From the Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
Received for publication, December 2, 2002, and in revised form, January 29, 2003
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ABSTRACT |
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To identify novel transporters in the kidney, we
have constructed an mRNA data base composed of 1000 overall clones
by random sequencing of a male rat kidney cDNA library. After a
BLAST search, ~40% of the clones were unknown and/or unannotated and
were screened by measuring the uptake of various compounds using
Xenopus oocytes. One clone stimulated the uptake of
Recently, several high throughput DNA sequencing approaches
have been performed, such as the human genome project, the mouse genome
project, and so on, in which many gene arrangements have been revealed
and submitted to the DDBJ/GenBankTM/EMBL Data Bank (1, 2).
However, for a large number of them, only the nucleic acid and amino
acid sequences have been clarified, with little information available
concerning their functions and tissue distributions. It is conceivable
to assume that there are many unknown transporters among these
unknown/unannotated genes. Although some transporters have been
isolated recently using the nucleotide sequences in the expressed
sequence tag data bases, most of them are members of known transporter
families (3, 4), suggesting difficulty in identifying novel transporter genes.
The random sequencing of a cDNA library, in which several thousand
clones are randomly picked and sequenced, is one of the promising
methods of establishing mRNA expression data bases (5). The
mRNAs in a data base constructed from the mouse kidney using this
method can be considered to be abundantly expressed and physiologically significant in vivo (6-8). However, ~70% of the clones
identified by this method were unknown cDNAs that were not
submitted to the DDBJ/GenBankTM/EMBL Data Bank or that were
submitted with only the base or amino acid sequences to the mouse
kidney cDNA data base (6). As the transporter-related mRNAs
share >2% of the known mRNAs in these studies (6, 7), some novel
transporter-related mRNAs might exist among unknown mRNAs.
Based on this hypothesis, we constructed an mRNA data base of male
rat kidney and screened unknown genes in the data base by measuring the
uptake of various compounds using Xenopus oocytes.
Consequently, we found a novel Na+-dependent
glucose transporter
(rNaGLT1)1 that transports
Glucose is reabsorbed in renal proximal tubules by several hexose
transporters (9). To date, two Na+-dependent
glucose transporters, SGLT1 (10) and SGLT2 (11, 12), with high and low
affinity for their substrates, respectively, have been isolated from
intestine or kidney. SGLT2 is predominantly expressed in the S1 segment
of proximal tubules, whereas SGLT1 is present in the S3 segment. It is
generally accepted that SGLT1 and SGLT2 work cooperatively during the
reabsorption of glucose in the proximal tubules. Although the severe
glucose/galactose malabsorption disease is caused by a single missense
mutation of the SGLT1 nucleotide sequence in the small intestine,
slight glycosuria is observed in patients with this disease (13). This finding indicates that SGLT1 may play a small part in the reabsorption of glucose in the kidney (9). In addition, there are few studies suggesting mutations of SGLT2 accompanying a severe inherited disease.
It has been speculated that Fanconi's syndrome or primary renal
glycosuria involves a malfunction of renal glucose transporters. Although Santer et al. (14) have suggested the involvement
of GLUT2 in Fanconi's syndrome, the mechanisms inducing renal
glycosuria remain largely to be clarified (9). In this study, we report the cDNA cloning of a novel Na+-dependent
glucose transporter (rNaGLT1) that would play a critical role in the
tubular reabsorption of glucose.
Materials--
Construction of a cDNA Library from Rat Kidney mRNA and
Its Screening--
Total RNA was extracted from the kidneys of
9-week-old male rats (n = 6) by the guanidine
isothiocyanate/CsCl ultracentrifugation method. The
poly(A)+ RNA was purified by chromatography on an
oligo(dT)-cellulose affinity column (Stratagene, La Jolla, CA) as
described previously (15). The cDNA library was constructed using
the Microdissection of Rat Nephrons--
Rat nephrons were
microdissected as described previously (16). Briefly, the left kidneys
of 7-week-old rats weighing 140-160 g were perfused and removed.
Slices were cut along the medullary axis and incubated with
collagenase. The tubules were microdissected to obtain the following
structures: glomerulus, proximal convoluted tubule, proximal straight
tubule, medullary thick ascending limb, cortical thick ascending limb,
cortical collecting duct, outer medullary collecting duct, and inner
medullary collecting duct. After microdissection, 20 glomeruli and 8 mm
of each dissected tubule segment were transferred into tubes to isolate
total RNA using an RNeasy® minikit (QIAGEN Inc., Hilden, Germany).
