described (23).<br />
Vesicles were resuspended <strong>in</strong> <strong>in</strong>travesicular buffer [100 mM KCl, 100<br />
mM mannitol, 12 mM Tris/HEPES, pH 7.5, 0.1 mM phenylmethylsulfonyl<br />
fluoride, 1 �g/ml pepstat<strong>in</strong> A, 2 ml/l protease cocktail stock<br />
(Sigma P2714, Sigma, St. Louis, MO) to a concentration of �1<br />
mg/ml]. Prote<strong>in</strong> was measured us<strong>in</strong>g the Bradford assay with BSA as<br />
AJP-<strong>Renal</strong> Physiol • VOL 292 • JANUARY 2007 • www.ajp<strong>renal</strong>.org<br />
F449<br />
a standard. BLMV were prepared at the same time from all groups to<br />
be compared <strong>in</strong> a specific experiment to m<strong>in</strong>imize variation among the<br />
preparations. <strong>Renal</strong> cortices from two to five animals were pooled for<br />
each BLMV preparation. Vesicle relative enrichment was estimated<br />
us<strong>in</strong>g ouaba<strong>in</strong>-<strong>in</strong>hibitable ATPase activity <strong>in</strong> BLMV compared with<br />
the homogenate (14). The relative enrichment did not vary significantly<br />
among preparations for the RU486 experiments (control 16 �<br />
2-, acidosis 27 � 9-, acidosis�RU486 18 � 3-fold <strong>in</strong>crease over<br />
homogenate; P � 0.3, ANOVA) or the chronic dexamethasone<br />
experiments (control 17 � 3-, dexamethasone 14 � 3-fold <strong>in</strong>crease<br />
over homogenate; P � 0.5, t-test). Vesicles frozen <strong>in</strong> liquid nitrogen<br />
were used for glutam<strong>in</strong>e transport assays.<br />
Glutam<strong>in</strong>e transport. For transport studies <strong>in</strong> <strong>renal</strong> <strong>cortical</strong> BLMV,<br />
Na � -dependent glutam<strong>in</strong>e uptake was measured at room temperature<br />
us<strong>in</strong>g a rapid mix<strong>in</strong>g/filtration technique described previously (23).<br />
Basolateral glutam<strong>in</strong>e transport was measured <strong>in</strong> cells grown to tight<br />
confluence on Transwell plates. Follow<strong>in</strong>g treatment, transport was<br />
measured <strong>in</strong> chol<strong>in</strong>e chloride or NaCl uptake buffer (<strong>in</strong> mM: 137 NaCl<br />
or chol<strong>in</strong>e Cl, 10 HEPES/Tris, pH 7.4, 4.7 KCl, 1.2 MgSO4, 1.2<br />
KH2PO4, 2.5 CaCl2) conta<strong>in</strong><strong>in</strong>g L-[ 3 H]glutam<strong>in</strong>e (4 �Ci/ml) and 50<br />
�M unlabeled L-glutam<strong>in</strong>e. Uptake was term<strong>in</strong>ated after 5 m<strong>in</strong> by<br />
wash<strong>in</strong>g with ice-cold NaCl uptake buffer without [ 3 H]glutam<strong>in</strong>e. Part<br />
of the cell lysate was counted <strong>in</strong> a TopCount sc<strong>in</strong>tillation spectrophotometer<br />
(Packard, Meriden, CT), and part was used for prote<strong>in</strong><br />
determ<strong>in</strong>ation. The rate of glutam<strong>in</strong>e transport was l<strong>in</strong>ear at 5 m<strong>in</strong> and<br />
was expressed as nanomoles per milligram prote<strong>in</strong> per m<strong>in</strong>ute. Where<br />
<strong>in</strong>dicated, 5 mM histid<strong>in</strong>e was added to the uptake buffer to allow<br />
identification of SNAT3-mediated transport <strong>in</strong> BLMV and GR101<br />
cells.<br />
Western blot analysis. Equal amounts of <strong>cortical</strong> BLMV (10 �g)<br />
were separated by SDS-PAGE on precast polyacrylamide gels (ISC<br />
BioExpress, Kaysville, UT) and transferred to polyv<strong>in</strong>ylidene difluoride<br />
membranes (Millipore, Bedford, MA). Blots were <strong>in</strong>cubated with<br />
a polyclonal antibody raised aga<strong>in</strong>st a SNAT3-GST fusion prote<strong>in</strong><br />
conta<strong>in</strong><strong>in</strong>g the NH2-term<strong>in</strong>al 71 am<strong>in</strong>o acids of SNAT3 followed by a<br />
horseradish peroxidase-conjugated goat anti-rabbit antibody (Rockland<br />
Immunochemicals, Gilbertsville, PA). SNAT3 prote<strong>in</strong> was detected<br />
by enhanced chemilum<strong>in</strong>escence (Upstate, Lake Placid, NY)<br />
follow<strong>in</strong>g the manufacturer’s <strong>in</strong>structions.<br />
Clon<strong>in</strong>g of the rat SNAT3 promoter. A rat genomic library (Clontech,<br />
Mounta<strong>in</strong> View, CA) was screened us<strong>in</strong>g a SNAT3-specific<br />
probe. One positive plaque was identified and isolated by limit<strong>in</strong>g<br />
dilution. The purified phage conta<strong>in</strong>ed an <strong>in</strong>sert of �12 kb, all of<br />
which represent a genomic sequence from the SNAT3 gene, based on<br />
comparison with the published rat genomic sequence (GenBank<br />
NW_047801.1). A 3.7-kb XhoI restriction fragment of the rat genomic<br />
clone that conta<strong>in</strong>s �2.4 kb of the 5�-flank<strong>in</strong>g sequence, exon I<br />
(untranslated) and �1.3 kb of <strong>in</strong>tron 1 was subcloned <strong>in</strong>to Bluescript<br />
and sequenced (Molecular Genetics Core Facility at the Pennsylvania<br />
State University College of Medic<strong>in</strong>e). The Transcription Factor<br />
Search program TFSearch (Yutaka Akiyama: “TFSEARCH: Search<strong>in</strong>g<br />
Transcription Factor B<strong>in</strong>d<strong>in</strong>g Sites”; http://www.rwcp.or.jp/<br />
papia/) was used to identify potential transcription factor b<strong>in</strong>d<strong>in</strong>g sites<br />
