Glucocorticoids have a role in renal cortical ... - Renal Physiology

Glucocorticoids have a role in renal cortical expression of 

the SNAT3 glutamine transporter during chronic 

metabolic acidosis 

Anne M. Karinch, Cheng-Mao Lin, QingHe Meng, Ming Pan and Wiley W. Souba 

Am J Physiol Renal Physiol 292:F448-F455, 2007. First published 5 September 2006; 

doi: 10.1152/ajprenal.00168.2006 

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Am J Physiol Renal Physiol 292: F448–F455, 2007. 

First published September 5, 2006; doi:10.1152/ajprenal.00168.2006. 

Glucocorticoids have a role in renal cortical expression of the SNAT3 

glutamine transporter during chronic metabolic acidosis 

Anne M. Karinch, Cheng-Mao Lin, QingHe Meng, Ming Pan, and Wiley W. Souba 

Department of Surgery, Milton S. Hershey Medical Center, The Pennsylvania 

State University College of Medicine, Hershey, Pennsylvania 

Submitted 15 May 2006; accepted in final form 27 August 2006 

Karinch AM, Lin C-M, Meng QH, Pan M, Souba WW. Glucocorticoids 

have a role in renal cortical expression of the SNAT3 

glutamine transporter during chronic metabolic acidosis. Am J Physiol 

Renal Physiol 292: F448–F455, 2007. First published September 5, 

2006; doi:10.1152/ajprenal.00168.2006.—Glucocorticoids are involved 

in many aspects of regulation of acid-base homeostasis, 

including the stimulation of renal ammoniagenesis during chronic 

metabolic acidosis. Plasma glutamine is the principal substrate for 

ammoniagenesis under these conditions. Expression of the System N 

glutamine transporter SNAT3 is increased in the renal proximal 

tubules during acidosis. In vivo studies in rats using 1) sham and 

adrenalectomized rats, 2) the glucocorticoid receptor antagonist 

RU486, and 3) dexamethasone treatment demonstrated involvement 

of glucocorticoids in regulation of SNAT3 expression. Adrenalectomy 

attenuated the acidosis-induced increase in renal cortical SNAT3 mRNA 

�40%, and treatment with dexamethasone (1 mg�kg�1�day�1 sc) partially 

reversed this effect. RU486 also blunted the acidosis-induced 

increase in SNAT3 expression �50%. Chronic dexamethasone treatment 

(0.1 mg�kg�1�day�1 sc, 6 days) of normal rats slightly increased SNAT3 

expression. In all cases, renal glutamine arteriovenous difference mirrored 

SNAT3 expression and activity in the proximal tubules, suggesting 

that SNAT3 regulates glutamine uptake during acidosis. These studies 

indicate that glucocorticoids regulate acid-base homeostasis during metabolic 

acidosis in part by regulating expression of the System N transporter 

SNAT3. 

dexamethasone; System N expression; ammonium chloride acidosis; 

RU486 

THE KIDNEY PLAYS A VITAL ROLE in maintaining acid-base homeostasis 

during chronic metabolic acidosis. Under acidotic 

conditions, a number of adaptive changes occur throughout the 

kidney, acting in concert to reduce the acid load and restore 

acid-base balance. The renal proximal convoluted tubule is the 

site of remarkable coordinated changes that result in increased 

ammoniagenesis that is sustained for the duration of acidosis 

(16, 30). These changes include enhanced uptake and metabolism 

of glutamine, the principal substrate for renal ammoniagenesis 

during acidosis (39). Asymmetric secretion from the 

proximal tubule of ammonium ions into the urine and bicarbonate 

ions into the blood functions to return acid-base balance 

toward normal. Ammonium and bicarbonate ions are both 

products of glutamine metabolism, a single molecule of glutamine 

producing two ammonium and two bicarbonate ions. 

Under normal conditions, the kidney releases low levels of 

glutamine into the blood whereas during acidosis �40% of the 

glutamine presented to the kidney in the blood is extracted (36, 

Address for reprint requests and other correspondence: W. W. Souba, 

College of Medicine, Ohio State University, 254 Meiling Hall, 370 West 9th 

Ave., Columbus, OH 43210 (e-mail: chip.souba@osumc.edu). 

F448 

38), so that the kidney becomes the body’s principal consumer 

of glutamine. 

Studies using adrenalectomized (ADX) rats suggest that 

glucocorticoids are involved in acid-base homeostasis during 

metabolic acidosis. This observation is consistent with elevated 

plasma corticosterone concentration during acidosis in rats (28, 

42). ADX rats that are challenged with an acid load have a 

reduced ability to increase renal ammoniagenesis (33, 46). 

During metabolic acidosis, the kidneys of these animals cannot 

increase glutamine uptake from the blood or increase glutamine 

metabolism to the same degree as adrenal-intact rats 

(46). Glucocorticoid treatment of ADX rats before induction of 

acidosis partially restores their ability to extract glutamine 

from the blood (46). Glucocorticoids also increase ammonia 

excretion in the intact animal (19) and ammonia production by 

perfused kidneys (44). These studies suggest that glucocorticoids 

play a direct or indirect role in regulation of acidosisinduced 

glutamine metabolism. 