Reverse Transcription (RT)-PCR Analysis--
Total RNA from the
dissected tubules or poly(A)+ RNA from rat tissues (brain,
heart, lung, liver, small intestine, spleen, kidney cortex, and kidney
medulla) was reverse-transcribed with random hexamers using Superscript
II reverse transcriptase (Invitrogen), followed by RNase H (Invitrogen)
digestion. These single-stranded DNA fragments were amplified with
primer sets specific for rNaGLT1, rSGLT1, rSGLT2, and rat
glyceraldehyde-3-phosphate dehydrogenase (rGAPDH), as shown in Table
I.
Northern Blot Analysis--
Five micrograms of total RNA from
rat kidney or 1 µg of poly(A)+ RNA from the eight tissues
was electrophoresed on 1% denaturing agarose gel containing
formaldehyde and transferred onto Hybond® N+
nylon membranes (Amersham Biosciences). The transferred RNAs were
linked to the nylon membrane by a UV cross-linker. The quality of the
RNA was assessed by ethidium bromide staining. After transfer, the
blots were hybridized under high stringency conditions (50% formamide,
5× saline/sodium phosphate/EDTA (20 × saline/sodium phosphate/EDTA = 3 M NaCl, 0.2 M
NaH2PO4, and 0.02 M EDTA, pH 7.4),
5× Denhardt's solution, 0.1% sodium dodecyl sulfate, and 10 µg/ml herring sperm DNA at 42 °C) with rNaGLT1, rSGLT1, rSGLT2, and rGAPDH cDNA fragments labeled with [ Polyclonal Antibody against rNaGLT1--
Polyclonal antibody was
raised against the synthetic peptide corresponding to the intracellular
domains near the C terminus (LPLDRKQEKSINSEGQ) of rNaGLT1. The peptide
was synthesized with cysteine for its N terminus (purity of 92.0% upon
high performance liquid chromatography; Sawady Technology Co., Ltd.,
Tokyo). A male Japanese White rabbit (2 kg) was immunized with 0.2 mg
of conjugates emulsified with Freund's complete adjuvant. Booster shots of conjugates emulsified with Freund's incomplete adjuvant were
injected every 2 weeks until the antibody was obtained. After each
booster shot, blood was collected, and antibody production was
determined by enzyme-linked immunosorbent assay. To verify the
specificity of antibody against rNaGLT1 protein, Xenopus
oocytes injected with rNaGLT1 cRNA were used in the immunohistochemical analysis. Capped cRNA from rNaGLT1 was transcribed from
NotI-linearized pBK-CMV containing rNaGLT1 cDNA with T3
RNA polymerase as described previously (17). Oocytes were injected with
water (50 nl) or rNaGLT1 cRNA (25 ng). Three days after injection,
oocytes were fixed with 4% paraformaldehyde in phosphate-buffered
saline. Fixed oocytes were embedded in O.C.T. compound (Sakura
Finetechnical, Tokyo) and rapidly frozen at Immunoblot Analysis--
Crude plasma membrane fractions were
prepared from rat kidney cortex as reported previously (18). The
brush-border and basolateral membrane fractions were obtained from rat
renal cortex as described previously (19, 20). To carry out immunoblot
analysis, the membrane fractions were solubilized in sample buffer (2%
SDS, 125 mM Tris, and 20% glycerol) in the presence or
absence of 50 mM dithiothreitol and heated at 95 °C for
10 min. The samples were separated by 10% SDS-PAGE and transferred to
polyvinylidene difluoride membranes (Hybond®) by semidry
electroblotting for 35 min. The blots were incubated with the purified
antibody (1:4000 dilution) and detected on x-ray film by enhanced
chemiluminescence with horseradish peroxidase-conjugated anti-rabbit
IgG and cyclic diacylhydrazides (Amersham Biosciences). To
confirm the specificity of the antibody, the antibody was absorbed with
an excess amount of peptide (5 µg/ml) used as immunogen and processed similarly.