<strong>in</strong> the 5�-flank<strong>in</strong>g region. A series of SNAT3 promoter fragments<br />
(5�-boundaries �2,414 �815, �132, �78 with a common 3� boundary<br />
at �20 with respect to the published rat SNAT3 cDNA sequence,<br />
GenBank AF273025) was amplified by PCR us<strong>in</strong>g the cloned phage<br />
DNA as a template and cloned <strong>in</strong>to the multiple clon<strong>in</strong>g site of the<br />
pCAT3-Basic reporter vector (Promega, Madison, WI). Reporter<br />
constructs were verified by sequenc<strong>in</strong>g by the Molecular Genetics<br />
Core Facility.<br />
Cell culture. LLC-PK1-GR101 (GR101) cells are porc<strong>in</strong>e epitheliallike<br />
LLC-PK1 cells that express rat glucocorticoid receptors (41).<br />
GR101 were grown <strong>in</strong> low-glucose DMEM (Invitrogen, Carlsbad,<br />
CA) conta<strong>in</strong><strong>in</strong>g 800 ng/ml hygromyc<strong>in</strong> B (Calbiochem, La Jolla, CA)<br />
to ma<strong>in</strong>ta<strong>in</strong> glucocorticoid receptor expression. These cells were<br />
generously provided by Dr. S. R. Price of Emory University.<br />
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F450 GLUCOCORTICOIDS AND RENAL SNAT3 EXPRESSION<br />
Transient transfection. GR101 cells were transiently transfected<br />
us<strong>in</strong>g Lipofect<strong>in</strong> (Invitrogen) follow<strong>in</strong>g the manufacturer’s protocol.<br />
After cotransfection with pCAT.SNAT3 constructs (1 �g) and<br />
pSV40-�-Gal (1 �g, Promega), cells were treated with control or<br />
dexamethasone (0.01 or 0.1 �M) medium and harvested after 48 h.<br />
The dexamethasone-responsive plasmid pGREtkCAT was used as a<br />
positive control for glucocorticoid responsiveness. This plasmid conta<strong>in</strong>s<br />
two <strong>in</strong>verted repeats of a glucocorticoid response element sequence<br />
followed by the thymid<strong>in</strong>e k<strong>in</strong>ase promoter that drives expression<br />
of the chloramphenicol acetyl transferase (CAT) gene.<br />
pGREtkCAT was provided by Dr. S. S. Simons, Jr., of National<br />
Institute of Diabetes and Digestive and Kidney Diseases, National<br />
Institutes of Health. CAT and �-galactosidase assays were carried out<br />
as described <strong>in</strong> the Promega protocols.<br />
Statistics. Data were analyzed us<strong>in</strong>g a t-test, paired t-test, or<br />
one-way analysis of variance followed by Bonferroni or Dunnett<br />
posttests for multiple comparisons, as appropriate.<br />
RESULTS<br />
ADX rats are less able than sham-operated rats to ma<strong>in</strong>ta<strong>in</strong><br />
acid-base homeostasis dur<strong>in</strong>g chronic metabolic acidosis. Five<br />
groups of rats were studied: 1) sham-operated, 2) ADX, 3)<br />
2-day acidotic sham-operated, 4) 2-day acidotic ADX, and 5)<br />
2-day acidotic ADX pretreated for 2 days and throughout<br />
acidosis with the synthetic glucocorticoid dexamethasone (1<br />
mg�kg �1 �day �1 sc). Table 1 shows the arterial (systemic) pH<br />
and bicarbonate concentration and <strong>renal</strong> glutam<strong>in</strong>e AV difference<br />
<strong>in</strong> each group of rats. The effect of ad<strong>renal</strong>ectomy on the<br />
acid-base status and <strong>renal</strong> glutam<strong>in</strong>e AV difference of normal<br />
and acidotic rats was similar to that previously reported (11,<br />
30, 46). After 2 days of <strong>in</strong>gest<strong>in</strong>g ammonium chloride, both<br />
sham and ADX rats became acidotic, but ADX rats were more<br />
severely acidotic than sham rats. Chronic acidosis also <strong>in</strong>creased<br />
glutam<strong>in</strong>e AV difference <strong>in</strong> both sham and ADX rats,<br />
but the <strong>in</strong>crease was significantly attenuated <strong>in</strong> ADX rats.<br />
Dexamethasone treatment improved the ability of acidotic<br />
ADX rats to compensate for the acid load, as <strong>in</strong>dicated by<br />
<strong>in</strong>creased blood pH and serum bicarbonate concentration. In<br />
addition, glutam<strong>in</strong>e AV difference <strong>in</strong> these rats reverted toward<br />
the level <strong>in</strong> acidotic sham rats.<br />
Plasma glutam<strong>in</strong>e concentration is altered by ad<strong>renal</strong>ectomy,<br />
metabolic acidosis, and dexamethasone. Figure 1 shows<br />
the arterial plasma glutam<strong>in</strong>e concentration of all animal<br />
groups <strong>in</strong> the studies presented <strong>in</strong> Figs. 1-3. Acidosis decreases<br />
Table 1. Selected blood parameters from sham-operated<br />
and ad<strong>renal</strong>ectomized rats<br />
Blood pH<br />
Blood<br />
Bicarbonate,<br />
mM<br />
<strong>Renal</strong><br />
Glutam<strong>in</strong>e AV<br />
Difference, mmol/l<br />
Sham 7.38�0.01 25.0�0.5 �0.026�0.009<br />
ADX 7.29�0.02 22.3�0.6 �0.030�0.014<br />
Sham�acidosis 7.22�0.02 a 16.9�0.4 a 0.173�0.020 a<br />
ADX�acidosis 7.17�0.02 a 14.9�0.5 ab 0.070�0.009 ac<br />
ADX�acidosis�Dex 7.30�0.02 d 20.4�0.8 d 0.138�0.015 e<br />
Values are means � SE; n � 12–27 animals/group. ADX, ad<strong>renal</strong>ectomized.<br />
Animals were treated as described <strong>in</strong> MATERIALS AND METHODS. Dexamethasone<br />
(Dex) concentration was 1 mg�kg �1 �day �1 . pH and bicarbonate<br />
