Department of Surgery, Milton S. Hershey Medical Center, The Pennsylvania State University College of Medicine, Hershey, PA 17033
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ABSTRACT |
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During chronic metabolic
acidosis, renal glutamine utilization increases markedly. We studied
the expression of the system N1 (SN1) amino acid transporter in the
kidney during chronic ammonium chloride acidosis in rats. Acidosis
caused a 10-fold increase in whole kidney SN1 mRNA level and a 100-fold
increase in the cortex. Acidosis increased Na+-dependent
glutamine uptake into basolateral and brush-border membrane vesicles
(BLMV and BBMV, respectively) isolated from rat cortex (BLMV, 219 ± 66 control vs. 651 ± 180 pmol · mg1 · min
1 acidosis;
BBMV, 1,112 ± 189 control vs. 1,652 ± 148 pmol · mg
1 · min
1 acidosis,
both P < 0.05). Na+-independent uptake was
unchanged by acidosis in BLMV and BBMV. The acidosis-induced increase
in Na+-dependent glutamine uptake was eliminated by
histidine, confirming transport by system N. SN1 protein was detected
only in BLMV and BBMV from acidotic rats. After recovery from acidosis,
SN1 mRNA and protein and Na+-dependent glutamine uptake
activity rapidly returned to control levels. These data provide
evidence that regulation of expression of the SN1 amino acid
transporter is part of the renal homeostatic response to acid-base imbalance.
renal acid-base homeostasis; basolateral membrane vesicle transport; brush-border membrane vesicle transport; ammonium chloride acidosis; system N1
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INTRODUCTION |
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AN ESSENTIAL
FUNCTION OF THE kidney is maintenance of acid-base homeostasis, a
role that assumes even greater importance under conditions of chronic
metabolic acidosis. During acidosis, ammoniagenesis is greatly
increased in the kidney, allowing excretion of protons in the form of
ammonium ions in the urine. Glutamine, the most abundant free amino
acid in the blood, is the precursor of 80-90% of the ammonia
produced during acidosis (37). Under normal conditions, renal uptake of glutamine from the blood is minimal. During chronic metabolic acidosis, however, renal extraction of glutamine increases markedly while extraction of other amino acids is virtually unchanged (34, 36). Enhanced glutamine uptake and metabolism by the kidney are the result of a coordinated series of events triggered by
the disruption of normal acid-base balance. The homeostatic response
involves alterations in interorgan glutamine flux, such that glutamine
release from skeletal muscle is doubled, the splanchnic bed shifts from
net glutamine uptake to glutamine release, and the kidney becomes the
major site of glutamine consumption (33). Intrarenal
alterations in glutamine utilization also occur. Ammoniagenesis and HCO-ketoglutarate
metabolism (4). Asymmetric secretion in the proximal
tubule of NH
Almost all the glutamine filtered at the glomerulus is reclaimed in the convoluted proximal tubule (35), leaving little margin for increased glutamine uptake from the tubular lumen during acidosis. The major portion of the increased renal glutamine taken up must therefore enter the tubular epithelium across the basolateral membrane from the blood (28, 34). Although it has been appreciated that glutamine transport must increase during acidosis (38), the carriers responsible for glutamine transport across the basolateral and brush-border membranes of kidney tubules have not been identified. System N1 (SN1) is an Na+-dependent amino acid transporter that transports glutamine, histidine, and asparagine in various tissues. Its activity was first characterized in rat liver, in which it is the principal transporter of glutamine (19), and subsequently system N-like activities were described in brain (10) and skeletal muscle (17). The cDNA for SN1 has recently been cloned (6, 11, 14), and the protein has been expressed in cells and Xenopus laevis oocytes, allowing functional study of the transporter (6, 11, 14). In addition to its expression in liver, brain, and skeletal muscle, SN1 mRNA is expressed at a relatively high level in the kidney (6, 14).
The studies reported here strongly suggest that SN1 is the glutamine transporter responsible for increased renal uptake of glutamine during acidosis and that its expression is regulated as part of the coordinated physiological response to an acid load.
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METHODS |
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Animals. Animals were treated in accordance with regulations of the Institutional Animal Use and Care Committee. Male Sprague-Dawley rats (250-300 g; Charles River, Wilmington, MA) 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 8 days. In some experiments, acidotic rats were returned to drinking tap water for up to 3 days of recovery. The rats readily adjusted to the acid load received. After an initial 2- to 3-day period of no weight gain, acidotic rats started to gain weight, although at a somewhat slower rate (~5 g/day) than that of control rats (~7 g/day). For most experiments reported here, rats were made acidotic for 7 days. In some experiments, animals were killed at shorter time points, and, in others, additional animals were killed at 8 days acidosis. In general, results for 7- or 8-day acidotic animals did not differ and have been pooled.
Rats were killed after being anesthetized with ketamine, and their kidneys were either freeze-clamped in tongs kept at the temperature of liquid nitrogen or immediately processed for isolation of basolateral or brush-border membrane vesicles (BLMV or BBMV, respectively). 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 with
an IL BG3 blood-gas machine (model 1420, Instrumentation Laboratory).
