1 Renal Division, Most amino acids filtered by the glomerulus are reabsorbed in
the kidney via specialized transport systems. Recently, the cDNA
encoding a high-affinity glutamate transporter, EAAC1, has been
isolated and shown to be expressed at high levels in the kidney. To determine the potential role of EAAC1 in renal acidic amino
acid reabsorption, the distribution of EAAC1 mRNA and protein in rat
kidney was examined. In situ hybridization revealed that EAAC1 mRNA is
expressed predominantly in S2 and S3 segments of the proximal tubules
and at low levels in the inner stripe of outer medulla and inner
medulla. Polyclonal antibodies raised against the carboxy terminus of
EAAC1 recognized a single band of ~70 kDa on Western blots of
membrane protein from kidney cortex and medulla. Immunofluorescence
microscopy revealed intense signals in the luminal membrane of S2 and
S3 segments and weaker signals in S1 segments, descending thin limbs of
long-loop nephrons, medullary thick ascending limbs, and distal
convoluted tubules. These results are consistent with EAAC1 encoding
the previously described apical high-affinity glutamate transporter in
the kidney that mediates reabsorption of acidic amino acids in tubules
beyond early proximal tubule S1 segments. Potential additional roles of
EAAC1 in acid/base balance, cell volume regulation, and amino acid
metabolism are discussed.
acidic amino acid; proximal tubule; loop of Henle; distal tubule; metabolic acidosis; glutamine; ammonia; volume regulation
TRANSPORT OF AMINO ACIDS across cell membranes is
mediated by different transport systems with overlapping substrate
specificities (5). The importance of amino acid transporters has been
defined in several aspects such as protein synthesis, regulation of
cellular metabolism, production of metabolic energy, cell growth, cell volume regulation, nerve transmission, and absorption of amino acids
from the lumen in polarized epithelia. In the kidney, these transporters are important in reabsorption of most amino acids that are
filtered at the glomeruli, thus leaving only small amounts excreted in
the final urine (33, 35). Reabsorption of amino acids occurs by
transport systems that are selective in terms of charge- and
stereoselectivity.
As for acidic amino acids, glutamate and aspartate are nearly
completely reabsorbed in the kidney, which results in fractional urinary excretions of ~0.2-0.5% for glutamate and
0.4-2.5% for aspartate (34). Early studies showed that more than
90% of the filtered glutamate is reabsorbed in the first third of the
proximal tubule (34). This process is mediated by a transport system that is electrogenic and accepts both aspartate and
L-glutamate (24, 36). On the
basis of rat renal brush-border membrane vesicle studies,
Na+- and
K+-coupled glutamate uptake
occurred via two distinct saturable processes, i.e., a high- and a
low-affinity transport system, with Michaelis constant
(Km) values of
0.016 and 3.6 mM, respectively (39). Interestingly,
Na+-coupled glutamate transport
has also been shown in a vesicle preparation enriched in rat renal
basolateral membranes (31).
The nephron segments distal to the proximal tubule were thought to
handle acidic amino acids differently. On the basis of in vivo and in
situ micropuncture studies, significant reabsorption of
glutamate between the proximal tubules and collecting ducts was found
to occur in deep cortical and juxtamedullary nephrons (7) but not in
superficial cortical nephrons (14). It was demonstrated that absorption
at this site lowers fractional amino acid excretion 2- to 40-fold when
there are increased filtered loads. Furthermore, these studies also
suggested that the acidic and neutral amino acids might be recycled
between the vasa recta and the loops of Henle in the inner medulla (7,
8), but the exact pathway is not clearly defined.
During the past few years, four mammalian high-affinity
Na+-dependent glutamate
transporters have been identified at the molecular level. These are
termed EAAC1 (16), GLT-1 (26), GLAST-1 (37), and EAAT4 (10). In the
central nervous system, these high-affinity glutamate transporters are
essential to prevent glutamate from reaching neurotoxic levels during
synaptic transmission. A study based on Northern analysis revealed that
EAAC1 is the only glutamate transporter that is widely expressed in
various tissues outside the nervous system including kidney and
intestine (16). This suggested that EAAC1 is involved in acidic amino
acid reabsorption in the kidney.