values are from arterial blood. Glutam<strong>in</strong>e <strong>renal</strong> arteriovenous (AV) difference<br />
is (arterial � <strong>renal</strong> ve<strong>in</strong>) plasma glutam<strong>in</strong>e concentration. a P � 0.001 vs. sham.<br />
b P � 0.05 vs. sham�acidosis. c P � 0.001 vs. sham�acidosis. d P � 0.001 vs.<br />
ADX�acidosis. e P � 0.01 vs. ADX�acidosis (ANOVA).<br />
AJP-<strong>Renal</strong> Physiol • VOL 292 • JANUARY 2007 • www.ajp<strong>renal</strong>.org<br />
Fig. 1. Effect of ad<strong>renal</strong>ectomy, acidosis, and dexamethasone on plasma<br />
glutam<strong>in</strong>e concentration. Plasma glutam<strong>in</strong>e concentration was measured <strong>in</strong><br />
arterial (aorta) and <strong>renal</strong> venous blood as described <strong>in</strong> MATERIALS AND METH-<br />
ODS. ADX, ad<strong>renal</strong>ectomized; Dex, dexamethasone treatment. The experimental<br />
groups and numbers of animals are the same <strong>in</strong> Figs. 1–3. Values are<br />
means � SE. a P � 0.05 vs. sham. b P � 0.01 vs. control. c P � 0.01 vs.<br />
ADX�acidosis�Dex. d P � 0.001 vs. sham�acidosis.<br />
(Fig. 1, A and B) and dexamethasone <strong>in</strong>creases (Fig. 1, A and<br />
C) plasma glutam<strong>in</strong>e concentration. The decl<strong>in</strong>e <strong>in</strong> plasma<br />
glutam<strong>in</strong>e dur<strong>in</strong>g acidosis is pr<strong>in</strong>cipally the result of greatly<br />
enhanced <strong>renal</strong> glutam<strong>in</strong>e utilization for ammoniagenesis.<br />
Plasma glutam<strong>in</strong>e <strong>in</strong>creases <strong>in</strong> animals treated with dexamethasone,<br />
reflect<strong>in</strong>g <strong>in</strong>creased glutam<strong>in</strong>e synthetase (GS) activity<br />
<strong>in</strong> muscle and lung (1, 27) and <strong>in</strong>creased proteolysis, pr<strong>in</strong>cipally<br />
<strong>in</strong> muscle (17). Treatment of acidotic rats with the<br />
glucocorticoid receptor antagonist RU486 did not alter systemic<br />
glutam<strong>in</strong>e concentration (Fig. 1B).<br />
Ad<strong>renal</strong>ectomy attenuates the acidosis-<strong>in</strong>duced <strong>in</strong>crease <strong>in</strong><br />
<strong>renal</strong> <strong>cortical</strong> SNAT3 mRNA and decreases <strong>renal</strong> glutam<strong>in</strong>e AV<br />
difference. Northern blot analysis was used to measure the<br />
relative levels of <strong>renal</strong> <strong>cortical</strong> SNAT3 mRNA <strong>in</strong> the same five<br />
groups as above and <strong>in</strong> ADX rats treated with dexamethasone<br />
(Fig. 2A). Metabolic acidosis significantly <strong>in</strong>creased SNAT3<br />
mRNA <strong>in</strong> both sham and ADX rats compared with sham and<br />
ADX rats not exposed to acidosis (P � 0.001 for both).<br />
However, ad<strong>renal</strong>ectomy blunted the acidosis-<strong>in</strong>duced <strong>in</strong>crease<br />
<strong>in</strong> SNAT3 mRNA (sham�acidosis 20.6 � 4.1 vs.<br />
ADX�acidosis 11.9 � 1.4 arbitrary units, P � 0.05) and<br />
dexamethasone partially reversed the effect of ad<strong>renal</strong>ectomy<br />
(15.9 � 3.4 arbitrary units). Glutam<strong>in</strong>e AV difference <strong>in</strong> these<br />
groups followed a similar pattern of response (Fig. 1B). Metabolic<br />
acidosis significantly <strong>in</strong>creased glutam<strong>in</strong>e AV difference<br />
<strong>in</strong> both sham and ADX rats compared with nonacidotic sham<br />
and ADX rats (sham P � 0.001; ADX P � 0.01). In ADX rats,<br />
acidosis-<strong>in</strong>duced glutam<strong>in</strong>e AV difference is attenuated compared<br />
with ad<strong>renal</strong>-<strong>in</strong>tact animals (sham�acidosis 0.16 � 0.03<br />
mmol/l vs. ADX�acidosis 0.08 � 0.01 mmol/l, P � 0.05).<br />
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Fig. 2. Effect of ad<strong>renal</strong>ectomy on <strong>renal</strong> <strong>cortical</strong> System N glutam<strong>in</strong>e transporter<br />
(SNAT3) mRNA expression (A) and <strong>renal</strong> glutam<strong>in</strong>e arteriovenous<br />
(AV) difference (B) <strong>in</strong> rats. Sham and ADX rats were treated as <strong>in</strong>dicated.<br />
Metabolic acidosis was <strong>in</strong>duced by addition of NH4Cl to the dr<strong>in</strong>k<strong>in</strong>g water for<br />
2 days. Dexamethasone treatment (1 mg�kg �1 �day �1 sc) was started 2 days<br />
before acidosis and cont<strong>in</strong>ued dur<strong>in</strong>g acidosis. A: Northern blots were hybridized<br />
as described <strong>in</strong> MATERIALS AND METHODS and relative SNAT3 mRNA,<br />
normalized to 18S ribosomal RNA, determ<strong>in</strong>ed us<strong>in</strong>g laser densitometry; n �<br />
11–23 rats/group. B: glutam<strong>in</strong>e AV difference was calculated as (arterial �<br />
<strong>renal</strong> venous) plasma glutam<strong>in</strong>e concentration; n � 11–20 rats/group. Values<br />
are means � SE. a P � 0.05 vs. sham�acidosis. b P � 0.001 vs. sham. c P �<br />
0.001 vs. ADX. d P � 0.01 vs. ADX (ANOVA with Bonferroni posttest).<br />
Dexamethasone partially reversed the repressive effect of ad<strong>renal</strong>ectomy<br />
(0.11 � 0.02 mmol/l). There was a trend for<br />
dexamethasone to <strong>in</strong>crease SNAT3 and glutam<strong>in</strong>e AV difference<br />