We reported that the acidosis-induced increase in renal 

glutamine uptake is accomplished by increased expression of 

the System N transporter in the proximal convoluted tubule of 

acidotic rats (23). This observation was recently confirmed by 

others (37). The System N transporter, previously known as 

SN1, has been renamed SNAT3 in recognition of its identity as 

a member of the SCL38 gene family of sodium-coupled neutral 

amino acid transporters (25). SNAT3 is located in the basolateral 

membrane of proximal tubule epithelial cells (37) and 

transports one glutamine molecule and one sodium ion in 

exchange for one proton (7, 10). The transporter mediates 

glutamine flux in both directions, depending on substrate 

gradients (7) and is associated with an uncoupled proton 

conductance that can serve to minimize the proton flux produced 

by a transport-coupled proton antiport (7, 10). 

We hypothesized that glucocorticoids enhance ammoniagenesis 

during metabolic acidosis in part by regulation of 

expression of the SNAT3 transporter. We studied regulation of 

renal cortical SNAT3 expression by glucocorticoids during 

chronic metabolic acidosis in vivo using normal and ADX rats 

and the glucocorticoid receptor antagonist RU486. Data from 

our studies suggest that glucocorticoids maintain acid-base 

homeostasis, in part, by altering renal glutamine uptake via 

regulation of expression of the SNAT3 transporter. 

MATERIALS AND METHODS 

Animals. Animals were treated in accordance with regulations of 

the Institutional Animal Use and Care Committee. Male Sprague- 

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. 

0363-6127/07 $8.00 Copyright © 2007 the American Physiological Society http://www.ajprenal.org 




GLUCOCORTICOIDS AND RENAL SNAT3 EXPRESSION 

Dawley rats (Charles River, Wilmington, MA), weighing 250–300 g, 

were maintained in a 12:12-h light-dark cycle with unrestricted access 

to rat chow and water or water containing 1.5% NH4Cl. Metabolic 

acidosis was maintained for varying lengths of time, up to 6 days. 

ADX (bilateral) rats and sham-operated controls purchased from 

Charles River were used for some experiments. Both sham and ADX 

rats were maintained on 0.9% saline substituted for their drinking 

water before experimentation. We followed a modification of the 

protocol of Welbourne and colleagues (46) that adjusts the NH4Cl 

concentration because ADX rats drink approximately twice the volume 

that intact rats drink. ADX rats were given 0.6% NH4Cl in saline, 

and sham acidotic rats were given 1.2% NH4Cl in saline so that they 

consumed approximately equal acid loads. ADX rats were exposed to 

acidosis for only 2 days because of significant mortality in acidotic 

ADX rats after 3 days. For some experiments, normal rats were 

treated acutely (5 mg/kg sc, once) or chronically (100 �g/rat sc, once 

a day for up to 6 days) with dexamethasone. The body weight of 

control rats increased steadily during the experimental period (259 � 

4gatday 0, 295 � 4gatday 6), whereas the body weight of 

chronically treated rats decreased for the first 3 days and then 

stabilized (255 � 3atday 0, 240 � 4gatday 6). 

Rats were killed after anesthesia with ketamine/acepromazine/ 

xylazine (80:1:12 mg/kg body wt). The kidneys were removed, and 

the renal cortex was immediately dissected out and processed for 

RNA isolation or membrane preparation. At the time of death, blood 

was drawn from the descending aorta and the renal vein for measurement 

of blood gases, pH, and plasma glutamine concentration. 

Measurement of blood gases, pH, and glutamine concentration. 

Arterial blood pH and PCO2 were measured using an IL BG3 bloodgas 

machine (model 1420, Instrumentation Laboratory, Lexington, 

� 

MA). Blood HCO3 concentration was automatically calculated from 

the measured pH and PCO2. Plasma glutamine levels were measured in 

triplicate by a modified spectrophotometric assay using a colorimetric 

assay kit (Boeringer Mannheim, Mannheim, Germany) adapted to a 

96-well plate format with a microplate spectrophotometer. Glutamine 

arteriovenous (AV) difference was calculated as (arterial � renal 

vein) plasma glutamine concentration. 

Kidney dissection. Kidney dissection was carried out under approximately 

threefold magnification. The decapsulated kidney was first cut 

lengthwise into two symmetrical halves. For dissection of the cortex, 

a thin slice (1.5–2 mm thick) was first cut from the curved outer 

surface. Additional cortex was dissected using fine scissors by trimming 

away the outer 1.5–2 mm, using the visible arcuate arteries as a 

guide to the junction of the cortex and medulla. 

Northern blot analysis. Total RNA was isolated from the renal 

cortex using the Totally RNA system (Ambion, Austin, TX) or from 

cultured cells using an RNeasy Mini kit (Qiagen, Valencia, CA). RNA 

was separated on a 1% formaldehyde gel, transferred to nylon membrane 

(Genescreen, New England Nuclear), and hybridized with a 

SNAT3-specific oligonucleotide probe. The SNAT3 oligonucleotide 

sequence was 5�-GTGCAGAAGGCTTCAGCAGTGTCAGGTTGG- 

3�. The oligonucleotide was radioactively 3�-end labeled using terminal 

transferase and �-[ 32P]dATP. For quantification of SNAT3 

mRNA, autoradiographs were scanned using a laser densitometer 

(Dynamic Biosystems). Glyceraldehyde-3 phosphate dehydrogenase 

(GAPDH) and �-actin were both found to be unsuitable for RNA 

loading normalization because acidosis increased mRNA levels of 

both. The 18S ribosomal RNA subunit was therefore used for normalization. 