Blood HCO renal vein) glutamine concentration/arterial
glutamine concentration and expressed as a percentage.
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 cortex, a thin slice (1.5-2 mm thick) was first cut from the curved outer surface. Additional cortex was dissected with 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. The outer and inner stripes of the outer medulla and the inner medulla are clearly discernible. These regions were dissected from the remainder of the kidney and carefully trimmed of visible remaining tissue from other regions.
Northern blot analysis.
Total RNA was isolated from whole kidney or dissected kidney regions by
using the Totally RNA system (Ambion, Austin, TX). No pooling of RNA
from different rats was necessary to obtain sufficient RNA for blots.
Twenty micrograms of RNA were separated on a 1% formaldehyde gel,
transferred to nylon membrane (Genescreen, New England Nuclear), and
hybridized with a SN1-specific oligonucleotide probe
(5'-GTGCAGAAGGCTTCAGCAGTGTCAGGTTGG-3') by using the method of Henderson
et al. (16). The oligonucleotide was radioactively 3'-end
labeled using terminal transferase. For quantification of SN1 mRNA,
autoradiographs were scanned with a laser densitometer (Dynamic
Biosystems). GAPDH and -actin were both found to be unsuitable for
RNA loading normalization. Acidosis increased mRNA levels of both GAPDH
and
-actin, and
-actin mRNA levels varied among kidney regions,
increasing from cortex to inner medulla. Therefore, raw scanning data
were used for relative quantitation of SN1 mRNA. Ethidium
bromide-stained gels showed that the RNA was intact and that loading
differences did not account for the wide variations observed in SN1
mRNA levels on the Northern blots. A cDNA closely related to SN1 has
recently been described and designated system N2 (SN2)
(22). The amino acid sequence of rat SN2 is 63% identical
to that of rat SN1, and SN2 mRNA is expressed in the kidney. The SN1
probe is complementary to a region of the SN1 cDNA in which the SN1 and
SN2 proteins differ completely. Basic Local Alignment Search Tool 2 sequence alignment found no significant similarity between the SN1
probe and the SN2 cDNA. Therefore, it is extremely unlikely that the
SN1 probe hybridizes with SN2 mRNA under the stringent conditions used
here for Northern blot analysis.
In situ hybridization. Kidneys were decapsulated and carefully frozen in an isopentane/dry ice bath. In situ hybridization was carried out as described by Campbell and Hess (5). Serial 20-µm sagittal sections were hybridized with 35S-labeled antisense and sense SN1 riboprobes synthesized by in vitro transcription from linearized Bluescript vector containing rat SN1 cDNA (a gift of Dr. Robert H. Edwards). Slides were exposed to X-ray film (Cronex 4, Sterling Diagnostic Imaging, Newark, DE) for 1 wk and then dipped in Kodak emulsion and developed 3-4 wk later. Slides were counterstained with hematoxylin/eosin. Sections from a total of 10 kidneys (5 from control and 5 from acidotic rats) were hybridized in two separate experiments. Hybridized sections were viewed with an Olympus B-Max 50 microscope, and images were captured by using a cooled charge-coupled device camera (Photometrics, Tucson, AZ) interfaced with IP Lab scientific imaging software (Scanalytics, Fairfax, VA).
Isolation of cortical BLMV.
BLMV were isolated from renal cortex by Percoll density gradient
centrifugation basically as described by Sacktor et al.
(31) and modified by Edwards et al. (9). All
steps were carried out on ice. Renal cortices were initially
homogenized in homogenization buffer using 10 strokes with a loose
plunger in a glass Dounce homogenizer, diluted with the same buffer and
processed with a polytron (2 bursts of 15-s duration at setting 6;
Brinkmann Instruments, Westbury, NY). The homogenization buffer
contained 250 mM sucrose, 10 mM triethanolamine, pH 7.6, 0.1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, and 2 ml/l
protease cocktail stock (P2714, Sigma, St. Louis, MO). The homogenate
was centrifuged at 2,500 g for 15 min at 4°C. The
resultant supernatant was centrifuged at 20,000 g for 20 min, and the upper fluffy layer of the pellet was resuspended in
homogenization buffer with 10 strokes of a tight Dounce plunger.
Percoll was added to 13% (vol/vol), and 36-ml gradients were
centrifuged at 48,000 g for 30 min. The top clear 6 ml of
the gradient were discarded, and the next 6 ml were harvested and
diluted with intravesicular buffer (100 mM KCl, 100 mM mannitol, 12 mM
Tris/HEPES, pH 7.5, protease inhibitors as above). The vesicles were
washed three times at 48,000 g for 15 min with
intravesicular buffer and resuspended in the same buffer to a
concentration of ~1 mg/ml. Protein was measured by using the Bradford
assay with BSA as a standard. BLMV were prepared in pairs of one
control and one 7-day acidotic rat (5 separate experiments) or one
control and one 2-day recovered rat (3 separate experiments). Renal
cortices from three to six animals were pooled for each BLMV
preparation. Freshly isolated or liquid nitrogen-frozen BLMV were used
for glutamine transport assays. Transport activity was similar in fresh
and frozen vesicles. Vesicle relative enrichment was estimated using
ouabain-inhibitable ATPase activity in BLMV compared with homogenate
(13). The enrichment factor was 23.0 ± 4.2 for
control vesicles (1.4 ± 0.2 vs. 29.5 ± 2.1 µmol
Pi · mg1 · 30 min
1, homogenate vs. BLMV) and 22.5 ± 4.5 for
acidotic vesicles (1.5 ± 0.3 vs. 31.7 ± 3.6 µmol
Pi · mg
1 · 30 min
1).