<strong>in</strong> ADX rats, although the <strong>in</strong>crease did not reach statistical<br />
significance. We reported that, <strong>in</strong> normal rats, <strong>in</strong>creased <strong>renal</strong><br />
glutam<strong>in</strong>e uptake dur<strong>in</strong>g chronic metabolic acidosis rats is<br />
associated with <strong>in</strong>creased expression of the System N transporter<br />
SNAT3 <strong>in</strong> <strong>renal</strong> proximal tubules (23). Figure 2 suggests<br />
that this association also occurs <strong>in</strong> ADX rats. We <strong>have</strong> used<br />
<strong>renal</strong> AV difference (arterial � <strong>renal</strong> venous glutam<strong>in</strong>e plasma<br />
concentration) as a broad <strong>in</strong>dicator of glutam<strong>in</strong>e uptake, while<br />
recogniz<strong>in</strong>g that this approach has limitations (see DISCUSSION).<br />
Inhibition of glucocorticoid activity depresses the acidosis<strong>in</strong>duced<br />
<strong>in</strong>crease <strong>in</strong> <strong>cortical</strong> SNAT3 expression and <strong>renal</strong><br />
glutam<strong>in</strong>e AV difference. The glucocorticoid antagonist RU486<br />
was used to further explore a <strong>role</strong> for glucocorticoids <strong>in</strong><br />
regulat<strong>in</strong>g <strong>renal</strong> <strong>cortical</strong> SNAT3 expression and <strong>renal</strong> glutam<strong>in</strong>e<br />
uptake. Rats were exposed to metabolic acidosis for 6<br />
days with or without adm<strong>in</strong>istration of RU486 (12.5 mg/kg sc,<br />
twice/day). Control rats were <strong>in</strong>jected with vehicle (ethanol).<br />
BLMV isolated from control, acidotic, and RU486-treated<br />
GLUCOCORTICOIDS AND RENAL SNAT3 EXPRESSION<br />
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F451<br />
acidotic rats were used to measure SNAT3-mediated glutam<strong>in</strong>e<br />
transport and for Western blot analysis. Acidosis <strong>in</strong>creased<br />
SNAT3 expression and glutam<strong>in</strong>e transport <strong>in</strong> BLMV (Fig. 3,<br />
A–C). RU486 treatment significantly attenuated acidosis<strong>in</strong>duced<br />
<strong>renal</strong> <strong>cortical</strong> SNAT3 mRNA (acidosis 1.0 � 0.08<br />
vs. acidosis�RU486, 0.5 � 0.1 arbitrary units, P � 0.05)<br />
(Fig. 3A), SNAT3 prote<strong>in</strong> (acidosis 4.05 � 0.75 vs.<br />
acidosis�RU486, 1.88 � 0.19 arbitrary units, P � 0.05)<br />
(Fig. 3B), and SNAT3-dependent transport <strong>in</strong> BLMV (acidosis<br />
409 � 20 pmol�mg �1 �m<strong>in</strong> �1 vs. acidosis�RU486,<br />
214 � 74 pmol�mg �1 �m<strong>in</strong> �1 , P � 0.05, repeated measures<br />
ANOVA) (Fig. 3C). <strong>Renal</strong> glutam<strong>in</strong>e AV difference was<br />
blunted but not significantly decreased by RU486 (acidosis<br />
0.24 � 0.02 mmol/l vs. acidosis�RU486, 0.18 � 0.03<br />
mmol/l) (Fig. 3D). Treatment of normal rats with RU486 did<br />
not affect SNAT3 mRNA or glutam<strong>in</strong>e AV difference (not<br />
shown).<br />
Chronic glucocorticoid treatment <strong>in</strong>creases <strong>renal</strong> <strong>cortical</strong><br />
SNAT3 expression. To obta<strong>in</strong> more direct evidence of a <strong>role</strong> for<br />
glucocorticoids <strong>in</strong> SNAT3 expression <strong>in</strong> the <strong>renal</strong> cortex, we<br />
treated normal rats with dexamethasone, acutely (5 mg/kg sc<br />
for 1–6 h) or chronically (100 �g/day sc for 1–6 days). Acute<br />
treatment with dexamethasone did not <strong>in</strong>crease <strong>renal</strong> <strong>cortical</strong><br />
SNAT3 mRNA. In chronically treated animals, SNAT3 mRNA<br />
<strong>in</strong>creased slowly to approximately twofold at day 3 (P � 0.05)<br />
and rema<strong>in</strong>ed at that level at day 6 (P � 0.001) (Fig. 4A).<br />
SNAT3 prote<strong>in</strong> and SNAT3-mediated glutam<strong>in</strong>e transport exhibited<br />
a small but not statistically significantly <strong>in</strong>crease (Fig.<br />
4, B and C). Glutam<strong>in</strong>e AV difference was modestly <strong>in</strong>creased<br />
at day 6 (�0.02 � 0.01 mmol/l control vs. 0.02 � 0.01 mmol/l<br />
day 6, P � 0.03, t-test). Dexamethasone stimulation of <strong>renal</strong><br />
GS activity <strong>in</strong> the dexamethasone-treated rats would tend to<br />
decrease apparent AV difference (see DISCUSSION), thus m<strong>in</strong>imiz<strong>in</strong>g<br />
the apparent <strong>in</strong>crease <strong>in</strong> glutam<strong>in</strong>e uptake.<br />
Dexamethasone <strong>in</strong>creases SNAT3 mRNA and SNAT3-mediated<br />
glutam<strong>in</strong>e transport but does not activate the SNAT3<br />
promoter <strong>in</strong> <strong>renal</strong> epithelial cells. SNAT3 mRNA and<br />
SNAT3-mediated basolateral glutam<strong>in</strong>e transport (control<br />
214 � 88 nmol�mg �1 �m<strong>in</strong> �1 , dexamethasone, 558 � 96<br />
nmol�mg �1 �m<strong>in</strong> �1 ) are <strong>in</strong>creased 50 (P � 0.01) and 250%<br />
(P � 0.01), respectively, by 0.01 �M dexamethasone treatment<br />
of glucocorticoid-responsive GR101 cells <strong>in</strong> culture<br />
(Fig. 5A). GR101 cells are porc<strong>in</strong>e epithelial-like LLC-PK1<br />
cells that express rat glucocorticoid receptors (41). To determ<strong>in</strong>e<br />
