Cloning and Characterization of a Novel Na+-dependent Glucose Transporter (NaGLT1) in Rat Kidney*

Naoshi Horiba, Satohiro Masuda, Ayako Takeuchi, Daisuke Takeuchi, Masahiro Okuda, and Ken-ichi InuiDagger

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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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. alpha -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

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 alpha -methyl-D-glucopyranoside (alpha -MeGlc) in an Na+-dependent manner.

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- alpha -[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, alpha -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.

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 lambda 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.

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 [alpha -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.


                              
View this table:
[in this window]
[in a new window]
 
Table I
Oligonucleotide sequences of PCR primers used for the determination of glucose transporters and rGAPDH by RT-PCR and Northern blotting
RT-PCR and Northern blotting were performed as described under "Experimental Procedures." Positions are from the rat sequence in the GenBank TM/EBI Data Bank, and the accession numbers are indicated.

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 -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).

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 DH5alpha 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).


                              
View this table:
[in this window]
[in a new window]
 
Table II
Nucleotide sequences of the primers and probes used for real-time PCR
Real-time PCR was performed as described under "Experimental Procedures." Positions are from the rat sequence in the GenBankTM/EBI Data Bank, and accession numbers are indicated.

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 alpha -[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.

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (38K):
[in this window]
[in a new window]
 
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 (triangle ) 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.


                              
View this table:
[in this window]
[in a new window]
 
Table III
Comparison of rNaGLT1 with hypothetical proteins
Homology was compared using GENETYX-MAC version 10.1. GenBankTM/EBI accession numbers are given. AA, amino acids.

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.


View larger version (49K):
[in this window]
[in a new window]
 
Fig. 2.   Northern blot analysis (A) and detection by PCR amplification (B) of rNaGLT1 mRNA in rat tissues. A, poly(A)+ RNA (1 µg) from the tissues indicated was electrophoresed, blotted, and hybridized with the specific probe for rNaGLT1 under high stringency conditions. B, poly(A)+ RNA (1 µg) from the tissues indicated was reverse-transcribed, and the cDNA synthesized was amplified using a set of primers specific for rNaGLT1. The PCR products were separated by electrophoresis on 1% agarose gels and stained with ethidium bromide.

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).


View larger version (89K):
[in this window]
[in a new window]
 
Fig. 3.   Detection of rNaGLT1, rSGLT1, and rSGLT2 mRNAs in microdissected renal nephron segments by RT-PCR and subsequent Southern blotting. Each PCR amplification (35 cycles) was performed using part of the reverse transcription reaction derived from 20 glomeruli or 8 mm of renal tubule. The cDNA synthesized was amplified using a set of primers for rNaGLT1, rSGLT1, rSGLT2, and rGAPDH. The PCR products were separated by electrophoresis on 1.5% agarose gels. The agarose gels were transferred onto a nylon membrane and hybridized with the [32P]dCTP-labeled rNaGLT1, rSGLT1, rSGLT2, and rGAPDH cDNAs as probes under high stringency conditions. Glm, glomerulus; PCT, proximal convoluted tubule; PST, proximal straight tubule; MAL, medullary thick ascending limb; CAL, cortical thick ascending limb; CCD, cortical collecting duct; OMCD, outer medullary collecting duct; IMCD, inner medullary collecting duct; -RT, without reverse transcriptase.

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).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   Immunoblot analysis of crude, brush-border, and basolateral membranes from rat kidney cortex with anti-rNaGLT1 antibody. Forty micrograms of each membrane was separated by SDS-PAGE under nonreducing (A and C) or reducing (B and D) conditions. A and B, the affinity-purified antiserum for rNaGLT1 was used as the primary antibody. C and D, the affinity-purified antiserum preabsorbed with the antigen peptide (5 µg/ml) of rNaGLT1 was used. Horseradish peroxidase-conjugated anti-rabbit IgG was used for detection of bound antibodies, and the strips of blots were visualized by chemiluminescence on x-ray film. The arrows indicate the positions of 97 and 50 kDa. BBM, brush-border membrane; BLM, basolateral membrane.


View larger version (52K):
[in this window]
[in a new window]
 
Fig. 5.   Expression of rNaGLT1 proteins in Xenopus oocytes. Oocytes were injected with water (50 nl; A) or in vitro transcribed rNaGLT1 cRNA (25 ng/oocyte; B and C). Three days after injection, oocytes were fixed, frozen, sectioned, and stained as described under "Experimental Procedures." A and B, the affinity-purified antiserum for rNaGLT1 was used as the primary antibody. C, the affinity-purified antiserum preabsorbed with the antigen peptide (50 µg/ml) of rNaGLT1 was used. Cy3-labeled anti-rabbit IgG was used for detection of bound antibodies.