Isolation of cortical BBMV.
BBMV were isolated from renal cortex by Mg2+ precipitation
basically as described by Edwards et al. (9). All steps
were carried out on ice. Renal cortices were homogenized with a
polytron (3 bursts of 20-s duration at setting 6) in 50 mM mannitol, 12 mM Tris/HEPES, pH 7.5, and protease inhibitors as above.
MgCl2 was added to a final concentration of 10 mM, and the
homogenate was stirred for 20 min. The homogenate was centrifuged for
10 min at 2,000 g, and the resultant supernatant was
centrifuged for 20 min at 35,000 g. The pellet was
resuspended in intravesicular buffer (as above) by using a glass Dounce
and centrifuged for 15 min at 35,000 g. The loosely packed
BBMV layer was gently washed off the pellet and recentrifuged an
additional three times as described. The BBMV were resuspended in
intravesicular buffer to a protein concentration of ~1 mg/ml and
stored in liquid nitrogen. Renal cortices from three animals were
pooled for each BBMV preparation. Vesicle relative enrichment was
estimated using alkaline phosphatase activity in BBMV compared with
homogenate, by using a commercially available kit (Sigma). The
enrichment factor was 12.6 ± 0.9 for control vesicles (332 ± 8 vs. 4,181 ± 210 units alkaline phosphatase activity · mg1 · 15 min
1,
homogenate vs. BBMV) and 13.3 ± 2.7 for acidotic vesicles
(290 ± 28 vs. 3,791 ± 403 units alkaline phosphatase
activity · mg
1 · 15 min
1).
Glutamine transport in renal cortical BLMV and BBMV.
For transport studies, Na+-dependent glutamine uptake was
measured in BLMV and BBMV from control or acidotic rats. Glutamine transport was evaluated at room temperature by a rapid
mixing/filtration technique described previously (24).
Uptake was initiated by mixing 10 µl of vesicles (~10 µg membrane
protein) with 40 µl Na+ or K+ uptake buffer
(75 mM NaCl or KCl, 100 mM mannitol, 12 mM Tris/HEPES, pH 7.9, 25 µM
glutamine containing a tracer amount of [3H]glutamine).
Uptake was terminated by addition of 1 ml ice-cold wash buffer (uptake
buffer without glutamine) followed by rapid filtration under vacuum
through a 0.45-µm membrane filter (GN-6 grid, Gelman Laboratory) and
four additional washes with 1 ml wash buffer. The filter was
incubated in 10 ml Scintisafe 30% for liquid scintillation counting
(Beckman LS 1801, Beckman Instruments, Palo Alto, CA). Preliminary
studies showed that glutamine transport was linear at 5 s under
these assay conditions. Therefore the 5-s time point was chosen for
transport experiments. Transport activity was expressed as picamoles
glutamine per milligram protein per minute by multiplying the 5-s
uptake by 12. L-[G-3H]glutamine was purchased
from PerkinElmer Life Sciences, Boston, MA. Where indicated, 5 mM
histidine, serine, or -(methylamino)isobutyric acid (MeAIB) were
added to the uptake buffer as inhibitors of glutamine transport to
allow identification of the transport systems present in BLMV and BBMV.
Western blot analysis. Equal amounts of cortical BLMV (10 µg) and BBMV (20 µg) were separated by SDS-PAGE on precast polyacrylamide gels (ISC BioExpress, Kaysville, UT) and transferred to polyvinylidene difluoride membrane (Millipore). Blots were incubated with primary and secondary (horseradish peroxidase-conjugated donkey-anti-rabbit, Jackson ImmunoResearch Laboratories, West Grove, PA) antibodies, and the SN1 protein was detected by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Piscataway, NJ) following the manufacturer's instructions. The antibody used to detect the SN1 transporter was a polyclonal antibody generously provided by Dr. Robert H. Edwards.
Statistics. Data were analyzed with paired Student's t-test or one-way analysis of variance followed by the Bonferroni or Dunnett posttests for multiple comparisons, as appropriate. In addition, a generalized linear model controlling for batch fixed effects was used to test for differences in Na+-dependent glutamine uptake in BLMV and BBMV in the inhibition profile experiments. When significant differences were detected, Duncan's multiple range test was used for post hoc analyses of uptake in the presence of MeAIB, serine, or serine plus histidine.