Real-time PCR--
Real-time PCR was performed using the ABI
PRISM 7700TM sequence detector (Applied Biosystems, Foster,
CA) as described previously (21). The specific primers, the TaqMan
probe, and the target sequence for real-time PCR are listed in Table
II. The cDNA fragments of the target
sequences were generated by RT-PCR with specific primers from rat
kidney total RNA. Each PCR product was ligated into the pGEM-T-Easy
vector (Promega, Madison, WI) and transformed into competent DH5 Functional Expression and Uptake Analysis of rNaGLT1 in Xenopus
oocytes--
Xenopus oocytes were injected with water (50 nl) or rNaGLT1 cRNA (25 ng). Three days after injection, the uptake
experiment was initiated by incubating oocytes at 25 °C in 500 µl
of uptake buffer (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2,
and 5 mM HEPES, pH 7.4) containing
Statistical Analysis--
Data are expressed as means ± S.E. Data were analyzed statistically by one-way analysis of variance
followed by Fisher's t test. Data from quantitative RT-PCR
analysis were analyzed statistically using Student's paired
t test with Bonferroni's correction.
A single cDNA clone encoding rNaGLT1 was isolated from the rat
kidney cDNA library and sequenced. The rNaGLT1 cDNA consists of
2173 bp with an open reading frame encoding a 484-amino acid protein
(calculated molecular mass of 51.7 kDa) and a poly(A)+
tail. Fig. 1A shows the
Kyte-Doolittle hydropathy analysis of rNaGLT1 (22). Membrane-spanning
regions and the N-terminal orientation were predicted using the TMpred program (31)2 and the SOSUI
program (32).3 On the basis
of physicochemical properties (i.e. hydrophobicity, charges,
and distribution) of amino acid residues and their sequences, an
11-transmembrane-spanning region model of rNaGLT1 was developed (Fig.
1B). The topology of rNaGLT1 includes five extracellular and
five intracellular loops; the N terminus is oriented extracellularly; and the 34-amino acid C terminus is oriented toward the cytoplasm.
-methyl-D-glucopyranoside and therefore was termed rat
Na+-dependent glucose transporter 1 (rNaGLT1).
The rNaGLT1 cDNA (2173 bp) has an open reading frame encoding a
484-amino acid protein, showing <22% homology to known SGLT and GLUT
glucose transporters.
-Methyl-D-glucopyranoside uptake
by rNaGLT1 cRNA-injected oocytes showed saturability, with an apparent
Km of 3.7 mM and a coupling ratio of
1:1 with Na+. rNaGLT1 mRNA was expressed predominantly
in the kidney upon Northern blot analysis and reverse
transcription-PCR. Reverse transcription-PCR in microdissected nephron
segments revealed that rNaGLT1 mRNA was primarily localized in the
proximal tubules. A clear signal corresponding to rNaGLT1 protein was
recognized in the brush-border (but not basolateral) membrane fraction
by immunoblot analysis. The rNaGLT1 mRNA level in the kidney was significantly higher than rat SGLT1 and SGLT2 mRNA levels. These findings suggest that rNaGLT1 is a novel
Na+-dependent glucose transporter with low
substrate affinity that mediates tubular reabsorption of glucose.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-methyl-D-glucopyranoside (
-MeGlc) in an
Na+-dependent manner.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-[U-14C]MeGlc (11.7 GBq/mmol),
D-[1-14C]galactose (2.07 GBq/mmol), and
D-[U-14C]mannose (11.6 GBq/mmol) were
obtained from Amersham Biosciences (Uppsala, Sweden).
D-[1-3H]Mannitol (629 GBq/mmol),
[3H]digoxin (584.6 GBq/mmol),
1-[3H]methyl-4-phenylpyridinium acetate (2886 GBq/mmol),
[6,7-3H]estrone sulfate (1609 GBq/mmol),
[14C]glycylsarcosine (1.848 GBq/mmol), and
L-[3H]leucine (1573 GBq/mmol) were obtained
from PerkinElmer Life Sciences. D-[3H]Glucose
(566.1 GBq/mmol), 2-[1,2-3H]deoxy-D-glucose
(2-DG; 925 GBq/mmol), and L-[2,5-3H]histidine
(1628 GBq/mmol) were purchased from Moravek Biochemicals, Inc. (Brea,
CA). [9-3H]Quinidine (740 GBq/mmol) was purchased from
American Radiolabeled Chemicals (St. Louis, MO).