Isolation of cortical basolateral membrane vesicles. Basolateral 

membrane vesicles (BLMV) were isolated from renal cortex by 

Percoll density gradient centrifugation as previously described (23). 

Vesicles were resuspended in intravesicular buffer [100 mM KCl, 100 

mM mannitol, 12 mM Tris/HEPES, pH 7.5, 0.1 mM phenylmethylsulfonyl 

fluoride, 1 �g/ml pepstatin A, 2 ml/l protease cocktail stock 

(Sigma P2714, Sigma, St. Louis, MO) to a concentration of �1 

mg/ml]. Protein was measured using the Bradford assay with BSA as 

AJP-Renal Physiol • VOL 292 • JANUARY 2007 • www.ajprenal.org 

F449 

a standard. BLMV were prepared at the same time from all groups to 

be compared in a specific experiment to minimize variation among the 

preparations. Renal cortices from two to five animals were pooled for 

each BLMV preparation. Vesicle relative enrichment was estimated 

using ouabain-inhibitable ATPase activity in BLMV compared with 

the homogenate (14). The relative enrichment did not vary significantly 

among preparations for the RU486 experiments (control 16 � 

2-, acidosis 27 � 9-, acidosis�RU486 18 � 3-fold increase over 

homogenate; P � 0.3, ANOVA) or the chronic dexamethasone 

experiments (control 17 � 3-, dexamethasone 14 � 3-fold increase 

over homogenate; P � 0.5, t-test). Vesicles frozen in liquid nitrogen 

were used for glutamine transport assays. 

Glutamine transport. For transport studies in renal cortical BLMV, 

Na � -dependent glutamine uptake was measured at room temperature 

using a rapid mixing/filtration technique described previously (23). 

Basolateral glutamine transport was measured in cells grown to tight 

confluence on Transwell plates. Following treatment, transport was 

measured in choline chloride or NaCl uptake buffer (in mM: 137 NaCl 

or choline Cl, 10 HEPES/Tris, pH 7.4, 4.7 KCl, 1.2 MgSO4, 1.2 

KH2PO4, 2.5 CaCl2) containing L-[ 3 H]glutamine (4 �Ci/ml) and 50 

�M unlabeled L-glutamine. Uptake was terminated after 5 min by 

washing with ice-cold NaCl uptake buffer without [ 3 H]glutamine. Part 

of the cell lysate was counted in a TopCount scintillation spectrophotometer 

(Packard, Meriden, CT), and part was used for protein 

determination. The rate of glutamine transport was linear at 5 min and 

was expressed as nanomoles per milligram protein per minute. Where 

indicated, 5 mM histidine was added to the uptake buffer to allow 

identification of SNAT3-mediated transport in BLMV and GR101 

cells. 

Western blot analysis. Equal amounts of cortical BLMV (10 �g) 

were separated by SDS-PAGE on precast polyacrylamide gels (ISC 

BioExpress, Kaysville, UT) and transferred to polyvinylidene difluoride 

membranes (Millipore, Bedford, MA). Blots were incubated with 

a polyclonal antibody raised against a SNAT3-GST fusion protein 

containing the NH2-terminal 71 amino acids of SNAT3 followed by a 

horseradish peroxidase-conjugated goat anti-rabbit antibody (Rockland 

Immunochemicals, Gilbertsville, PA). SNAT3 protein was detected 

by enhanced chemiluminescence (Upstate, Lake Placid, NY) 

following the manufacturer’s instructions. 

Cloning of the rat SNAT3 promoter. A rat genomic library (Clontech, 

Mountain View, CA) was screened using a SNAT3-specific 

probe. One positive plaque was identified and isolated by limiting 

dilution. The purified phage contained an insert of �12 kb, all of 

which represent a genomic sequence from the SNAT3 gene, based on 

comparison with the published rat genomic sequence (GenBank 

NW_047801.1). A 3.7-kb XhoI restriction fragment of the rat genomic 

clone that contains �2.4 kb of the 5�-flanking sequence, exon I 

(untranslated) and �1.3 kb of intron 1 was subcloned into Bluescript 

and sequenced (Molecular Genetics Core Facility at the Pennsylvania 

State University College of Medicine). The Transcription Factor 

Search program TFSearch (Yutaka Akiyama: “TFSEARCH: Searching 

Transcription Factor Binding Sites”; http://www.rwcp.or.jp/ 

papia/) was used to identify potential transcription factor binding sites 

in the 5�-flanking region. A series of SNAT3 promoter fragments 

(5�-boundaries �2,414 �815, �132, �78 with a common 3� boundary 

at �20 with respect to the published rat SNAT3 cDNA sequence, 

GenBank AF273025) was amplified by PCR using the cloned phage 

DNA as a template and cloned into the multiple cloning site of the 

pCAT3-Basic reporter vector (Promega, Madison, WI). Reporter 

constructs were verified by sequencing by the Molecular Genetics 

Core Facility. 

Cell culture. LLC-PK1-GR101 (GR101) cells are porcine epitheliallike 

LLC-PK1 cells that express rat glucocorticoid receptors (41). 

GR101 were grown in low-glucose DMEM (Invitrogen, Carlsbad, 

CA) containing 800 ng/ml hygromycin B (Calbiochem, La Jolla, CA) 

to maintain glucocorticoid receptor expression. These cells were 

generously provided by Dr. S. R. Price of Emory University. 