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RESULTS |
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Effect of chronic metabolic acidosis on selected physiological
parameters.
Chronic metabolic acidosis was induced in rats by adding 1.5%
NH4Cl to their drinking water. This is a well-characterized model of chronic metabolic acidosis in rats (15, 26, 36). Selected physiological parameters were measured in control, acidotic, and 7-day acidotic rats that were returned to drinking tap water for 1 or 3 days. Decreased blood pH (7.40 ± 0.01 control vs. 7.34 ± 0.02 acidosis, P < 0.05) and HCO4.0 ± 3.7%).
Chronic metabolic acidosis increases renal expression of the SN1
mRNA.
Northern blot analysis was used to determine the effect of acidosis on
expression of SN1 amino acid transporter mRNA in whole kidney. Figure
1A is a representative
Northern blot of whole kidney RNA isolated from control, acidotic, and
1- and 3-day recovered acidotic rats. The result of scans of
blots from three separate experiments is shown in Fig. 1B.
In acidotic rats, the level of SN1 mRNA is ~10-fold higher than in
controls (P < 0.001). The response to an acid load is
rapid; within 24 h of induction of acidosis, the SN1 mRNA level is
already approximately fourfold that of controls (not shown). When the
rats were returned to drinking tap water, SN1 mRNA approached control
levels after 24 h.
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Chronic metabolic acidosis alters the localization of SN1 RNA in
rat kidney.
In situ hybridization was used to localize SN1 mRNA in the rat kidney
and to determine whether the distribution was altered in response to
acidosis. Figure 2D shows
autoradiographs of sagittal sections of kidneys from two control and
two acidotic rats, hybridized with an antisense SN1 riboprobe. There is
strong hybridization in the outer stripe of both control and acidotic
kidneys. No hybridization is detected in the inner stripe or inner
medulla. A response to acidosis is evident in the renal cortex in which
expression of SN1 mRNA is induced (arrows). Equivalent regions of
cortex of control and acidotic kidney (Fig. 2, A and
B, respectively) hybridized with the SN1 riboprobe are
shown. Silver grains indicating the presence of SN1 mRNA are
distributed over tubular structures in Fig. 2B but are
absent from Fig. 2A. Induction of SN1 mRNA expression in the
cortex was not uniform but tended to occur in patches distributed throughout the cortex. Distribution of silver grains shows that SN1
mRNA is expressed in proximal tubules but is not expressed in distal
tubules or glomeruli (Fig. 2C) or collecting ducts or blood
vessels (not shown). No specific hybridization was seen on sections
hybridized with a sense SN1 riboprobe.
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Chronic metabolic acidosis increases glutamine transport in renal cortical BLMV. Two lines of evidence suggested to us that the SN1 transporter is present in the basolateral membrane of cells. First, studies of glutamine uptake and utilization conclude that the markedly increased glutamine extraction during metabolic acidosis reflects augmented uptake from the blood across the basolateral membrane (36). Second, in the liver, SN1 is located in the basolateral membrane of hepatocytes rather than in the canalicular membrane (22). Because the largest induction in SN1 mRNA occurs in the cortex and because we expected the transporter to be in the basolateral membrane, we first isolated BLMV from renal cortices of normal, acidotic and 2-day-recovered rats and used these vesicles for glutamine uptake studies and for Western blot analysis.
Na+-dependent glutamine uptake activity in BLMV from acidotic rats was increased approximately threefold compared with control BLMV (219 ± 66 control vs. 651 ± 180 pmol · mg
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Chronic metabolic acidosis increases glutamine transport in renal
cortical BBMV.
Under normal conditions, >95% of glutamine filtered at the glomerulus
is reabsorbed in the proximal convoluted tubule (35). Therefore, we also isolated brush-border (luminal surface of tubule epithelia) membranes (BBMV) from the cortices of control and 7-day acidotic rats. Na+-dependent glutamine transport was
increased in BBMV from acidotic rats (1,112 ± 189 control vs.
1,652 ± 148 pmol · mg1 · min
1 acidosis,
P < 0.05; Fig. 4B).
Na+-independent uptake was not altered by acidosis
(323 ± 30 control vs. 249 ± 14 pmol · mg
1 · min
1 acidosis,
P = NS). Na+-dependent glutamine uptake was
not inhibited by 5 mM histidine in control BBMV (1,112 ± 189 without histidine vs. 853 ± 66 pmol · mg
1 · min
1 plus
histidine, P = NS) compared with ~50% inhibition in
acidotic BBMV (1,652 ± 148 without histidine vs. 800 ± 49 pmol · mg
1 · min
1 plus
histidine, P < 0.05; Fig. 4D). As in BLMV,
histidine eliminated the acidosis-induced increase in uptake,
suggesting that chronic acidosis selectively increases system N
transport activity in cortical brush-border membranes as well as in
basolateral membranes.
Chronic metabolic acidosis increases SN1 transporter protein in
renal BLMV and BBMV.
Western blot analysis was carried out with BLMV isolated from control,
acidotic, and 2-day-recovered rats (Fig.
