Regulation of expression of the SN1 transporter during renal adaptation to chronic metabolic acidosis in rats

Anne M. Karinch, Cheng-Mao Lin, Christopher L. Wolfgang, Ming Pan, and Wiley W. Souba

Department of Surgery, Milton S. Hershey Medical Center, The Pennsylvania State University College of Medicine, Hershey, PA 17033


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 · mg-1 · 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> production are greatly increased by augmented flux through mitochondrial glutaminase and glutamate dehydrogenase (GDH) (23) and subsequent alpha -ketoglutarate metabolism (4). Asymmetric secretion in the proximal tubule of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> ions into the tubular lumen and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> ions into the venous blood is critical for maintenance of acid-base balance.

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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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 (Boehringer Mannheim, Mannheim, Germany) adapted to a 96-well plate format with a microplate spectrophotometer. Glutamine extraction was calculated as (arterial - 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 beta -actin were both found to be unsuitable for RNA loading normalization. Acidosis increased mRNA levels of both GAPDH and beta -actin, and beta -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 · mg-1 · 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 · mg-1 · 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 alpha -(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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration (24.3 ± 0.53 control vs. 20.9 ± 0.89 mM acidosis, P < 0.05) confirmed that the rats were acidotic. The degree of acidosis was mild, reflecting the action of renal and extrarenal compensatory mechanisms that minimize acid-base imbalance. After 7 days of acidosis, the kidney, which extracts relatively little glutamine from the plasma under normal conditions, becomes a major organ of glutamine consumption (2.8 ± 2.4 glutamine extraction control vs. 35.6 ± 6.5% extraction acidosis, P < 0.01). These acidosis-induced changes in blood parameters rapidly returned to, or overshot, control values within 24 h of the return of the animals to drinking tap water, and glutamine uptake changed to glutamine release (pH, 7.45 ± 0.01; HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration, 30.30 ± 0.68 mM; glutamine extraction, -4.0 ± 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.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of chronic metabolic acidosis and recovery from acidosis on the expression of system N1 (SN1) mRNA in rat whole kidney. A: Northern blot of kidney total RNA (20 µg) hybridized with an SN1-specific oligonucleotide probe and the ethidium bromide-stained gel before transfer. The blot contains kidney RNA from 8 rats in 1 experiment. B: relative level of SN1 mRNA determined from densitometric scans of Northern blots of 3 separate experiments. Values are means ± SE of 10-22 animals. ***P < 0.001 vs. control (ANOVA).

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.


View larger version (129K):
[in this window]
[in a new window]
 
Fig. 2.   Localization of SN1 mRNA in kidney of control and 7-day acidotic rats. Sagittal sections of kidney of control and acidotic rats were hybridized with a 35S-labeled antisense SN1 riboprobe as described in METHODS. A: outer cortex of kidney of control rat. Few silver grains are visible. B: outer cortex of kidney of acidotic rat, equivalent to the region shown in A. Note the concentration of silver grains over tubules (arrows). C: cortex of acidotic kidney showing silver grains over proximal tubules (arrows) but not over glomerulus (G) or distal tubules (*). A-C: magnification = 20 µm. D: autoradiographs of hybridized kidney sections of 2 control and 2 acidotic rats. Slides were exposed to film for 7 days. Arrows, cortex.

To verify the regional differences in SN1 mRNA expression, Northern blot analysis was carried out by using total RNA isolated from the cortex, outer and inner stripes of the outer medulla, and the inner medulla of control and acidotic rats (Fig. 3A). Dramatic induction of SN1 mRNA occurs in the cortex, in which expression is almost undetectable in control kidney but achieves a high level during acidosis (Fig. 3B). It is difficult to obtain an accurate measurement of the increase of induction in the cortex because of the vanishingly small amount present in control cortex. Under control conditions, the SN1 mRNA level is highest in the outer stripe in which, in response to acidosis, the level of SN1 mRNA increases about ninefold. A low level of SN1 mRNA is apparent in the inner stripe (Fig. 3A). However, in situ hybridization shows that SN1 mRNA is not expressed in the inner stripe (Fig. 2D), so the SN1 mRNA detected on the Northern blot probably represents outer stripe contamination during dissection. SN1 mRNA is not expressed in the inner medulla.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of chronic metabolic acidosis on regional expression of SN1 mRNA. A: Northern blot of total RNA (20 µg) isolated from dissected regions of kidney from a control (C) and 7-day acidotic (A) rat. The blot was hybridized with an SN1-specific probe. Also shown is the ethidium bromide-stained gel before transfer. B: regional increase of induction of SN1 mRNA in kidney regions in response to metabolic acidosis, on the basis of the densitometric scan of the blot in A.

A cDNA closely related to SN1 has recently been described and designated SN2 (22). The amino acid sequence of rat SN2 is 63% identical to rat SN1, and SN2 mRNA is expressed in kidney. All Northern blots presented in this paper were hybridized with an SN1-specific oligonucleotide probe complementary to a region of the SN1 cDNA in which the SN1 and SN2 proteins differ completely (see METHODS). The expression and induction shown here on Northern blots represent only SN1 mRNA.