This study was undertaken to investigate the nephron distribution and
cellular localization of EAAC1 in rat kidney both at the mRNA and
protein levels. The results indicate that the distribution of EAAC1
mRNA and protein correlates with the functionally defined distribution
of the previously described apical acidic amino acid transporter. Our
data provide a fundamental basis for understanding the mechanism of
acidic amino acid reabsorption in different nephron segments and the
potential role of glutamate in renal amino acid metabolism and other
cell functions.
Northern analysis.
Poly(A)+ RNA was prepared from
different regions of male Sprague-Dawley rat kidney, i.e., superficial
cortex, deep cortex, outer and inner stripe of outer medulla, inner
medulla, and papilla. About 3 µg of each sample was separated on a
1% agarose gel in the presence of 2.2 M formaldehyde and blotted onto
a nitrocellulose filter. The filter was hybridized at 42°C with a
32P-labeled probe synthesized from
the full-length rat EAAC1 cDNA (15) and washed in 0.1× SSC
(1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) + 0.1% sodium dodecyl sulfate (SDS) at 65°C.
In situ hybridization. The basic
procedure for in situ hybridization was as previously described (16).
Briefly, rat kidneys were perfusion fixed with 4% paraformaldehyde in
phosphate-buffered saline (PBS), postfixed in the same solution at
4°C, and immersed in 30% ice-cold sucrose for 48 h. Two serial
cryosections of ~4 µm were prepared and hybridized at 42°C with
35S-labeled sense or antisense RNA
probes synthesized from nucleotides 500-1751 of rat EAAC1 cDNA.
The probe was degraded by partial hydrolysis to ~100 nucleotides in
length. After washing at 50°C the air-dried slides were dipped into
Kodak NTB2 emulsion and developed 2-3 wk later.
To localize EAAC1 mRNA in different segments of the kidney proximal
tubules, adjacent sections were used for immunocytochemistry using the
antibodies which recognize specific antigens in each segment and the
immunoperoxidase staining method. S1 segments were identified using
antibody against the facilitated glucose transporter GLUT-2 (38), S2
segments by an anti-carbonic anhydrase IV antibody (1), and S3 segments
by anti-ectoadenosinetriphosphatase (anti-ecto-ATPase) antibody (30).
Immunoblotting. The monospecific
polyclonal anti-EAAC1 antibodies used in the immunoblot and
immunocytochemistry experiments were raised against a peptide
corresponding to amino acids 511-524 of the carboxy terminus of
EAAC1 from rat, as previously described (27). For immunoblot, the
protein was extracted from the kidney cortex and medulla of male
Sprague-Dawley rats (200-250 g). The samples were homogenized in a
solution containing 250 mM sucrose, 10 mM triethanolamine, 1 µg/ml
leupeptin, and 0.1 mg/ml phenylmethylsulfonyl fluoride (pH 7.6) and
centrifuged at 1,000 g for 10 min. The
supernatant was saved and recentrifuged at 17,000 g for 20 min. The pelleted membrane
fractions were suspended in the same solution, measured for protein
concentrations, and solubilized at 60°C for 15 min in Laemmli
sample buffer. SDS-polyacrylamide gel electrophoresis was performed by
minigel electrophoresis using a 10% polyacrylamide gel, and the gel
was transferred to a nitrocellulose membrane by electroblotting. The
membrane was blocked for 1 h with 5% nonfat dried milk powder + PBS-T
(0.1% Tween 20 in PBS) and exposed to primary antibody diluted in
0.1% milk powder + PBS-T for 1 h. The secondary antibody was a donkey
anti-rabbit immunoglobulin G (IgG) conjugated to horseradish peroxidase
(Amersham Life Science, Arlington Heights, IL). The antigen-antibody
complexes were detected by autoradiography using the enhanced
chemiluminescence method (ECL Western blotting analysis system,
Amersham Life Science).
Immunocytochemistry.