F450 GLUCOCORTICOIDS AND RENAL SNAT3 EXPRESSION 

Transient transfection. GR101 cells were transiently transfected 

using Lipofectin (Invitrogen) following the manufacturer’s protocol. 

After cotransfection with pCAT.SNAT3 constructs (1 �g) and 

pSV40-�-Gal (1 �g, Promega), cells were treated with control or 

dexamethasone (0.01 or 0.1 �M) medium and harvested after 48 h. 

The dexamethasone-responsive plasmid pGREtkCAT was used as a 

positive control for glucocorticoid responsiveness. This plasmid contains 

two inverted repeats of a glucocorticoid response element sequence 

followed by the thymidine kinase promoter that drives expression 

of the chloramphenicol acetyl transferase (CAT) gene. 

pGREtkCAT was provided by Dr. S. S. Simons, Jr., of National 

Institute of Diabetes and Digestive and Kidney Diseases, National 

Institutes of Health. CAT and �-galactosidase assays were carried out 

as described in the Promega protocols. 

Statistics. Data were analyzed using a t-test, paired t-test, or 

one-way analysis of variance followed by Bonferroni or Dunnett 

posttests for multiple comparisons, as appropriate. 

RESULTS 

ADX rats are less able than sham-operated rats to maintain 

acid-base homeostasis during chronic metabolic acidosis. Five 

groups of rats were studied: 1) sham-operated, 2) ADX, 3) 

2-day acidotic sham-operated, 4) 2-day acidotic ADX, and 5) 

2-day acidotic ADX pretreated for 2 days and throughout 

acidosis with the synthetic glucocorticoid dexamethasone (1 

mg�kg �1 �day �1 sc). Table 1 shows the arterial (systemic) pH 

and bicarbonate concentration and renal glutamine AV difference 

in each group of rats. The effect of adrenalectomy on the 

acid-base status and renal glutamine AV difference of normal 

and acidotic rats was similar to that previously reported (11, 

30, 46). After 2 days of ingesting ammonium chloride, both 

sham and ADX rats became acidotic, but ADX rats were more 

severely acidotic than sham rats. Chronic acidosis also increased 

glutamine AV difference in both sham and ADX rats, 

but the increase was significantly attenuated in ADX rats. 

Dexamethasone treatment improved the ability of acidotic 

ADX rats to compensate for the acid load, as indicated by 

increased blood pH and serum bicarbonate concentration. In 

addition, glutamine AV difference in these rats reverted toward 

the level in acidotic sham rats. 

Plasma glutamine concentration is altered by adrenalectomy, 

metabolic acidosis, and dexamethasone. Figure 1 shows 

the arterial plasma glutamine concentration of all animal 

groups in the studies presented in Figs. 1-3. Acidosis decreases 

Table 1. Selected blood parameters from sham-operated 

and adrenalectomized rats 

Blood pH 

Blood 

Bicarbonate, 

mM 

Renal 

Glutamine AV 

Difference, mmol/l 

Sham 7.38�0.01 25.0�0.5 �0.026�0.009 

ADX 7.29�0.02 22.3�0.6 �0.030�0.014 

Sham�acidosis 7.22�0.02 a 16.9�0.4 a 0.173�0.020 a 

ADX�acidosis 7.17�0.02 a 14.9�0.5 ab 0.070�0.009 ac 

ADX�acidosis�Dex 7.30�0.02 d 20.4�0.8 d 0.138�0.015 e 

Values are means � SE; n � 12–27 animals/group. ADX, adrenalectomized. 

Animals were treated as described in MATERIALS AND METHODS. Dexamethasone 

(Dex) concentration was 1 mg�kg �1 �day �1 . pH and bicarbonate 

values are from arterial blood. Glutamine renal arteriovenous (AV) difference 

is (arterial � renal vein) plasma glutamine concentration. a P � 0.001 vs. sham. 

b P � 0.05 vs. sham�acidosis. c P � 0.001 vs. sham�acidosis. d P � 0.001 vs. 

ADX�acidosis. e P � 0.01 vs. ADX�acidosis (ANOVA). 


Fig. 1. Effect of adrenalectomy, acidosis, and dexamethasone on plasma 

glutamine concentration. Plasma glutamine concentration was measured in 

arterial (aorta) and renal venous blood as described in MATERIALS AND METH- 

ODS. ADX, adrenalectomized; Dex, dexamethasone treatment. The experimental 

groups and numbers of animals are the same in Figs. 1–3. Values are 

means � SE. a P � 0.05 vs. sham. b P � 0.01 vs. control. c P � 0.01 vs. 

ADX�acidosis�Dex. d P � 0.001 vs. sham�acidosis. 

(Fig. 1, A and B) and dexamethasone increases (Fig. 1, A and 

C) plasma glutamine concentration. The decline in plasma 

glutamine during acidosis is principally the result of greatly 

enhanced renal glutamine utilization for ammoniagenesis. 

Plasma glutamine increases in animals treated with dexamethasone, 

reflecting increased glutamine synthetase (GS) activity 

in muscle and lung (1, 27) and increased proteolysis, principally 

in muscle (17). Treatment of acidotic rats with the 

glucocorticoid receptor antagonist RU486 did not alter systemic 

glutamine concentration (Fig. 1B). 