[14C]Ciprofloxacin hydrochloride (2.55 GBq/mmol)
was a gift from Bayer AG (Leverkusen-Bayerwerk, Germany).
Phlorizin,
-MeGlc, D-glucose, L-glucose,
D-galactose, D-mannose, D-fructose,
and D-mannitol were purchased from Nacalai Tesque, Inc.
(Kyoto, Japan). Phloretin and 2-DG were obtained from Sigma. All other
chemicals were of the highest purity available.
ZaP Express cDNA synthesis kit (Stratagene) according
to the manufacturer's instructions. After conversion from phage to
plasmid, 1000 colonies were randomly chosen from this cDNA library
and sequenced using the RISA-384 multicapillary DNA sequencing system
(Shimadzu, Kyoto) from the 5'-end with primer T3. The resulting
sequences were submitted to a BLAST search of the
DDBJ/GenBankTM/EMBL Data Bank and the Protein Data Bank.
Four-hundred unknown clones from the rat kidney cDNA library were
subjected to a BLAST search. These clones were screened for the
transport activity of nine compounds using Xenopus oocytes.
-32P]dCTP
(29.6 TBq/mmol; Amersham Biosciences). Each probe was obtained by
RT-PCR amplification of rat kidney total RNA as described above (Table
I). After hybridization, the blots were
washed three times with 2× SSC (20× SSC = 3 M NaCl
and 0.3 M sodium citrate, pH 7.0) containing 0.1% SDS at
42 °C for 10 min and then twice with 0.5× SSC and 0.1% SDS at
42 °C for 30 min. The dried membranes were exposed to the imaging
plates of a Fujix BIO-imaging BAS-2000 II analyzer (Fuji Photo
Film Co., Tokyo, Japan). To compare the expression levels of
rNaGLT1 and rSGLT1 or rSGLT2, the experimental conditions (such as the
quantity of labeled probes, the wash conditions, and the exposure time)
were kept uniform.
Oligonucleotide sequences of PCR primers used for the determination of
glucose transporters and rGAPDH by RT-PCR and Northern blotting
20 °C. Tissues
were cut into 12-µm-thick sections, mounted on glass slides, and
covered with 10% goat serum for 1 h. The covered sections were
incubated with anti-rNaGLT1 serum (1:500 dilution) with or without
preabsorption by the synthesized antigen peptide (50 µg/ml) for
1 h and then incubated with Cy3-labeled anti-rabbit IgG (Caltag
Laboratories, San Francisco, CA) for 1 h. These sections were
examined with a BX-50-FLA fluorescence microscope (Olympus, Tokyo) at
magnification ×100. Images were captured with a DP-50 CCD camera
(Olympus) using Studio Lite software (Olympus).
cells (Invitrogen). The concentrations of the purified plasmid DNA were
measured by spectrophotometry, and corresponding copy numbers were
calculated. Serial dilutions of the respective plasmid DNA were used as
standards to make calibration curves. PCR amplification was performed
in a total volume of 20 µl containing 5 µl of cDNA sample, 1 µM each primer, 0.2 µM TaqMan probe, and 10 µl of TaqMan Universal PCR Master Mix (Applied Biosystems). rGAPDH mRNA was also measured as an internal control with
TaqMan® rodent GAPDH control reagents (Applied
Biosystems).
Nucleotide sequences of the primers and probes used for real-time PCR
-[14C]MeGlc or radiolabeled sugar analogs (37 kBq/ml)
in the presence or absence of unlabeled inhibitors for 1 h unless
otherwise indicated. Uptake in the absence of Na+ or at a
lowered Na+ concentration was measured by substituting NaCl
with choline chloride and by adding valinomycin. The uptake reaction
was terminated by adding 2 ml of ice-cold uptake buffer, followed by
washing the oocytes five times with 2 ml of the buffer. After the wash, each oocyte was dissolved with 300 µl of 10% SDS. Radioactivity was
determined by adding 3 ml of ACSII (Amersham Biosciences) to
each solubilized oocyte in a liquid scintillation counter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Hydropathy plot (A) and
deduced amino acid sequence (B) of rNaGLT1.
A, shown is a Kyte-Doolittle hydropathy plot with a window
of 13 amino acid residues. Numbers indicate putative
membrane-spanning regions. B, potential N-linked
glycosylation sites are indicated by asterisks. Potential
protein kinase C ( ) and protein kinase A (
) phosphorylation sites
are indicated.