Immunocytochemistry was performed as previously described (27) with
some modifications. Briefly, the rat kidneys were perfusion fixed with
4% paraformaldehyde in PBS, postfixed in the same solution at 4°C
overnight, and infiltrated with 30% sucrose in PBS. Thin 4-µm
cryosections were treated with 1% SDS for 5 min to expose antigenic
sites (2), rinsed three times with PBS, and preincubated with 5%
normal goat serum for 15 min. Sections were incubated overnight at
4°C with anti-EAAC1 antibody (0.2 µg/ml IgG), washed three times
with PBS, and then incubated for 1 h with goat anti-rabbit
indocarbocyanine Cy3 (1:100; Jackson ImmunoResearch, West Grove, PA).
Specificity of the staining was tested by using anti-EAAC1 antibody
preadsorbed with immunizing peptide at 50 µg/ml. Sections were
examined and photographed on a Nikon FXA photomicroscope, using Kodak
Ektachrome 400 film. The possible autofluorescence was determined by
switching filters and was negligible.
Localization of EAAC1 mRNA in rat
kidney. Northern analysis using full-length EAAC1 as
the probe revealed strong hybridization to a 4.2-kb band for almost
every region of the rat kidney except the renal papilla (Fig.
1). The signals in the superficial and deep
cortex and in the outer stripe of outer medulla were stronger than
those in the inner stripe of outer medulla and inner medulla. The
weaker 2.7-kb band, which is likely the result of the use of an
internal polyadenylation site (15), exhibited the same distribution in
the kidney.
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References
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Fig. 1.
Distribution of EAAC1 mRNA in rat kidney by high-stringency Northern
analysis. Equal loading poly(A)+
RNA (3 µg/lane) from different regions of rat kidney: superficial
cortex (SC), deep cortex (DC), outer stripe of outer medulla (OS),
inner stripe of outer medulla (IS), inner medulla (IM), and papilla
(PAP).
In situ hybridization was initially used to investigate the expression of EAAC1 mRNA in different nephron segments in the rat kidney. Figure 2 shows a low-magnification view and demonstrates dense hybridization of the antisense EAAC1 cRNA probe to tubules of both cortex and outer stripe of outer medulla. Consistent with Northern analysis, a weak signal was also evident in inner stripe of outer medulla and in inner medulla. The pattern of hybridization in these regions suggest that EAAC1 is expressed in thick ascending limbs and/or thin descending limbs of loops of Henle.
|
Figure 3A shows a low-power micrograph of the hybridization pattern of EAAC1 antisense cRNA probe in cortex and outer stripe of outer medulla. Figure 3, B and C, shows the adjacent sections stained with the S2-specific carbonic anhydrase IV antibody and the S3-specific anti-ecto-ATPase antibody, respectively. The strong in situ hybridization signal in the cortex was present both in tubules that were immunopositive and immunonegative for the S2-specific antibody. The signal in the medullary rays was present in tubules that were immunopositive for the S3 antibody, consistent with a previous study of rabbit kidney (22).
|
Localization of EAAC1 protein in rat kidney. To establish that the antibody used in this study specifically recognizes EAAC1 protein, we first performed Western blot analysis of crude membrane fractions from cortex and whole medulla of rat kidney. Figure 4 shows that the antibody recognized a single band of ~70 kDa in both regions, with stronger signal detected in cortex than in medulla. The apparent molecular mass of labeled protein is consistent with the molecular mass of the glycosylated protein predicted from the cDNA clone (16) and the molecular mass previously reported for total rat brain homogenates (27).
|
The cellular localization of EAAC1 protein was further investigated by indirect immunofluorescence using affinity-purified antibodies applied to rat kidney frozen sections. Staining for this transporter was detected in different nephron segments with different intensities, whereas no significant staining was observed using antibody preadsorbed with peptide antigen (not shown). The results are shown in Fig. 5.
|
Cortex. EAAC1 was detected in the apical membrane of cells from the proximal tubule. However, the different proximal tubule segments were labeled with different intensities. Staining for EAAC1 was strongest in S2 segments, whereas S1 segments, identified by connection to a glomeruli, showed weaker staining (Fig. 5, A and B). Moderate labeling was also detected in distal convoluted tubules (Fig. 5, A and C).