Adrenalectomy attenuates the acidosis-induced increase in 

renal cortical SNAT3 mRNA and decreases renal glutamine AV 

difference. Northern blot analysis was used to measure the 

relative levels of renal cortical SNAT3 mRNA in the same five 

groups as above and in ADX rats treated with dexamethasone 

(Fig. 2A). Metabolic acidosis significantly increased SNAT3 

mRNA in both sham and ADX rats compared with sham and 

ADX rats not exposed to acidosis (P � 0.001 for both). 

However, adrenalectomy blunted the acidosis-induced increase 

in SNAT3 mRNA (sham�acidosis 20.6 � 4.1 vs. 

ADX�acidosis 11.9 � 1.4 arbitrary units, P � 0.05) and 

dexamethasone partially reversed the effect of adrenalectomy 

(15.9 � 3.4 arbitrary units). Glutamine AV difference in these 

groups followed a similar pattern of response (Fig. 1B). Metabolic 

acidosis significantly increased glutamine AV difference 

in both sham and ADX rats compared with nonacidotic sham 

and ADX rats (sham P � 0.001; ADX P � 0.01). In ADX rats, 

acidosis-induced glutamine AV difference is attenuated compared 

with adrenal-intact animals (sham�acidosis 0.16 � 0.03 

mmol/l vs. ADX�acidosis 0.08 � 0.01 mmol/l, P � 0.05). 




Fig. 2. Effect of adrenalectomy on renal cortical System N glutamine transporter 

(SNAT3) mRNA expression (A) and renal glutamine arteriovenous 

(AV) difference (B) in rats. Sham and ADX rats were treated as indicated. 

Metabolic acidosis was induced by addition of NH4Cl to the drinking water for 

2 days. Dexamethasone treatment (1 mg�kg �1 �day �1 sc) was started 2 days 

before acidosis and continued during acidosis. A: Northern blots were hybridized 

as described in MATERIALS AND METHODS and relative SNAT3 mRNA, 

normalized to 18S ribosomal RNA, determined using laser densitometry; n � 

11–23 rats/group. B: glutamine AV difference was calculated as (arterial � 

renal venous) plasma glutamine concentration; n � 11–20 rats/group. Values 

are means � SE. a P � 0.05 vs. sham�acidosis. b P � 0.001 vs. sham. c P � 

0.001 vs. ADX. d P � 0.01 vs. ADX (ANOVA with Bonferroni posttest). 

Dexamethasone partially reversed the repressive effect of adrenalectomy 

(0.11 � 0.02 mmol/l). There was a trend for 

dexamethasone to increase SNAT3 and glutamine AV difference 

in ADX rats, although the increase did not reach statistical 

significance. We reported that, in normal rats, increased renal 

glutamine uptake during chronic metabolic acidosis rats is 

associated with increased expression of the System N transporter 

SNAT3 in renal proximal tubules (23). Figure 2 suggests 

that this association also occurs in ADX rats. We have used 

renal AV difference (arterial � renal venous glutamine plasma 

concentration) as a broad indicator of glutamine uptake, while 

recognizing that this approach has limitations (see DISCUSSION). 

Inhibition of glucocorticoid activity depresses the acidosisinduced 

increase in cortical SNAT3 expression and renal 

glutamine AV difference. The glucocorticoid antagonist RU486 

was used to further explore a role for glucocorticoids in 

regulating renal cortical SNAT3 expression and renal glutamine 

uptake. Rats were exposed to metabolic acidosis for 6 

days with or without administration of RU486 (12.5 mg/kg sc, 

twice/day). Control rats were injected with vehicle (ethanol). 

BLMV isolated from control, acidotic, and RU486-treated 



F451 

acidotic rats were used to measure SNAT3-mediated glutamine 

transport and for Western blot analysis. Acidosis increased 

SNAT3 expression and glutamine transport in BLMV (Fig. 3, 

A–C). RU486 treatment significantly attenuated acidosisinduced 

renal cortical SNAT3 mRNA (acidosis 1.0 � 0.08 

vs. acidosis�RU486, 0.5 � 0.1 arbitrary units, P � 0.05) 

(Fig. 3A), SNAT3 protein (acidosis 4.05 � 0.75 vs. 

acidosis�RU486, 1.88 � 0.19 arbitrary units, P � 0.05) 

(Fig. 3B), and SNAT3-dependent transport in BLMV (acidosis 

409 � 20 pmol�mg �1 �min �1 vs. acidosis�RU486, 

214 � 74 pmol�mg �1 �min �1 , P � 0.05, repeated measures 

ANOVA) (Fig. 3C). Renal glutamine AV difference was 

blunted but not significantly decreased by RU486 (acidosis 

0.24 � 0.02 mmol/l vs. acidosis�RU486, 0.18 � 0.03 

mmol/l) (Fig. 3D). Treatment of normal rats with RU486 did 

not affect SNAT3 mRNA or glutamine AV difference (not 

shown). 

Chronic glucocorticoid treatment increases renal cortical 

SNAT3 expression. To obtain more direct evidence of a role for 

glucocorticoids in SNAT3 expression in the renal cortex, we 

treated normal rats with dexamethasone, acutely (5 mg/kg sc 

for 1–6 h) or chronically (100 �g/day sc for 1–6 days). Acute 

treatment with dexamethasone did not increase renal cortical 

SNAT3 mRNA. In chronically treated animals, SNAT3 mRNA 

increased slowly to approximately twofold at day 3 (P � 0.05) 

and remained at that level at day 6 (P � 0.001) (Fig. 4A). 