whether glucocorticoids <strong>in</strong>creased SNAT3 expression via<br />
<strong>in</strong>creased transcription of the SNAT3 gene, we cloned and<br />
sequenced 2.4 kb of the 5�-flank<strong>in</strong>g region of the rat SNAT3<br />
gene from a rat genomic library. Analysis of the 5�-flank<strong>in</strong>g<br />
sequence us<strong>in</strong>g the TFSearch program identified a number of<br />
potential glucocorticoid response elements. The promoter conta<strong>in</strong>s<br />
no TATA or CCAAT box sequences, but a GC-rich<br />
region conta<strong>in</strong><strong>in</strong>g three potential SP1 sites is present, �100<br />
bases upstream from the 5�-end of exon I. A series of pCAT<br />
reporter vectors conta<strong>in</strong><strong>in</strong>g up to 2.4 kb of SNAT3 5�-flank<strong>in</strong>g<br />
sequence was used to transiently transfect GR101 cells. The<br />
transfected cells were <strong>in</strong>cubated <strong>in</strong> the presence or absence of<br />
0.01 or 0.1 �M dexamethasone for 48 h. Data from 0.01 and<br />
0.1 �M dexamethasone were comb<strong>in</strong>ed because there was no<br />
difference between them. The promoterless expression vector<br />
pCAT3-Basic showed negligible CAT activity, whereas the<br />
pCAT.SNAT3 vector conta<strong>in</strong><strong>in</strong>g the longest promoter segment<br />
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F452 GLUCOCORTICOIDS AND RENAL SNAT3 EXPRESSION<br />
Fig. 3. Effect of glucocorticoid <strong>in</strong>hibition on<br />
<strong>renal</strong> <strong>cortical</strong> SNAT3 mRNA (A), SNAT3<br />
basolateral membrane vesicle (BLMV) prote<strong>in</strong><br />
(B), SNAT3-mediated glutam<strong>in</strong>e transport<br />
(C), and <strong>renal</strong> glutam<strong>in</strong>e AV difference<br />
(D) <strong>in</strong> rats. Rats were exposed to NH4Cl<br />
acidosis for 6 days <strong>in</strong> the presence or absence<br />
of the glucocorticoid receptor antagonist<br />
RU486 (12.5 mg/kg twice a day). A: relative<br />
SNAT3 mRNA was determ<strong>in</strong>ed us<strong>in</strong>g Northern<br />
blot analysis; n � 8–12 rats/group. B:<br />
relative SNAT3 prote<strong>in</strong> was measured by<br />
Western blot analysis <strong>in</strong> isolated <strong>renal</strong> <strong>cortical</strong><br />
BLMV; n � 8 vesicle preparations/group.<br />
C: SNAT3-mediated glutam<strong>in</strong>e transport was<br />
measured <strong>in</strong> the same vesicles as <strong>in</strong> B. D:<br />
glutam<strong>in</strong>e AV difference was calculated as<br />
(arterial � <strong>renal</strong> venous) glutam<strong>in</strong>e concentration;<br />
n � 8–20 rats/group. Values are<br />
means � SE. Detailed procedures are described<br />
<strong>in</strong> MATERIALS AND METHODS. a P �<br />
0.001 vs. control. b P � 0.05 vs. acidosis<br />
(ANOVA).<br />
tested (�2.4 kb) exhibited a 15-fold <strong>in</strong>crease <strong>in</strong> basal activity,<br />
<strong>in</strong>dicat<strong>in</strong>g that this 5�-flank<strong>in</strong>g region of the SNAT3 promoter<br />
conta<strong>in</strong>s a functional promoter. The active promoter constructs<br />
did not, however, respond to dexamethasone treatment (Fig.<br />
5B). The pCAT.SNAT3 vector conta<strong>in</strong><strong>in</strong>g only the proximal 78<br />
bases of the flank<strong>in</strong>g region was transcriptionally <strong>in</strong>active,<br />
presumably because three Sp1 b<strong>in</strong>d<strong>in</strong>g sites between �101 and<br />
�78 are absent from this construct. Glucocorticoid responsive-<br />
Fig. 4. Effect of chronic dexamethasone treatment<br />
on <strong>renal</strong> <strong>cortical</strong> SNAT3 mRNA (A), SNAT3<br />
BLMV prote<strong>in</strong> (B), SNAT3-mediated glutam<strong>in</strong>e<br />
transport (C), and <strong>renal</strong> glutam<strong>in</strong>e AV difference<br />
(D) <strong>in</strong> rats. Rats were treated with dexamethasone<br />
(0.1 mg/day sc) for 1–6 days as <strong>in</strong>dicated. A: relative<br />
SNAT3 mRNA was determ<strong>in</strong>ed us<strong>in</strong>g Northern<br />
blot analysis; n � 9–11 rats/group. B: relative<br />
SNAT3 prote<strong>in</strong> was measured by Western blot analysis<br />
<strong>in</strong> isolated <strong>renal</strong> <strong>cortical</strong> BLMV; n � 5 vesicle<br />
preparations/group. C: SNAT3-mediated glutam<strong>in</strong>e<br />
transport was measured <strong>in</strong> the same vesicles as <strong>in</strong> B.<br />
D: glutam<strong>in</strong>e AV difference was calculated as (arterial<br />
� <strong>renal</strong> venous) plasma glutam<strong>in</strong>e concentration.<br />
Values are means � SE; n ��20 rats/group.<br />
Detailed procedures are described <strong>in</strong> MATERIALS AND<br />
METHODS. a P � 0.05 vs. control. b P � 0.001 vs.<br />
control [ANOVA (A) with Dunnett’s posttest; t-test<br />
(D)].<br />
AJP-<strong>Renal</strong> Physiol • VOL 292 • JANUARY 2007 • www.ajp<strong>renal</strong>.org<br />
ness of the GR101 cells was verified by transfection with the<br />
dexamethasone-responsive pGREtkCAT plasmid that conta<strong>in</strong>s<br />
two <strong>in</strong>verted glucocorticoid response elements followed by the<br />
thymid<strong>in</strong>e k<strong>in</strong>ase promoter. CAT activity of pGREtkCAT<br />