5A) and BBMV from control and
acidotic rats (Fig. 5B). The molecular mass of SN1,
predicted from the cDNA, is 56 kDa. Under control conditions, no SN1 protein is detected in BLMV or BBMV. However, these
membranes in acidotic rats contain high levels of transporter protein.
After 2 days of recovery from acidosis, SN1 protein is barely
detectable in BLMV membranes. As noted above, SN2, a cDNA closely
related to SN1, has recently been described (22). The
antibody used for the Western blot analyses shown in Fig. 5 was raised
against the NH2 terminus of SN1 (6) in which
the amino acid sequences of SN1 and SN2 differ completely
(22).
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Renal cortical BLMV and BBMV contain system ASC transport activity.
To determine what transport system(s) is responsible for "normal"
glutamine transport, we also measured transport in the presence of 5 mM
MeAIB (transported by system A) and 5 mM serine (transported by system
ASC) (Fig. 6). The inhibition profiles
for BLMV (Fig. 6A) and BBMV (Fig. 6B) are quite
similar. The failure of MeAIB to inhibit glutamine uptake indicates
that system A is not a major basolateral membrane carrier of glutamine
in the renal cortex. Figure 6B suggests that it may play a
role in the brush border during acidosis but not under control
conditions. This is consistent with in situ hybridization data
(30) showing that ATA2 mRNA is not expressed in the renal
cortex of normal rats. Serine reduced transport in the BLMV and BBMV of
both control and acidotic animals. Inhibition by cysteine, also
transported by system ASC, was similar to serine (not shown). These
results suggest that system ASC is, at least in part, responsible for
cortical glutamine transport under normal conditions. The combination
of serine and histidine, as expected, reduces normal transport and
eliminates the difference between control and acidotic membrane
vesicles (Fig. 6).
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DISCUSSION |
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Our laboratory has been studying the regulation of the altered glutamine metabolism that is characteristic of pathophysiological states for years (20, 25). The central role of glutamine metabolism in the coordinated response to acid loading has been the subject of much research. During chronic metabolic acidosis, renal glutamine extraction increases from <3% to up to 35% (Ref. 36 and this study), greatly exceeding the extraction of any other amino acid (34, 36). However, although it has been appreciated that increased glutamine transport into renal tubule cells is a necessary feature of the homeostatic response to acidosis (38), few studies have specifically examined the effect of acidosis on renal membrane transport of glutamine (12, 15, 21, 40).
Three Na+-dependent amino acid transport systems transport glutamine across cell membranes. These are system A, system ASC (ATB0), and system N and were originally described on the basis of functional transport criteria. In recent years, cDNAs corresponding to these activities have been cloned and their tissue distribution determined (3, 6, 11, 14, 30, 39). System A (ATA2) (30), system ASC (ASCT2) (39), and system N (SN1) (6) mRNA are all expressed in rat kidney. In the studies reported here, we examine the expression of the SN1 amino acid transporter in the rat kidney and demonstrate its role in the renal homeostatic response to chronic metabolic acidosis.
In this study, the level of SN1 mRNA in whole kidney increased ~10-fold in response to acidosis and in situ hybridization, and Northern blot analysis of dissected kidney regions identified the renal cortex as the specific site of increased SN1 expression. Hybridization occurred specifically in the epithelial cells of the proximal convoluted tubules that make up ~80% of cortical cells (27). In control animals, SN1 mRNA is confined to the outer stripe and medullary rays that are projections of the outer stripe into the renal cortex. The outer stripe consists predominantly of straight proximal tubules, the thick ascending limb of distal tubules, and collecting ducts. The bulk of filtered glutamine is reabsorbed by epithelial cells lining the proximal convoluted tubule (35) so that filtrate entering the straight segment of the proximal tubule is depleted of glutamine. The pattern of SN1 expression in control animals may reflect the means of supplying this region of the kidney with glutamine for normal cell function. The deep cortex and outer stripe are also the site of glutamine synthase expression (32), so in this region, SN1 may function to export newly synthesized glutamine. The inner regions of the kidney in which SN1 is not expressed may be served by system A, which is expressed in the renal medulla (30).
Na+-dependent glutamine transport was increased approximately threefold in BLMV from acidotic animals compared with controls and 50% in BBMV. Elimination of the acidosis-induced increase in transport activity in both BLMV and BBMV by histidine suggests that SN1 is the induced transporter. This conclusion is supported by the detection of SN1 protein in BLMV and BBMV that exhibit induced Na+-dependent uptake activity and the failure to detect SN1 in BLMV and BBMV lacking the induced activity (control and recovered animals). Two earlier studies that examined the effect of chronic acidosis on renal basolateral and brush-border glutamine uptake had differing results (12, 40). Windus et al. (40) observed a twofold increase in Na+-dependent uptake of glutamine into BLMV isolated from renal cortex of chronically acidotic dogs compared with BLMV from control dogs but found no difference in transport into BBMV. On the other hand, Foreman et al. (12) found no difference in glutamine transport in renal BLMV of acidotic and control rats but observed an increase in transport into acidotic BBMV. In the latter study, BLMV were isolated from whole kidney.