SNAT3 protein and SNAT3-mediated glutamine transport exhibited 

a small but not statistically significantly increase (Fig. 

4, B and C). Glutamine AV difference was modestly increased 

at day 6 (�0.02 � 0.01 mmol/l control vs. 0.02 � 0.01 mmol/l 

day 6, P � 0.03, t-test). Dexamethasone stimulation of renal 

GS activity in the dexamethasone-treated rats would tend to 

decrease apparent AV difference (see DISCUSSION), thus minimizing 

the apparent increase in glutamine uptake. 

Dexamethasone increases SNAT3 mRNA and SNAT3-mediated 

glutamine transport but does not activate the SNAT3 

promoter in renal epithelial cells. SNAT3 mRNA and 

SNAT3-mediated basolateral glutamine transport (control 

214 � 88 nmol�mg �1 �min �1 , dexamethasone, 558 � 96 

nmol�mg �1 �min �1 ) are increased 50 (P � 0.01) and 250% 

(P � 0.01), respectively, by 0.01 �M dexamethasone treatment 

of glucocorticoid-responsive GR101 cells in culture 

(Fig. 5A). GR101 cells are porcine epithelial-like LLC-PK1 

cells that express rat glucocorticoid receptors (41). To determine 

whether glucocorticoids increased SNAT3 expression via 

increased transcription of the SNAT3 gene, we cloned and 

sequenced 2.4 kb of the 5�-flanking region of the rat SNAT3 

gene from a rat genomic library. Analysis of the 5�-flanking 

sequence using the TFSearch program identified a number of 

potential glucocorticoid response elements. The promoter contains 

no TATA or CCAAT box sequences, but a GC-rich 

region containing three potential SP1 sites is present, �100 

bases upstream from the 5�-end of exon I. A series of pCAT 

reporter vectors containing up to 2.4 kb of SNAT3 5�-flanking 

sequence was used to transiently transfect GR101 cells. The 

transfected cells were incubated in the presence or absence of 

0.01 or 0.1 �M dexamethasone for 48 h. Data from 0.01 and 

0.1 �M dexamethasone were combined because there was no 

difference between them. The promoterless expression vector 

pCAT3-Basic showed negligible CAT activity, whereas the 

pCAT.SNAT3 vector containing the longest promoter segment 





Fig. 3. Effect of glucocorticoid inhibition on 

renal cortical SNAT3 mRNA (A), SNAT3 

basolateral membrane vesicle (BLMV) protein 

(B), SNAT3-mediated glutamine transport 

(C), and renal glutamine AV difference 

(D) in rats. Rats were exposed to NH4Cl 

acidosis for 6 days in the presence or absence 

of the glucocorticoid receptor antagonist 

RU486 (12.5 mg/kg twice a day). A: relative 

SNAT3 mRNA was determined using Northern 

blot analysis; n � 8–12 rats/group. B: 

relative SNAT3 protein was measured by 

Western blot analysis in isolated renal cortical 

BLMV; n � 8 vesicle preparations/group. 

C: SNAT3-mediated glutamine transport was 

measured in the same vesicles as in B. D: 

glutamine AV difference was calculated as 

(arterial � renal venous) glutamine concentration; 

n � 8–20 rats/group. Values are 

means � SE. Detailed procedures are described 

in MATERIALS AND METHODS. a P � 

0.001 vs. control. b P � 0.05 vs. acidosis 

(ANOVA). 

tested (�2.4 kb) exhibited a 15-fold increase in basal activity, 

indicating that this 5�-flanking region of the SNAT3 promoter 

contains a functional promoter. The active promoter constructs 

did not, however, respond to dexamethasone treatment (Fig. 

5B). The pCAT.SNAT3 vector containing only the proximal 78 

bases of the flanking region was transcriptionally inactive, 

presumably because three Sp1 binding sites between �101 and 

�78 are absent from this construct. Glucocorticoid responsive- 

Fig. 4. Effect of chronic dexamethasone treatment 

on renal cortical SNAT3 mRNA (A), SNAT3 

BLMV protein (B), SNAT3-mediated glutamine 

transport (C), and renal glutamine AV difference 

(D) in rats. Rats were treated with dexamethasone 

(0.1 mg/day sc) for 1–6 days as indicated. A: relative 

SNAT3 mRNA was determined using Northern 

blot analysis; n � 9–11 rats/group. B: relative 

SNAT3 protein was measured by Western blot analysis 

in isolated renal cortical BLMV; n � 5 vesicle 

preparations/group. C: SNAT3-mediated glutamine 

transport was measured in the same vesicles as in B. 

D: glutamine AV difference was calculated as (arterial 

� renal venous) plasma glutamine concentration. 

Values are means � SE; n ��20 rats/group. 

Detailed procedures are described in MATERIALS AND 

METHODS. a P � 0.05 vs. control. b P � 0.001 vs. 

control [ANOVA (A) with Dunnett’s posttest; t-test 

(D)]. 


ness of the GR101 cells was verified by transfection with the 

dexamethasone-responsive pGREtkCAT plasmid that contains 

two inverted glucocorticoid response elements followed by the 

thymidine kinase promoter. CAT activity of pGREtkCAT 

increased approximately fivefold in response to 100 nM dexamethasone. 