<strong>in</strong>creased approximately fivefold <strong>in</strong> response to 100 nM dexamethasone.<br />
The SNAT3 promoter constructs used <strong>in</strong> these<br />
studies (except the <strong>in</strong>active �78-bp construct) are activated by<br />
<strong>in</strong>cubation <strong>in</strong> acidic medium. CAT activity of the constructs<br />
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Fig. 5. Dexamethasone <strong>in</strong>creases SNAT3 expression and transport activity <strong>in</strong><br />
GR101 cells but does not activate the SNAT3 promoter <strong>in</strong> transiently transfected<br />
cells. A: GR101 cells were grown on regular culture dishes (RNA<br />
isolation) or Transwell plates (transport) with or without 0.01 �M dexamethasone<br />
for 48 h. SNAT3 mRNA was determ<strong>in</strong>ed from Northern blot analysis<br />
and SNAT3-mediated transport measured as described <strong>in</strong> MATERIALS AND<br />
METHODS. Values are means � SE from 4–6 separate experiments carried out<br />
<strong>in</strong> duplicate or triplicate. a P � 0.01. B: GR101 cells were transiently transfected<br />
with pCAT vectors carry<strong>in</strong>g SNAT3 promoter fragments of various<br />
lengths and with the glucocorticoid-responsive plasmid pGREtkCAT, as <strong>in</strong>dicated.<br />
Cells were <strong>in</strong>cubated with or without dexamethasone (0.1 or 0.01 �M)<br />
for 48 h. Data were normalized for transfection efficiency by cotransfection<br />
with pSV-�-Gal. Normalized chloramphenicol acetyl transferase (CAT) activity<br />
is expressed relative to the control �2,414 SNAT3 promoter construct<br />
(100%). Transfection and CAT and �-galactosidase activity assays were<br />
carried out as described <strong>in</strong> MATERIALS AND METHODS. Values are means � SE<br />
of 6 separate experiments with triplicate or duplicate determ<strong>in</strong>ations.<br />
<strong>in</strong>creases approximately twofold <strong>in</strong> GR101 and LLC-PK1 cells<br />
<strong>in</strong> response to acidosis (not shown).<br />
DISCUSSION<br />
The plasma concentration of glucocorticoids is <strong>in</strong>creased <strong>in</strong><br />
response to metabolic acidosis (28, 42), result<strong>in</strong>g <strong>in</strong> activation<br />
or suppression of a variety of activities or pathways that act <strong>in</strong><br />
concert to ma<strong>in</strong>ta<strong>in</strong> blood pH with<strong>in</strong> strict limits. Early studies<br />
us<strong>in</strong>g ADX dogs and rats identified the kidneys as an important<br />
site of glucocorticoid action dur<strong>in</strong>g metabolic acidosis. Of<br />
particular <strong>in</strong>terest to us was the defect <strong>in</strong> <strong>renal</strong> glutam<strong>in</strong>e<br />
uptake and <strong>in</strong>duction of ammoniagenesis exhibited by ADX<br />
animals (11, 18, 33, 46). Dur<strong>in</strong>g acidosis, SNAT3 transporter<br />
expression and SNAT3-mediated glutam<strong>in</strong>e transport activity<br />
are <strong>in</strong>duced <strong>in</strong> the proximal tubules (23, 37), the site of<br />
<strong>in</strong>creased glutam<strong>in</strong>e metabolism and ammoniagenesis (16, 35).<br />
We hypothesized that glucocorticoids regulate acid-base homeostasis<br />
dur<strong>in</strong>g acidosis, <strong>in</strong> part, by <strong>in</strong>creas<strong>in</strong>g glutam<strong>in</strong>e<br />
availability for ammoniagenesis through upregulation of<br />
SNAT3 transporter expression.<br />
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In our studies us<strong>in</strong>g sham-operated and ADX rats, <strong>renal</strong><br />
<strong>cortical</strong> SNAT3 mRNA was <strong>in</strong>creased by metabolic acidosis <strong>in</strong><br />
sham animals. This response was attenuated <strong>in</strong> ADX animals,<br />
and treatment of ADX animals with dexamethasone partially<br />
reversed the effect of ad<strong>renal</strong>ectomy (Fig. 1A), suggest<strong>in</strong>g that<br />
glucocorticoids <strong>have</strong> a <strong>role</strong> <strong>in</strong> acidosis-<strong>in</strong>duced activation of<br />
SNAT3 expression. The observation that the glucocorticoid<br />
receptor antagonist RU486 also blunts the acidosis-<strong>in</strong>duced<br />
<strong>in</strong>crease <strong>in</strong> SNAT3 expression (Fig. 2) provides additional<br />
evidence of glucocorticoid <strong>in</strong>volvement. Dexamethasone also<br />
<strong>in</strong>creased SNAT3 expression <strong>in</strong> the absence of acidosis, although<br />
the <strong>in</strong>crease was much smaller than that <strong>in</strong>duced by<br />
acidosis. In all the <strong>in</strong> vivo studies reported here, <strong>renal</strong> glutam<strong>in</strong>e<br />
AV difference mirrored SNAT3 expression, re<strong>in</strong>forc<strong>in</strong>g<br />
the suggestion that glutam<strong>in</strong>e uptake and expression of SNAT3<br />
<strong>in</strong> the <strong>renal</strong> cortex are l<strong>in</strong>ked. These results are consistent with<br />
previous observations that glucocorticoids play a <strong>role</strong> <strong>in</strong> acidbase<br />
homeostasis <strong>in</strong> part via regulation of <strong>renal</strong> glutam<strong>in</strong>e<br />
utilization (33, 46).<br />