The amino acid sequence of rNaGLT1 shares <22% homology with known SGLT and GLUT glucose transporters and might belong to a new family of membrane transporters. Comparison of the amino acid sequence using the BLAST program revealed that rNaGLT1 shares a high degree of amino acid homology with three hypothetical proteins, of which only primary structures were clarified without any functional characterization (Table III). Compared with known proteins, rNaGLT1 is slightly homologous to the multidrug efflux transporter and the glucose/galactose transporter of Xylella fastidiosa in part (Table III). In addition, the rNaGLT1 amino acid sequence does not contain any conserved domain when searched against the Smart4 and Pfam5 libraries.
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We examined the tissue distribution of rNaGLT1 mRNA transcripts by
Northern blot analysis (Fig.
2A). Under high stringency conditions, a full-length rNaGLT1 probe was hybridized with mRNA transcripts from rat kidney cortex and medulla. For PCR analysis of
rNaGLT1 mRNA expression, a set of specific primers for the cDNA
of rNaGLT1 was used. As shown in Fig. 2B, a PCR product of the expected size for rNaGLT1 was found in rat brain, liver, lung, kidney cortex, and kidney medulla.
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To examine the distribution of rNaGLT1 in the kidney, we performed
RT-PCR analysis of the microdissected nephron segments. With RT and
subsequent PCR and Southern blotting, a signal with the predicted size
of 468 bp for rNaGLT1 was detected in the proximal convoluted and
straight tubules (Fig. 3). Faint signals
with the same size were detected in the medullary thick ascending limb and cortical collecting duct. Similarly, rSGLT1 and rSGLT2 were also
detected primarily in the proximal convoluted and straight tubules
(Fig. 3).
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To clarify the membrane localization of rNaGLT1 protein, we performed
immunoblot analysis of rNaGLT1 in rat kidney crude membranes, brush-border membranes, and basolateral membranes using
affinity-purified antibody against rNaGLT1. Under nonreducing
conditions without dithiothreitol, an immunoreactive protein with an
apparent molecular mass of 97 kDa was strongly expressed in the
brush-border membrane fraction, slightly expressed in the crude
membrane fraction, and faintly expressed in the basolateral membrane
fraction (Fig. 4A). Under
reducing conditions with 50 mM dithiothreitol, a signal was
detected at ~50 kDa in the brush-border membrane fraction (Fig.
4B). The band in the crude membrane fraction was slightly larger than that in the brush-border membrane fraction. The
immunoreactive bands in the brush-border and crude membranes were
completely abolished when the antibody was preabsorbed with the antigen
peptide (5 µg/ml) (Fig. 4, C and D), suggesting
that the positive bands observed in the brush-border and crude
membranes were specific for rNaGLT1. The oocytes expressing rNaGLT1
displayed strong signals along the plasma membranes and weak signals in
the cytoplasm, although oocytes injected with water exhibited no
labeling of the membranes or cytoplasm (Fig.
5, A and B). In
addition, the signals were completely abolished when the antibody was
preabsorbed with the antigen peptide (50 µg/ml) (Fig.
5C).
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To characterize the transport function of rNaGLT1, the accumulation of
various sugar analogs was measured in Xenopus oocytes injected with rNaGLT1 cRNA (Fig. 6). The
uptake of -MeGlc and D-glucose was significantly
increased, but the other analogs tested showed little or no uptake in
rNaGLT1 cRNA-injected compared with water-injected oocytes. The
substrate specificity of rNaGLT1 was tested by inhibition experiments
in which inhibition of the uptake of
-[14C]MeGlc (2 mM for 1 h) was determined in the presence of various sugar analogs (30 mM), phlorizin (25 µM), or
phloretin (50 µM) (Fig. 7).
-MeGlc, D-glucose, and phlorizin completely inhibited the uptake. The uptake of
-[14C]MeGlc was strongly
inhibited by 2-DG and slightly inhibited by fructose and phloretin,
whereas L-glucose, 3-O-methylglucose, galactose,
and mannose had little or no effect.