The SNAT3 promoter constructs used in these 

studies (except the inactive �78-bp construct) are activated by 

incubation in acidic medium. CAT activity of the constructs 




Fig. 5. Dexamethasone increases SNAT3 expression and transport activity in 

GR101 cells but does not activate the SNAT3 promoter in transiently transfected 

cells. A: GR101 cells were grown on regular culture dishes (RNA 

isolation) or Transwell plates (transport) with or without 0.01 �M dexamethasone 

for 48 h. SNAT3 mRNA was determined from Northern blot analysis 

and SNAT3-mediated transport measured as described in MATERIALS AND 

METHODS. Values are means � SE from 4–6 separate experiments carried out 

in duplicate or triplicate. a P � 0.01. B: GR101 cells were transiently transfected 

with pCAT vectors carrying SNAT3 promoter fragments of various 

lengths and with the glucocorticoid-responsive plasmid pGREtkCAT, as indicated. 

Cells were incubated with or without dexamethasone (0.1 or 0.01 �M) 

for 48 h. Data were normalized for transfection efficiency by cotransfection 

with pSV-�-Gal. Normalized chloramphenicol acetyl transferase (CAT) activity 

is expressed relative to the control �2,414 SNAT3 promoter construct 

(100%). Transfection and CAT and �-galactosidase activity assays were 

carried out as described in MATERIALS AND METHODS. Values are means � SE 

of 6 separate experiments with triplicate or duplicate determinations. 

increases approximately twofold in GR101 and LLC-PK1 cells 

in response to acidosis (not shown). 

DISCUSSION 

The plasma concentration of glucocorticoids is increased in 

response to metabolic acidosis (28, 42), resulting in activation 

or suppression of a variety of activities or pathways that act in 

concert to maintain blood pH within strict limits. Early studies 

using ADX dogs and rats identified the kidneys as an important 

site of glucocorticoid action during metabolic acidosis. Of 

particular interest to us was the defect in renal glutamine 

uptake and induction of ammoniagenesis exhibited by ADX 

animals (11, 18, 33, 46). During acidosis, SNAT3 transporter 

expression and SNAT3-mediated glutamine transport activity 

are induced in the proximal tubules (23, 37), the site of 

increased glutamine metabolism and ammoniagenesis (16, 35). 

We hypothesized that glucocorticoids regulate acid-base homeostasis 

during acidosis, in part, by increasing glutamine 

availability for ammoniagenesis through upregulation of 

SNAT3 transporter expression. 



F453 

In our studies using sham-operated and ADX rats, renal 

cortical SNAT3 mRNA was increased by metabolic acidosis in 

sham animals. This response was attenuated in ADX animals, 

and treatment of ADX animals with dexamethasone partially 

reversed the effect of adrenalectomy (Fig. 1A), suggesting that 

glucocorticoids have a role in acidosis-induced activation of 

SNAT3 expression. The observation that the glucocorticoid 

receptor antagonist RU486 also blunts the acidosis-induced 

increase in SNAT3 expression (Fig. 2) provides additional 

evidence of glucocorticoid involvement. Dexamethasone also 

increased SNAT3 expression in the absence of acidosis, although 

the increase was much smaller than that induced by 

acidosis. In all the in vivo studies reported here, renal glutamine 

AV difference mirrored SNAT3 expression, reinforcing 

the suggestion that glutamine uptake and expression of SNAT3 

in the renal cortex are linked. These results are consistent with 

previous observations that glucocorticoids play a role in acidbase 

homeostasis in part via regulation of renal glutamine 

utilization (33, 46). 

We have used renal AV difference as a broad indicator of 

glutamine uptake. AV difference is a net measurement that is 

a function of arterial glutamine concentration, renal GS activity, 

and renal blood flow. 1) Figure 1 shows the arterial 

glutamine concentration in all experimental groups. 2) GS is 

present in the outer medulla of the kidney (35). GS activity, 

mRNA, and protein in the kidney are unchanged (8, 35, 47) by 

chronic acidosis so that the impact of renal glutamine synthesis 

on AV difference in acidotic rats is negligible. Glucocorticoids 

increase GS activity and gene expression in muscle and lung 

(1). In the kidney, glucocorticoids have no effect (1) or modestly 

increase (26, 43) GS activity. The effect of increased GS 

activity would be to increase the renal venous concentration of 

glutamine, thus decreasing the apparent glutamine uptake in 

those groups treated with dexamethasone. 3) Renal blood flow 

or glomerular filtration rate either is not altered (15, 24, 32) or 

is increased (45, 46) by metabolic acidosis. Welbourne and 

colleagues (45, 46) reported that renal plasma flow in ADX and 

acidotic ADX rats decreased compared with normal animals 

and increased in acidotic rats and in ADX rats supplemented 

with glucocorticoid. They studied experimental groups similar 

to ours and observed a pattern of renal glutamine AV differences 

in these groups the same as that shown in Fig. 2, except 

for their ADX�acidosis�glucocorticoid group, where the AV 

difference recovered to or above the sham acidosis level, 

whereas we observe only partial recovery. This difference may 

result from the use of different glucocorticoids and doses in our 

studies. In those studies, the pattern of glutamine extraction 

(renal plasma flow � AV difference) and AV difference for 

each group was the same, suggesting that, in these experimental 

conditions, AV difference broadly reflects glutamine extraction. 