We <strong>have</strong> used <strong>renal</strong> AV difference as a broad <strong>in</strong>dicator of<br />
glutam<strong>in</strong>e uptake. AV difference is a net measurement that is<br />
a function of arterial glutam<strong>in</strong>e concentration, <strong>renal</strong> GS activity,<br />
and <strong>renal</strong> blood flow. 1) Figure 1 shows the arterial<br />
glutam<strong>in</strong>e concentration <strong>in</strong> all experimental groups. 2) GS is<br />
present <strong>in</strong> the outer medulla of the kidney (35). GS activity,<br />
mRNA, and prote<strong>in</strong> <strong>in</strong> the kidney are unchanged (8, 35, 47) by<br />
chronic acidosis so that the impact of <strong>renal</strong> glutam<strong>in</strong>e synthesis<br />
on AV difference <strong>in</strong> acidotic rats is negligible. <strong>Glucocorticoids</strong><br />
<strong>in</strong>crease GS activity and gene expression <strong>in</strong> muscle and lung<br />
(1). In the kidney, glucocorticoids <strong>have</strong> no effect (1) or modestly<br />
<strong>in</strong>crease (26, 43) GS activity. The effect of <strong>in</strong>creased GS<br />
activity would be to <strong>in</strong>crease the <strong>renal</strong> venous concentration of<br />
glutam<strong>in</strong>e, thus decreas<strong>in</strong>g the apparent glutam<strong>in</strong>e uptake <strong>in</strong><br />
those groups treated with dexamethasone. 3) <strong>Renal</strong> blood flow<br />
or glomerular filtration rate either is not altered (15, 24, 32) or<br />
is <strong>in</strong>creased (45, 46) by metabolic acidosis. Welbourne and<br />
colleagues (45, 46) reported that <strong>renal</strong> plasma flow <strong>in</strong> ADX and<br />
acidotic ADX rats decreased compared with normal animals<br />
and <strong>in</strong>creased <strong>in</strong> acidotic rats and <strong>in</strong> ADX rats supplemented<br />
with glucocorticoid. They studied experimental groups similar<br />
to ours and observed a pattern of <strong>renal</strong> glutam<strong>in</strong>e AV differences<br />
<strong>in</strong> these groups the same as that shown <strong>in</strong> Fig. 2, except<br />
for their ADX�acidosis�glucocorticoid group, where the AV<br />
difference recovered to or above the sham acidosis level,<br />
whereas we observe only partial recovery. This difference may<br />
result from the use of different glucocorticoids and doses <strong>in</strong> our<br />
studies. In those studies, the pattern of glutam<strong>in</strong>e extraction<br />
(<strong>renal</strong> plasma flow � AV difference) and AV difference for<br />
each group was the same, suggest<strong>in</strong>g that, <strong>in</strong> these experimental<br />
conditions, AV difference broadly reflects glutam<strong>in</strong>e extraction.<br />
ADX animals are deficient <strong>in</strong> both glucocorticoids and<br />
m<strong>in</strong>eralocorticoids. Both glucocorticoids and m<strong>in</strong>eralocorticoids<br />
act <strong>in</strong> the kidney to <strong>in</strong>crease elim<strong>in</strong>ation of protons <strong>in</strong> the<br />
ur<strong>in</strong>e of acidotic animals, but their sites and mechanisms of<br />
action differ. <strong>Glucocorticoids</strong> <strong>in</strong>crease acid excretion <strong>in</strong> the<br />
form of ammonia excretion <strong>in</strong> response to an acid load with<br />
little effect on ur<strong>in</strong>e acidification, whereas m<strong>in</strong>eralocorticoids<br />
<strong>in</strong>crease acid secretion and acidify the ur<strong>in</strong>e with a limited<br />
effect on ammoniagenesis (20, 48). Glucocorticoid receptors<br />
are enriched <strong>in</strong> the proximal tubule, whereas m<strong>in</strong>eralocorticoid<br />
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F454 GLUCOCORTICOIDS AND RENAL SNAT3 EXPRESSION<br />
receptors are enriched <strong>in</strong> more distal regions (12, 29, 34). The<br />
differential distribution of these receptors is consistent with the<br />
restriction of ammoniagenesis <strong>in</strong>duction to the proximal tubules.<br />
In addition, other acidosis-regulated transporters demonstrate<br />
a <strong>role</strong> for glucocorticoids, but not m<strong>in</strong>eralocorticoids,<br />
<strong>in</strong> the response to acidosis (2, 5, 13). For these reasons, we<br />
<strong>have</strong> focused on glucocorticoid regulation of SNAT3 expression<br />
<strong>in</strong> the studies reported here. However, a <strong>role</strong> for m<strong>in</strong>eralocorticoids<br />
is not excluded.<br />
The studies presented here provide some <strong>in</strong>sight <strong>in</strong>to the<br />
mechanism(s) by which glucocorticoids regulate SNAT3 expression.<br />
The effects of H � and glucocorticoids cannot be<br />
separated <strong>in</strong> normal rats <strong>in</strong> vivo because the plasma concentration<br />
of glucocorticoids <strong>in</strong>creases <strong>in</strong> response to acidosis (28,<br />
42). Experiments us<strong>in</strong>g ADX rats provide <strong>in</strong>sight <strong>in</strong>to the<br />
relative <strong>role</strong>s of glucocorticoids and H � <strong>in</strong> regulat<strong>in</strong>g <strong>cortical</strong><br />
SNAT3 expression. A comparison of Figs. 2A, 3A, and 4A<br />
shows that expression of SNAT3 mRNA is more strongly<br />
<strong>in</strong>duced by H � than by dexamethasone <strong>in</strong> both ADX and<br />