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The saturation of -MeGlc uptake in rNaGLT1-injected oocytes is
demonstrated in Fig. 8A. Based
on Eadie-Hofstee plot analysis, the apparent Km and
Vmax values for
-MeGlc were calculated to be 3.71 ± 0.09 mM and 136.3 ± 17.0 pmol/oocyte/h, respectively. The Na+ dependence of
-MeGlc uptake in rNaGLT1-injected oocytes was examined by measuring
-[14C]MeGlc uptake as a function of extracellular
[Na+] with the membrane voltage clamped to zero (equal
internal and external [K+] in the presence of 7 µM valinomycin). In the rNaGLT1 cRNA-injected oocytes,
the
-MeGlc uptake was stimulated by the extracellular Na+ in a concentration-dependent manner,
although the uptake was not saturated at Na+ concentrations
between 0 and 96 mM (Fig. 8B). Hill plot
analyses using the data in Fig. 8 (A and B) gave
coefficients of 1.06 and 1.00, respectively, indicating that the
Na+/glucose coupling ratio for rNaGLT1 is 1:1 (Fig. 8,
C and D).
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The results of Northern blotting of rNaGLT1, rSGLT1, and rSGLT2 in the
kidney cortex or medulla are shown in Fig.
9. The relative quantity of rNaGLT1,
rSGLT1, and rSGLT2 was estimated by densitometry. rNaGLT1 mRNA was
expressed more abundantly in the cortex than in the medulla, and its
expression level was higher than that of rSGLT1 or rSGLT2. To compare
the expression levels of rNaGLT1 and rSGLT mRNAs precisely, we
performed a quantitative RT-PCR analysis (Fig.
10). The mRNA levels of rNaGLT1
both in the cortex (Fig. 10A) and medulla (Fig.
10B) were significantly higher than those of rSGLT1 or
rSGLT2.
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DISCUSSION |
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We have isolated and characterized a cDNA encoding a novel
Na+-dependent glucose transporter (rNaGLT1)
predominantly expressed in rat kidney. rNaGLT1 transports -MeGlc in
an Na+-dependent manner with low substrate
affinity, with the apparent Km for
-MeGlc being
3.7 mM (Fig. 8). It is known that there are high and low
affinity types of Na+-dependent glucose
transporters, whose Km values are 0.35 and 6 mM, respectively, in rabbit kidney (23). rNaGLT1 was
suggested to be one of the low affinity-type
Na+-dependent glucose transporters because the
characteristics of rNaGLT1 are similar to those rSGLT2 in several ways,
as follows: 1) its mRNA was abundantly expressed in the kidney
cortex and primarily in the proximal convoluted and straight tubules
(Figs. 2 and 3); 2) rNaGLT1 was expressed in the brush-border membranes of proximal tubules (Fig. 4); 3) galactose was not recognized as its
substrate (Fig. 6); and 4) the coupling ratio of sodium to glucose was
1:1 (Fig. 8) (11). Despite such similarity to rSGLT2, rNaGLT1 shares a
low degree of amino acid sequence homology with SGLTs or GLUTs. Our
previous report demonstrated that the Vmax for
low affinity-type Na+-dependent glucose
transport activity in renal brush-border membrane vesicles was
significantly decreased in the 5/6 nephrectomized rats, an
animal model of chronic renal failure, compared with the sham-operated
controls, although the expression level of SGLT2 mRNA was
maintained in 5/6 nephrectomized rats (24). The reason for this
discrepancy of low affinity-type glucose transport in the 5/6
nephrectomized rats remains unknown and could be accounted for by
rNaGLT1. Therefore, rNaGLT1 would be another low affinity-type Na+-dependent glucose transporter and belongs
to a new gene family in view of its structure.
A signal corresponding to rNaGLT1 was recognized in the crude membrane fraction, and it was concentrated further in the brush-border membrane fraction by immunoblot analysis under nonreducing conditions (Fig. 4A). This indicates that rNaGLT1 was localized in the brush-border membranes of the proximal tubule. However, a faint band was also detected in the basolateral membrane fraction. The basolateral membrane fraction was purified by Percoll density gradient centrifugation (18). Although this method is useful for obtaining the basolateral membrane fraction, slight contamination of brush-border membranes cannot be avoided. Therefore, the faint band shown in the basolateral membrane fraction might be due to the contamination of brush-border membranes. The approximate molecular mass of the rNaGLT1 signal is 97 kDa (Fig. 4A). This is markedly larger than the molecular mass predicted from the amino acid sequence (51.7 kDa). However, when the crude and brush-border membrane fractions were reduced by dithiothreitol, a signal was detected at ~50 kDa, which is comparable to the predicted size of rNaGLT1 (Fig. 4B). These findings indicate that rNaGLT1 might form homodimer or a complex with other proteins.
rNaGLT1 mRNA was expressed abundantly in the kidney cortex (Fig.