ADX animals are deficient in both glucocorticoids and 

mineralocorticoids. Both glucocorticoids and mineralocorticoids 

act in the kidney to increase elimination of protons in the 

urine of acidotic animals, but their sites and mechanisms of 

action differ. Glucocorticoids increase acid excretion in the 

form of ammonia excretion in response to an acid load with 

little effect on urine acidification, whereas mineralocorticoids 

increase acid secretion and acidify the urine with a limited 

effect on ammoniagenesis (20, 48). Glucocorticoid receptors 

are enriched in the proximal tubule, whereas mineralocorticoid 





receptors are enriched in more distal regions (12, 29, 34). The 

differential distribution of these receptors is consistent with the 

restriction of ammoniagenesis induction to the proximal tubules. 

In addition, other acidosis-regulated transporters demonstrate 

a role for glucocorticoids, but not mineralocorticoids, 

in the response to acidosis (2, 5, 13). For these reasons, we 

have focused on glucocorticoid regulation of SNAT3 expression 

in the studies reported here. However, a role for mineralocorticoids 

is not excluded. 

The studies presented here provide some insight into the 

mechanism(s) by which glucocorticoids regulate SNAT3 expression. 

The effects of H � and glucocorticoids cannot be 

separated in normal rats in vivo because the plasma concentration 

of glucocorticoids increases in response to acidosis (28, 

42). Experiments using ADX rats provide insight into the 

relative roles of glucocorticoids and H � in regulating cortical 

SNAT3 expression. A comparison of Figs. 2A, 3A, and 4A 

shows that expression of SNAT3 mRNA is more strongly 

induced by H � than by dexamethasone in both ADX and 

adrenal-intact rats. Figure 2A demonstrates that the role of 

glucocorticoids in regulating SNAT3 expression is not permissive 

because SNAT3 mRNA is induced by acidosis in ADX 

rats that lack endogenous glucocorticoids. The data in Fig. 2A 

also suggest that, for SNAT3 mRNA, the inductive effects of 

H � and glucocorticoids may be additive. There is no evidence 

of synergistic interaction between H � and glucocorticoids in 

the induction of SNAT3 expression. 

The ability of RU486 to partially block the stimulation of 

SNAT3 expression by acidosis suggests the involvement of the 

glucocorticoid receptor in the regulation of SNAT3 gene transcription. 

Dexamethasone treatment increased SNAT3 mRNA 

in control, ADX, and acidotic ADX rats (Figs. 2A and 4A), 

although to a lesser degree than chronic acidosis. The 5�flanking 

region of the SNAT3 gene contains several potential 

glucocorticoid response elements that could be involved in 

glucocorticoid activation of SNAT3 transcription. However, 

transcriptionally active reporter expression vectors containing 

up to 2.4 kb of the SNAT3 5�-flanking sequence did not 

respond to dexamethasone treatment when transiently transfected 

into glucocorticoid-responsive GR101 cells (Fig. 5B). 

The lack of response suggests that glucocorticoids do not 

regulate SNAT3 directly by binding of the activated glucocorticoid 

receptor to a response element within the promoter, but 

indirectly perhaps by interaction with or induction of an unknown 

factor or by message stabilization. It is also possible 

that an essential glucocorticoid-regulatory element lies outside 

the cloned 2.4 kb of promoter used for transfection. 

In addition to SNAT3, expression of a number of enzymes 

and transporters important in the response to acid-base imbalance 

is regulated in the kidney by both H � and glucocorticoids 

(3, 4, 21, 31). In some cases, for example, the gluconeogenic 

enzyme phosphoenolpyruvate carboxykinase and the Na � /H � 

antiporter NHE3, the mechanism of glucocorticoid action has 

been elucidated in considerable detail and has proven to be 

very complex. Both acidosis and glucocorticoids regulate 

phosphoenolpyruvate carboxykinase transcription through 

complex interactions of nuclear factors with two independent 

domains in the promoter (9). Regulation of NHE3 involves 

both transcription and transcription-independent mechanisms 

(trafficking, phosphorylation) that differ in acute and chronic 

acidosis (6, 22, 40). Regulatory mechanisms for other acidosis- 

and glucocorticoid-regulated transporters have yet to be elucidated. 

The studies reported here demonstrate that glucocorticoids, 

in addition to H � , directly or indirectly regulate expression of 

the SNAT3 transporter during chronic metabolic acidosis. In 

addition, the close association between SNAT3 expression and 

renal glutamine uptake as reflected by AV difference under a 

number of experimental conditions makes a strong argument 

for a central role for SNAT3 in the adaptive ammoniagenic 

response to acidosis. When rats are chronically exposed to 

metabolic acidosis, a series of compensatory adaptations occur 

in the kidney so that blood pH and bicarbonate concentration 

are restored to normal values within 7 days despite continued 

acid ingestion (30). Ammoniagenesis, however, remains elevated 

despite the normalization of acid-base status, suggesting 

that factors other than H � are responsible for maintenance of 

activation of adaptive processes, including SNAT3 upregulation 

and renal glutamine uptake. Blood glucocorticoids, in 

contrast, remain elevated during acidosis (28, 42). Because of 

their widespread actions, glucocorticoids may be important in 

orchestrating the systemic and renal response to metabolic 

acidosis and in maintaining activation of key homeostatic 

activities. 

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