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-1 · min-1 acidosis, P < 0.05; Fig. 4A). Na+-independent glutamine uptake activity, on the other hand, was unchanged by acidosis [250 ± 72 control vs. 180 ± 34 pmol · mg-1 · min-1 acidosis, P = not significant (NS)]. After 2 days of recovery from acidosis, Na+-dependent transport activity was not different from control activity (286 ± 103 pmol · mg-1 · min-1, P = NS; Fig. 4A). The presence of 5 mM histidine (also transported by system N) was used to determine the proportion of Na+-dependent uptake in BLMV mediated by system N. Na+-dependent glutamine uptake was not inhibited by 5 mM histidine in control BLMV (235 ± 165 without histidine vs. 176 ± 81 pmol ·mg-1 · min-1 plus histidine, P = NS) compared with 85% inhibition in acidotic BLMV (747 ± 394 without histidine vs. 111 ± 48 pmol · mg-1 · min-1 plus histidine, P < 0.05; Fig. 4C). Therefore, histidine eliminated the acidosis-induced increase in uptake, suggesting that chronic acidosis selectively increases system N transport activity in cortical basolateral membranes.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of chronic metabolic acidosis on Na+-dependent glutamine transport in basolateral and brush-border membrane vesicles (BLMV and BBMV, respectively). The initial rate of Na+-dependent glutamine (25 µM) uptake was measured by a rapid mixing-filtration technique as described in METHODS. Uptake was measured for 5 s, multiplied by 12, and expressed as pmol · mg-1 · min-1. A: glutamine uptake in BLMV isolated from cortices of control, 7 day-acidotic, and 2 day-recovered rats. *P < 0.05 vs. control (ANOVA). B: glutamine uptake in BBMV isolated from cortices of control and acidotic rats. *P < 0.05 vs. control (paired Student's t-test). C: inhibition by 5 mM histidine of glutamine uptake into BLMV from control and acidotic rats. Open bars, without histidine; hatched bars, plus histidine. *P < 0.05 vs. acidosis without histidine (ANOVA). D: inhibition by 5 mM histidine of glutamine uptake into BBMV from control and acidotic rats. Open bars, without histidine; hatched bars, plus histidine. *P < 0.05 vs. acidosis without histidine (ANOVA). Values are means ± SE of the number of vesicle preparations that are shown in each bar.

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 · mg-1 · 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).


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of chronic metabolic acidosis on the level of SN1 transporter protein in cortical BLMV and BBMV. A: Western blot analysis of BLMV (10 µg) isolated from cortices of control, 7-day acidotic, and 2-day-recovered rats. Three BLMV preparations are shown in each group. The BLMV preparations in each group are some of those used for the transport activity determinations shown in Figs. 4 and 6. B: Western blot analysis of BBMV (20 µg) isolated from cortices of control and 7-day acidotic rats. Three BBMV preparations are shown in each group. The BBMV preparations are those used for the transport activity determinations shown in Figs. 4 and 6. SN1 was detected with a polyclonal antibody raised against the NH2 terminus of SN1.

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).


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 6.   Inhibition profiles of glutamine uptake in cortical BLMV and BBMV from control and acidotic rats. Na+-dependent glutamine uptake was measured in BLMV (A) and BBMV (B) in the presence of no inhibitor (N), MeAIB (M), serine (S), or serine plus histidine (S+H) (all at 5 mM). Three BLMV and BBMV preparations are shown in each group (except for 2 S+H in BLMV). No significant differences were detected within BLMV groups: P = 0.091, control; P = 0.100, acidosis. For BBMV, *P <=  0.05 vs. no inhibitor for each group (Duncan's multiple range test).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (29), promoting both removal of H+ from cell to tubular lumen and translocation of reabsorbed and newly synthesized HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> ions to the venous blood. Other alterations include rapid induction of the expression and activity of enzymes involved in glutamine utilization. These include mitochondrial glutaminase [phosphate-dependent glutaminase (PDG)] (8), GDH (32, 41), and phosphoenolpyruvate carboxykinase (4), enzymes involved in ammoniagenesis and oxidation of alpha -ketoglutarate with formation of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> ions. As we report for SN1, induction of these enzymes occurs exclusively in the proximal convoluted tubule. The specific mechanisms leading to increased activity of these enzymes vary. For example, PDG and GDH mRNA are stabilized in response to increased intracellular H+ concentration via binding of zeta -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<UP><SUB>3</SUB><SUP>−</SUP></UP> ions are added to the blood. In the aggregate, the presence of SN1 transporters in both basolateral and brush-border membranes of proximal tubule epithelia during acidosis contributes to maintenance of acid/base homeostasis.

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<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration increased, plasma pH increased, and ammonia excretion and renal glutamine extraction returned to control levels (26). This study illustrates the adaptation to chronic acidosis made possible by increased renal ammoniagenesis. Increased expression of the SN1 glutamine transporter plays an essential adaptive role by supplying additional glutamine, the critical substrate for increased ammoniagenesis.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

9.   Edwards, RM, Stack E, and Trizna W. alpha -Ketoglutarate transport in rat renal brush-border and basolateral membrane vesicles. J Pharmacol Exp Ther 281: 1059-1064, 1997[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

23.   Nissim, I, Yudkoff M, and Segal S. Metabolism of glutamine and glutamate by rat renal tubules. J Biol Chem 260: 13955-13967, 1985[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 283(5):F1011-F1019
0363-6127/02 $5.00 Copyright © 2002 the American Physiological Society