Expression of SN1 mRNA is rapidly induced in rat kidney after exposure
to an acid load, increasing fourfold within 24 h and 10-fold after
7 days. Induction of SN1 expression and transport activity was rapidly
reversed on withdrawal of NH4Cl and return to tap water. A
similar pattern of response to acidosis has been established for a
variety of physiological parameters and enzyme activities (7,
26). The rapid alterations in SN1 expression parallel those of
other changes that make up the coordinated homeostatic response to
disturbed acid-base balance. Activity of other transporters that
facilitate adaptation to an acid load is elevated in proximal tubules
during chronic acidosis. These include the apical
Na+/H+ exchanger NHE3 (42) and the
basolateral Na+3HCO
-ketoglutarate with formation of HCO
-crystallin/reduced
NADP:quinone reductase to a pH-response element (an 8-base AU sequence)
present in the 3'-untranslated region (UTR) (7). In
contrast, transcription of the phosphoenolpyruvate
carboxykinase gene is increased (18). The rapid
increase in SN1 mRNA and protein level suggests that a
pretranslational mechanism is involved in acidosis-induced
augmented glutamine transport activity. Analysis of the nucleotide
sequence of the 3'-UTR of SN1 shows that sequences corresponding to the pH response element in the 3'-UTR of PDG and GDH (7) are
not present. However, further studies are required to determine whether mRNA stabilization and/or transcription activation causes the observed
elevated SN1 mRNA concentration. Other regulatory mechanisms may also
be involved.
We have demonstrated that chronic metabolic acidosis induces the
expression of the system N transporter SN1 in the epithelia of cortical
convoluted proximal tubules and that the transporter is present in both
basolateral and brush-border membranes of these cells. Associated with
the presence of the transporter protein is increased
Na+-dependent glutamine transport. Expression of SN1 in
cultured cells (6) and X. laevis oocytes
(11, 14) has allowed functional characterization of
SN1-mediated glutamine uptake. The SN1 transporter is an antiporter
that exchanges one proton for one glutamine molecule and one
Na+ ion (2). The direction of exchange is
reversible so that SN1 can mediate glutamine influx or efflux depending
on the gradients for H+ or Na+ ions. These
characteristics have implications for the activity of SN1 during
acidosis. If proximal tubule intracellular pH is lower than
extracellular pH, as reported by Alpern and Chambers (1),
glutamine uptake from both the blood and the glomerular filtrate will
be facilitated. Under normal conditions, >95% of filtered glutamine
is reabsorbed across the proximal tubule cell brush border
(35) so that total glutamine uptake via this route has
little room for increase during acidosis, despite the potential for
increased uptake provided by additional SN1 transporters. However,
during acidosis, glutamine reabsorbed by SN1 transporters in the brush
border is accompanied by proton secretion into the urine. Early studies
of renal glutamine uptake and utilization conclude that increased
uptake from the blood across the basolateral membrane causes the
markedly elevated glutamine extraction observed during metabolic
acidosis (36). The antiporter activity of SN1 also
dictates the secretion of a proton to the blood for each glutamine
molecule taken up across the basolateral membrane. This action would
seem to decrease the efficiency of removal of protons from the body,
although the net effect is to remove H+ because two
ammonium ions are produced from each glutamine molecule metabolized and
two HCO
Parry and Brosnan (26) studied glutamine metabolism in the
kidney during induction of and recovery from chronic NH4Cl
acidosis in rats. Acid-base status returned toward normal after 2 days of exposure to NH4Cl when ammonia excretion had increased
to a maximal level. The elevated rate of ammonia excretion was
maintained, along with normal acid-base status, until the rats were
returned to drinking tap water. Within 1 day of recovery from acidosis, plasma HCO
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ACKNOWLEDGEMENTS |
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The authors are grateful to Dr. Steven W. Levison for assistance in capturing the in situ hybridization images and to Dr. Christopher S. Hollenbeak for assistance with the statistical analysis.
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FOOTNOTES |
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This research was supported by National Heart, Lung, and Blood Institute Grant HL-44986 (W. W. Souba).
Address for reprint requests and other correspondence: W. W. Souba, Dept. of Surgery, Milton S. Hershey Medical Ctr., Pennsylvania State Univ. College of Medicine, PO Box 850, H051, Hershey, PA 17033 (E-mail:wsouba{at}psu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajprenal.00106.2002
Received 19 March 2002; accepted in final form 13 July 2002.
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REFERENCES |
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1.
Alpern, RJ,
and
Chambers M.
Cell pH in the rat proximal convoluted tubule.
J Clin Invest
78:
502-510,
1986[ISI][Medline].
2.
Broer, A,
Albers A,
Setiawan I,
Edwards RH,
Chaudry FA,
Lang F,
Wagner CA,
and
Broer S.
Regulation of the glutamine transporter SN1 by extracellular pH and intracellular sodium ions.