Outer stripe of outer medulla. EAAC1 was detected in the apical membrane of the straight (S3) part of proximal tubules with similar intensity as in S2 segments (Fig. 5D). No staining was detected in collecting ducts (Fig. 5D).
Inner stripe of outer medulla. Figure 5, E and F, shows the transitional zone between S3 segments and thin descending limbs of short-loop and long-loop nephrons, respectively. The staining shows evidence for internephron heterogeneity with respect to the expression pattern of EAAC1, because a significant labeling was evident only in thin descending limbs of long-loop but not short-loop nephrons. Figure 5G shows vascular bundles that contain descending limbs of short loops of Henle surrounded by vasa recta, as well as adjacent thin descending limbs of long loop of Henle. Labeling was evident only in thin descending limbs of long loops of Henle but not in vascular bundles. Another segment that expresses EAAC1 at an intermediate level in this region is the medullary thick ascending limbs (Fig. 5F).
Inner medulla. Figure 5H shows the frontier between inner stripe of outer medulla and inner medulla. EAAC1 labeling was identified only in thin descending limbs of long loops of Henle and medullary thick ascending limb, whereas the ascending thin limb is negative. The staining was not evident along the inner medullary collecting duct.
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DISCUSSION |
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The evidence presented in this report and in our previous studies (15, 16) indicates that EAAC1 encodes the apical, high-affinity glutamate transporter in the kidney. We showed that expression of rat EAAC1 in Xenopus oocytes induces a high-affinity uptake of DL-aspartate and L-glutamate with an apparent Km of 14 ± 2 µM and that transport is coupled to the cotransport of Na+ and the countertransport of K+ (16, 18, 25). These data are in accordance with the high-affinity transport system reported from rat renal proximal tubular brush-border membrane vesicles (24, 39). In the present study, we show that expression of EAAC1 in the proximal tubule is much higher in the brush-border membranes of S2 and S3 segments than in S1 segments, thus indicating that acidic amino acid reabsorption in the S2 and S3 segments is mediated by the high-affinity transporter EAAC1.
The localization of EAAC1 in the late portion of proximal tubules indicates that the reabsorptive mechanism for acidic amino acids is similar to that for other solutes. For example, the low-affinity, high-capacity glucose transporter SGLT2 is located in the S1 segment to reabsorb the majority of filtered D-glucose (17), whereas the remaining glucose is further reabsorbed by the high-affinity, low-capacity glucose transporter SGLT1 in the S3 segment (20). In the case of acidic amino acid reabsorption, the first third of the rat proximal convoluted tubule is responsible for reabsorption of >90% of filtered glutamate under normal conditions (34). This process is likely mediated by a low-affinity, high-capacity transport system (39) that has not yet been cloned. The high-affinity, low-capacity transporter EAAC1 will participate in final reabsorption of acidic amino acids that may escape the early part of the proximal tubule, especially when the filtered load of glutamate is increased (3, 34).
As stated above, there is internephron heterogeneity for amino acid reabsorption distal to the proximal tubules. The localization of EAAC1 expression distal to the proximal tubules reveals additional nephron segments where acidic amino acids are further reabsorbed, i.e., along the entire length of the thin descending limbs of long-looped nephrons, the thick ascending limbs, and distal convoluted tubules. The absence of EAAC1 expression in thin descending limbs of short-looped nephrons and collecting ducts is in agreement with previous data showing that there is no amino acid reabsorption in these segments of the kidney (14).