2), and its level was ~3-fold higher in the cortex and 5-fold higher
in the medulla compared with rSGLT2 mRNA (Fig. 10). Similar to
rSGLT2, the uptake activity of -[14C]MeGlc in rNaGLT1
cRNA-injected oocytes is 2.5-4-fold higher than that in water-injected
oocytes (11, 12). The low glucose uptake activity shown in
rNaGLT1-expressing oocytes might be accounted for by the non-saturable
extracellular concentration of Na+, for which the apparent
Km was estimated as 202 mM, and/or by
poor ability to express rNaGLT1.
The inhibitory effects of glucose analogs on the uptake of -MeGlc in
rNaGLT1-injected oocytes are almost comparable to those of
rSGLT2, except for phloretin and 2-DG (Fig. 7) (11, 12). In general,
phloretin and 2-DG are known to inhibit GLUT-mediated glucose transport
(25, 26). However, phloretin slightly inhibits glucose transport in rat
renal brush-border membrane vesicles (27). In addition, as the
concentration of 2-DG (30 mM) induces ATP depletion and
cytotoxicity (28), the inhibition of
-MeGlc uptake by 2-DG might be
caused by a nonspecific disturbance of the activity. Taken together,
the decrease in rNaGLT1-mediated uptake in the presence of phloretin
and 2-DG is considered not to contradict previous findings of renal
Na+-dependent glucose transport.
The rNaGLT1 mRNA was detected only in the kidney cortex and medulla by Northern blot analysis (Fig. 2A). On the other hand, the amplified products corresponding to rNaGLT1 were detected not only in the kidney, but also in the brain, lung, and liver by RT-PCR analysis. rNaGLT1 mRNA was expressed primarily in the kidney, but rNaGLT1 itself or its homolog also might be expressed slightly in several other tissues. Previous reports showed that the brain and lung, as well as the intestine and kidney, possess activity for Na+-dependent glucose transport (29, 30). It remains to be clarified whether rNaGLT1 or its analog takes part in glucose transport in such tissues or not.
In this study, we successfully isolated rNaGLT1 from a rat kidney
mRNA data base constructed by the random sequencing technique. This
might be the first demonstration of the cloning of a novel transporter
by the random sequencing of a cDNA library coupled with the
functional expression technique. Although many genes have recently been
submitted to public data bases such as the DDBJ/GenBankTM/EMBL Data Bank, ~40% of the genes in the
rat kidney data base are unknown. In addition, theoretically, a clone
from the random sequence data base is expressed abundantly in
vivo (6-8). Therefore, this method should be useful for the
cloning of novel critical transporters. The physiological role of
rNaGLT1, especially in correlation with SGLTs and GLUTs, remains to be
clarified. Studies using diabetic and nephropathic animal models or
gene knockout animals might be useful for the determination of the
pathophysiological role of rNaGLT1.
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FOOTNOTES |
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* This work was supported by Grant-in-aid for Research on Human Genome, Tissue Engineering, and Food Biotechnology H12-Genome-019 from the Ministry of Health, Labor, and Welfare of Japan and by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB089802.
To whom correspondence should be addressed. Tel.: 81-75-751-3577;
Fax: 81-75-751-4207; E-mail: inui@kuhp.kyoto-u.ac.jp.
Published, JBC Papers in Press, February 14, 2003, DOI 10.1074/jbc.M212240200
2 Available at www.expasy.ch.
3 Available at www.sosui.proteome.bio.taut.ac.jp.
4 Available at smart.embl-heidelberg.de/.
5 Available at pfam.wust1.edu/.
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ABBREVIATIONS |
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The abbreviations used are:
rNaGLT1, rat
Na+-dependent glucose transporter 1;
-MeGlc,
-methyl-D-glucopyranoside;
rSGLT, rat
sodium-dependent glucose transporter;
GLUT, glucose
transporter;
2-DG, 2-deoxy-D-glucose;
RT, reverse
transcription;
rGAPDH, rat glyceraldehyde-3-phosphate
dehydrogenase.
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