J Physiol
5391:
3-14,
2002.
3.
Broer, A,
Brookes N,
Ganapathy V,
Dimmer KS,
Wagner CA,
Lang F,
and
Broer S.
The astroglial ASCT2 amino acid transporter as a mediator of glutamine efflux.
J Neurochem
73:
2184-2194,
1999[ISI][Medline].
4.
Burch, HB,
Narins C,
Chu C,
Fagiolo S,
McCarthy W,
and
Lowry OH.
Distribution along the rat nephron of three enzymes of gluconeogenesis in acidosis and starvation.
Am J Physiol Renal Fluid Electrolyte Physiol
235:
F246-F253,
1978
5.
Campbell, DB,
and
Hess EJ.
Cerebellar circuitry is activated during convulsive episodes in the tottering tg/tg mutant mouse.
Neuroscience
85:
773-783,
1998[ISI][Medline].
6.
Chaudhry, FA,
Reimer RJ,
Krizaj D,
Barber D,
Storm-Mathisen J,
Cohenhagen DR,
and
Edwards RH.
Molecular analysis of system N suggests novel physiological roles in nitrogen metabolism and synaptic transmission.
Cell
99:
769-780,
1999[ISI][Medline].
7.
Curthoys, NP,
and
Gstraunthaler G.
Mechanism of increased renal gene expression during metabolic acidosis.
Am J Physiol Renal Physiol
281:
F381-F390,
2001
8.
Curthoys, NP,
and
Lowry OH.
The distribution of glutaminase isoenzymes in the various structures of the nephron in normal, acidotic, and alkalotic rat kidney.
J Biol Chem
248:
162-168,
1973
9.
Edwards, RM,
Stack E,
and
Trizna W.
-Ketoglutarate transport in rat renal brush-border and basolateral membrane vesicles.
J Pharmacol Exp Ther
281:
1059-1064,
1997
10.
Ennis, SR,
Kawai N,
Ren XD,
Abdelkarim GE,
and
Keep RF.
Glutamine uptake at the blood-brain barrier is mediated by N-system transport.
J Neurochem
71:
2565-2572,
1998[ISI][Medline].
11.
Fei, YJ,
Sugawara M,
Nakanisji T,
Wang H,
Prasad PD,
Leibach FH,
and
Ganapathy V.
Primary structure, genomic organization, and functional and electrogenic characteristics of human system N1, a Na+- and H+-coupled glutamine transporter.
J Biol Chem
275:
23707-23717,
2000
12.
Foreman, JW,
Reynolds RA,
Ginkinger K,
and
Stanton S.
Effect of acidosis on glutamine transport by isolated rat renal brush-border and basolateral-membrane vesicles.
Biochem J
212:
713-720,
1983[ISI][Medline].
13.
Fujita, M,
Matsui H,
Nagano K,
and
Nakao M.
Asymmetric distribution of ouabain-sensitive ATPase activity in rat intestinal membrane.
Biochim Biophys Acta
233:
404-408,
1971[ISI][Medline].
14.
Gu, S,
Roderick HL,
Camacho P,
and
Jiang JX.
Identification and characterization of an amino acid transporter expressed differentially in liver.
Proc Natl Acad Sci USA
97:
3230-3235,
2000
15.
Harrison, D,
Joshi S,
Carter P,
and
Welbourne TC.
Renal brush border glutamine transport: comparison between in situ and isolate membrane vesicle uptake.
Biochim Biophys Acta
902:
301-306,
1987[ISI][Medline].
16.
Henderson, GS,
Conary JT,
Davidson JM,
Stewart SJ,
House FS,
and
McCurley TL.
A reliable method for Northern blot analysis using synthetic oligonucleotide probes.
Biotechniques
10:
190-197,
1991[ISI][Medline].
17.
Hundal, HS,
Rennie MJ,
and
Watt PW.
Characteristics of L-glutamine transport in perfused rat skeletal muscle.
J Physiol
393:
283-305,
1987[Abstract].
18.
Hwang, JJ,
and
Curthoys NP.
Effect of acute alterations in acid-base balance on rat renal glutaminase and phosphoenolpyruvate carboxykinase gene expression.
J Biol Chem
266:
9392-9396,
1991
19.
Kilberg, MS,
Handlogten ME,
and
Christensen HN.
Characteristics of an amino acid transport system in rat liver for glutamine, asparagine, histidine, and closely related analogs.
J Biol Chem
255:
4011-4019,
1980
20.
Lukaszewicz, G,
Abcower SF,
Labow BI,
and
Souba WW.
Glutamine synthetase gene expression in the lungs of endotoxin-treated and adrenalectomized rats.
Am J Physiol Lung Cell Mol Physiol
273:
L1182-L1190,
1997
21.
McFarlane, N,
and
Alleyne GAO
Transport of glutamine by rat kidney brush-border membrane vesicles.
Biochem J
182:
295-300,
1979[ISI][Medline].
22.
Nakanishi, T,
Ramesh K,
Fei YJ,
Hatanaka T,
Sugawara M,
Martindale RG,
Leibach FH,
Prasad PD,
and
Ganapathy V.