Regulation of acidic amino acid reabsorption in the kidney has not been
clearly demonstrated. An early microperfusion study showed that
reabsorption of glutamate in the proximal tubule is influenced only to
a minor or negligible extent by acute alteration in cellular metabolism
(36). However, it is well known that acute metabolic acidosis results
in a fall in cellular glutamate content both in vivo (6, 13) and in
LLC-PK1 cells (32). This results
in increased mitochondrial phosphate-dependent glutaminase activity and
increased ammoniagenesis (12, 40). Recent studies showed that reduced
cellular glutamate in
LLC-PK1-F+
cells during metabolic acidosis is caused by at least two pathways: 1) enhanced intracellular glutamate
removal due to acidosis-induced activation of glutamate dehydrogenase,
and 2) reduced cellular glutamate
influx due to inhibition of -glutamyl transpeptidase phosphate-independent glutaminase in the proximal tubule (9, 29) by the
reduced extracellular bicarbonate concentration (23, 40). However,
metabolic acidosis increased glutamate uptake in
LLC-PK1-F+
cells (23). This might be explained by the direct effect of lower
extracellular pH on transport activity, consistent with a previous
study that showed that glutamate is transported by EAAC1 in its
protonated form (42). Additional in vivo studies to investigate the
effects of metabolic acidosis on both EAAC1 and the low-affinity
glutamate transporter in the proximal tubule will be necessary to
understand the role of glutamate transport in renal ammoniagenesis and
its effect on intracellular acidification.
Expression of EAAC1 in segments other than proximal tubules raises the question of whether glutamate might be used as a metabolic fuel in these segments. A previous study showed that glutamate can be metabolized and oxidized to produce CO2 in thick ascending limbs (19). However, it has been shown that glutamate is not the preferred substrate to maintain cellular ATP content in thick ascending limbs and to energize transepithelial Na+ transport (4, 41). This can be attributed to the high metabolic expenditure required to absorb glutamate (coupling to the cotransport of Na+ and H+ and to the countertransport of K+).
Recently, it has been shown that hypertonic stress activates the
high-affinity glutamate transport system
in the bovine
renal epithelial cell line NBL-1 (11). In contrast to the neutral amino
acids transport system A, which
appears to be activated through a regulatory protein (28),
upregulation of glutamate transporters in hypertonic medium is
accompanied by an increase in EAAC1 mRNA levels and is dependent on
protein synthesis. Expression of EAAC1 in thin descending limbs of
long-looped nephrons suggests a potential role for acidic amino acids
in cell volume regulation in the medullary interstitium. An
immunohistochemical study using monospecific antibodies for free
glutamate and aspartate also showed that these amino acids are
accumulated in the medullary interstitium (21). Although there is no
evidence that the concentration of glutamate or aspartate alone is
sufficiently high to account for the increased osmolality inside the
cell, the sum of these acidic amino acids and others may allow their
contribution as significant organic osmolytes in cells of the renal
medulla.
In conclusion, we have examined the tubular distribution and cellular localization of the high-affinity glutamate transporter EAAC1 in the rat kidney at both the mRNA and protein levels. The predominant localization of EAAC1 in the apical membrane of S2 and S3 segments of the proximal tubule supports the view that EAAC1 is responsible for glutamate reabsorption in these segments. In addition, the expression of EAAC1 in thin descending limbs of long-looped nephrons, medullary thick ascending limbs, and distal convoluted tubules is in agreement with previous physiological studies showing significant glutamate reabsorption distal to the proximal tubules and indicates that EAAC1 is possibly involved in glutamate recycling and cell volume regulation in the inner medulla.
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ACKNOWLEDGEMENTS |
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This work was supported by the National Institutes of Health Grants DK-43171 and NS-32001 (to M. A. Hediger) and DK-42956 (to D. Brown), by a Juvenile Diabetes Foundation International Fellowship (to W.-S. Lee), by an International Human Frontier Science Program Organization Long-Term Fellowship (to Y. Kanai), and by research fellowship grant from Siriraj-China Medical Board, Mahidol University, Bangkok, Thailand (to C. Shayakul).
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FOOTNOTES |
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Present addresses: Y. Kanai, Department of Pharmacology and Toxicology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181, Japan; and W.-S. Lee, Graduate Institute of Medical Sciences, Taipei Medical College, 250 Wu-Hsin Street, Taipei 110, Taiwan, ROC.
Address for reprint requests: M. A. Hediger, Harvard Institutes of Medicine, 77 Ave. Louis Pasteur, Boston, MA 02115.
Received 18 February 1997; accepted in final form 31 July 1997.
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