ad<strong>renal</strong>-<strong>in</strong>tact rats. Figure 2A demonstrates that the <strong>role</strong> of<br />
glucocorticoids <strong>in</strong> regulat<strong>in</strong>g SNAT3 expression is not permissive<br />
because SNAT3 mRNA is <strong>in</strong>duced by acidosis <strong>in</strong> ADX<br />
rats that lack endogenous glucocorticoids. The data <strong>in</strong> Fig. 2A<br />
also suggest that, for SNAT3 mRNA, the <strong>in</strong>ductive effects of<br />
H � and glucocorticoids may be additive. There is no evidence<br />
of synergistic <strong>in</strong>teraction between H � and glucocorticoids <strong>in</strong><br />
the <strong>in</strong>duction of SNAT3 expression.<br />
The ability of RU486 to partially block the stimulation of<br />
SNAT3 expression by acidosis suggests the <strong>in</strong>volvement of the<br />
glucocorticoid receptor <strong>in</strong> the regulation of SNAT3 gene transcription.<br />
Dexamethasone treatment <strong>in</strong>creased SNAT3 mRNA<br />
<strong>in</strong> control, ADX, and acidotic ADX rats (Figs. 2A and 4A),<br />
although to a lesser degree than chronic acidosis. The 5�flank<strong>in</strong>g<br />
region of the SNAT3 gene conta<strong>in</strong>s several potential<br />
glucocorticoid response elements that could be <strong>in</strong>volved <strong>in</strong><br />
glucocorticoid activation of SNAT3 transcription. However,<br />
transcriptionally active reporter expression vectors conta<strong>in</strong><strong>in</strong>g<br />
up to 2.4 kb of the SNAT3 5�-flank<strong>in</strong>g sequence did not<br />
respond to dexamethasone treatment when transiently transfected<br />
<strong>in</strong>to glucocorticoid-responsive GR101 cells (Fig. 5B).<br />
The lack of response suggests that glucocorticoids do not<br />
regulate SNAT3 directly by b<strong>in</strong>d<strong>in</strong>g of the activated glucocorticoid<br />
receptor to a response element with<strong>in</strong> the promoter, but<br />
<strong>in</strong>directly perhaps by <strong>in</strong>teraction with or <strong>in</strong>duction of an unknown<br />
factor or by message stabilization. It is also possible<br />
that an essential glucocorticoid-regulatory element lies outside<br />
the cloned 2.4 kb of promoter used for transfection.<br />
In addition to SNAT3, expression of a number of enzymes<br />
and transporters important <strong>in</strong> the response to acid-base imbalance<br />
is regulated <strong>in</strong> the kidney by both H � and glucocorticoids<br />
(3, 4, 21, 31). In some cases, for example, the gluconeogenic<br />
enzyme phosphoenolpyruvate carboxyk<strong>in</strong>ase and the Na � /H �<br />
antiporter NHE3, the mechanism of glucocorticoid action has<br />
been elucidated <strong>in</strong> considerable detail and has proven to be<br />
very complex. Both acidosis and glucocorticoids regulate<br />
phosphoenolpyruvate carboxyk<strong>in</strong>ase transcription through<br />
complex <strong>in</strong>teractions of nuclear factors with two <strong>in</strong>dependent<br />
doma<strong>in</strong>s <strong>in</strong> the promoter (9). Regulation of NHE3 <strong>in</strong>volves<br />
both transcription and transcription-<strong>in</strong>dependent mechanisms<br />
(traffick<strong>in</strong>g, phosphorylation) that differ <strong>in</strong> acute and chronic<br />
acidosis (6, 22, 40). Regulatory mechanisms for other acidosis-<br />
and glucocorticoid-regulated transporters <strong>have</strong> yet to be elucidated.<br />
The studies reported here demonstrate that glucocorticoids,<br />
<strong>in</strong> addition to H � , directly or <strong>in</strong>directly regulate expression of<br />
the SNAT3 transporter dur<strong>in</strong>g chronic metabolic acidosis. In<br />
addition, the close association between SNAT3 expression and<br />
<strong>renal</strong> glutam<strong>in</strong>e uptake as reflected by AV difference under a<br />
number of experimental conditions makes a strong argument<br />
for a central <strong>role</strong> for SNAT3 <strong>in</strong> the adaptive ammoniagenic<br />
response to acidosis. When rats are chronically exposed to<br />
metabolic acidosis, a series of compensatory adaptations occur<br />
<strong>in</strong> the kidney so that blood pH and bicarbonate concentration<br />
are restored to normal values with<strong>in</strong> 7 days despite cont<strong>in</strong>ued<br />
acid <strong>in</strong>gestion (30). Ammoniagenesis, however, rema<strong>in</strong>s elevated<br />
despite the normalization of acid-base status, suggest<strong>in</strong>g<br />
that factors other than H � are responsible for ma<strong>in</strong>tenance of<br />
activation of adaptive processes, <strong>in</strong>clud<strong>in</strong>g SNAT3 upregulation<br />
and <strong>renal</strong> glutam<strong>in</strong>e uptake. Blood glucocorticoids, <strong>in</strong><br />
contrast, rema<strong>in</strong> elevated dur<strong>in</strong>g acidosis (28, 42). Because of<br />
their widespread actions, glucocorticoids may be important <strong>in</strong><br />
orchestrat<strong>in</strong>g the systemic and <strong>renal</strong> response to metabolic<br />
acidosis and <strong>in</strong> ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g activation of key homeostatic<br />
activities.<br />
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