Cloning and functional characterization of a new subtype of the amino acid transport system N.
Am J Physiol Cell Physiol
281:
C1757-C1768,
2001
23.
Nissim, I,
Yudkoff M,
and
Segal S.
Metabolism of glutamine and glutamate by rat renal tubules.
J Biol Chem
260:
13955-13967,
1985
24.
Pacitti, AJ,
Austgen TR,
and
Souba WW.
Adaptive regulation of alanine transport in hepatic plasma membrane vesicles (HPMVs) from the endotoxin-treated rat.
J Surg Res
51:
46-53,
1991[ISI][Medline].
25.
Pacitti, JA,
Inoue Y,
and
Souba WW.
Tumor necrosis factor stimulates amino acid transport in plasma membrane vesicles from rat liver.
J Clin Invest
91:
474-483,
1993[ISI][Medline].
26.
Parry, DM,
and
Brosnan JT.
Glutamine metabolism in the kidney during induction of, and recovery from, metabolic acidosis in the rat.
Biochem J
174:
387-396,
1978[ISI][Medline].
27.
Pfaller, W,
and
Rittinger M.
Quantitative morphology of the rat kidney.
Int J Biochem
12:
17-22,
1980[ISI][Medline].
28.
Pilkington, LA,
Young TK,
and
Pitts RF.
Properties of renal luminal and antiluminal transport of plasma glutamine.
Nephron
7:
51-60,
1970[ISI][Medline].
29.
Presig, PA,
and
Alpern RJ.
Chronic metabolic acidosis causes an adaptation in the apical membrane Na/H antiporter and basolateral Na(HCO3)3 symporter in the rat proximal tubule.
J Clin Invest
82:
1445-1453,
1988[ISI][Medline].
30.
Reimer, RJ,
Chaudhry FA,
Gray AT,
and
Edwards RH.
Amino acid transport system A resembles system N in sequence but differs in mechanism.
Proc Natl Acad Sci USA
97:
7715-7720,
2000
31.
Sacktor, B,
Rosenbloom IL,
Liang CT,
and
Cheng L.
Sodium gradient- and sodium plus potassium gradient-dependent L-glutamate uptake in renal basolateral membrane vesicles.
J Membr Biol
60:
63-71,
1981[ISI][Medline].
32.
Schoolwerth, AC,
deBoer PAJ,
Moorman AFM,
and
Lamers WH.
Changes in mRNAs for enzymes of glutamine metabolism in kidney and liver during ammonium chloride acidosis.
Am J Physiol Renal Fluid Electrolyte Physiol
267:
F400-F406,
1994
33.
Schrock, H,
and
Goldstein L.
Interorgan relationships for glutamine metabolism in normal and acidotic rats.
Am J Physiol Endocrinol Metab
240:
E519-E525,
1981
34.
Shalhoub, R,
Webber W,
Glabman S,
Canessa-Fischer M,
Klein J,
deHaas J,
and
Piyys RF.
Extraction of amino acids from and their addition to renal blood plasma.
Am J Physiol
204:
181-186,
1963
35.
Silbernagl, S.
Tubular reabsorption of L-glutamine studied by free-flow micropuncture and microperfusion of rat kidney.
Int J Biochem
12:
9-16,
1980[ISI][Medline].
36.
Squires, EJ,
Hall DE,
and
Brosnan JT.
Arteriovenous differences for amino acids and lactate across kidneys of normal and acidotic rats.
Biochem J
160:
125-128,
1976[ISI][Medline].
37.
Stone, WJ,
Balagura S,
and
Pitts RF.
Diffusion equilibrium for ammonia in the kidney of the acidotic dog.
J Clin Invest
46:
1603-1608,
1967[ISI][Medline].
38.
Tamarappoo, BK,
Joshi S,
and
Welbourne TC.
Interorgan glutamine flow regulation in metabolic acidosis.
Miner Electrolyte Metab
16:
322-330,
1990[ISI][Medline].
39.
Utsunomiya-Tate, N,
Endou H,
and
Kanai Y.
Cloning and functional characterization of a system ASC-like Na+-dependent neutral amino acid transporter.
J Biol Chem
271:
14883-14890,
1996
40.
Windus, DW,
Cohn DE,
Klahr S,
and
Hammerman MR.
Glutamine transport in renal basolateral vesicles from dogs with metabolic acidosis.
Am J Physiol Renal Fluid Electrolyte Physiol
246:
F78-F86,
1984
41.
Wright, PA,
and
Knepper MA.
Glutamate dehydrogenase activities in microdissected rat nephron segments: effects of acid-base loading.
Am J Physiol Renal Fluid Electrolyte Physiol
259:
F53-F59,
1990
42.
Wu, MS,
Biemesderfer D,
Giebisch G,
and
Aronson PS.
Role of NHE3 in mediating renal brush border Na+-H+ exchange. Adaptation to metabolic acidosis.
J Biol Chem
271:
32749-32752,
1996