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 alpha -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 alpha -[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). alpha -MeGlc, D-glucose, and phlorizin completely inhibited the uptake. The uptake of alpha -[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.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 6.   Uptake of sugar analogs by Xenopus oocytes injected with rNaGLT1 cRNA. Uptake assays were performed with oocytes incubated at 25 °C for 1 h in buffer containing sugar analogs (2 mM, 37 kBq/ml) 3 days after injection of water (50 nl; white bars) or in vitro transcribed rNaGLT1 cRNA (25 ng/oocyte; gray bars). Each bar represents the mean ± S.E. of 8-10 oocytes. *, p < 0.05, significantly different from water-injected oocytes (Student's unpaired t test).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 7.   Effects of sugar analogs, phlorizin, and phloretin on alpha -[14C]MeGlc uptake by Xenopus oocytes. The uptake of alpha -MeGlc (2 mM, 37 kBq/ml) by oocytes was measured for 1 h at 25 °C in the absence and presence of sugar analogs (30 mM), phlorizin (50 µM), or phloretin (25 µM). White bars, oocytes injected with water (50 nl); gray bars, oocytes injected with in vitro transcribed rNaGLT1 cRNA (25 ng/oocyte). Each bar represents the mean ± S.E. of 8-10 oocytes. *, p < 0.05, significantly different from control values (Fisher's t test). 3-OMG, 3-O-methylglucose.

The saturation of alpha -MeGlc uptake in rNaGLT1-injected oocytes is demonstrated in Fig. 8A. Based on Eadie-Hofstee plot analysis, the apparent Km and Vmax values for alpha -MeGlc were calculated to be 3.71 ± 0.09 mM and 136.3 ± 17.0 pmol/oocyte/h, respectively. The Na+ dependence of alpha -MeGlc uptake in rNaGLT1-injected oocytes was examined by measuring alpha -[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 alpha -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).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 8.   Concentration and Na+ dependence of rNaGLT1-mediated alpha -MeGlc uptake by Xenopus oocytes. A, the uptake of alpha -MeGlc by oocytes injected with rNaGLT1 cRNA (25 ng/oocyte) was assayed for 1 h at 25 °C in incubation buffer at various concentrations (0.5-15 mM), and the uptake measured in water-injected oocytes was subtracted. The inset shows an Eadie-Hofstee plot of the uptake. B, the uptake of alpha -MeGlc by oocytes injected with rNaGLT1 cRNA (25 ng/oocyte) was assayed for 1 h at 25 °C in incubation buffer at various Na+ concentrations (0-96 mM), and the uptake measured in water-injected oocytes was subtracted. The inset shows an Eadie-Hofstee plot of the uptake. C, shown is a Hill (log to log) plot of the data from Fig. 8A. D, shown is a Hill plot of the data from Fig. 8B. Each point represents the mean ± S.E. of 8-10 oocytes. The apparent Km values were obtained from three independent experiments.

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.


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 9.   Northern blot analysis of rNaGLT1, rSGLT1, and rSGLT2 in the kidney cortex and medulla. A, 5 µg of total RNA from the kidney cortices and medullas of four rats was electrophoresed, blotted, and hybridized with the probes for rNaGLT1, rSGLT1, rSGLT2, and rGAPDH under high stringency conditions. B, the amounts of mRNA were quantified by densitometry and normalized to rGAPDH. Each bar represents the mean ± S.E. of four rats.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 10.   Quantification of rNaGLT1, rSGLT1, and rSGLT2 mRNAs in the kidney cortex (A) and medulla (B) by real-time PCR. Total cellular RNA was extracted from rat kidney cortex (A) and medulla (B), and the extracted RNA was reverse-transcribed. The rNaGLT1, rSGLT1, and rSGLT2 mRNA levels were determined by real-time PCR using an ABI PRISM 7700TM sequence detector. Each bar represents the mean ± S.E. of four rats. *, p < 0.05, significantly different from rNaGLT1 (Student's paired t test with Bonferroni's correction).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have isolated and characterized a cDNA encoding a novel Na+-dependent glucose transporter (rNaGLT1) predominantly expressed in rat kidney. rNaGLT1 transports alpha -MeGlc in an Na+-dependent manner with low substrate affinity, with the apparent Km for alpha -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 alpha -[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 alpha -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 alpha -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.

    FOOTNOTES

* 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.

Dagger 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/.

    ABBREVIATIONS

The abbreviations used are: rNaGLT1, rat Na+-dependent glucose transporter 1; alpha -MeGlc, alpha -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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Collins, F. S., Patrinos, A., Jordan, E., Chakravarti, A., Gesteland, R., and Walters, L. (1998) Science 282, 682-689[Abstract/Free Full Text]
2. RIKEN Genome Exploration Research Group Phase II Team FANTOM Consortium. (2001) Nature 409, 685-690[CrossRef][Medline] [Order article via Infotrieve]
3. Allikmets, R., Gerrard, B., Glavac, D., Ravnik-Glavac, M., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., Modi, W., and Dean, M. (1995) Mamm. Genome 6, 114-117[Medline] [Order article via Infotrieve]
4. Phay, J. E., Hussain, H. B., and Moley, J. F. (2000) Surgery 128, 946-951[CrossRef][Medline] [Order article via Infotrieve]
5. Okubo, K., Hori, N., Matoba, R., Niiyama, T., Fukushima, A., Kojima, Y., and Matsubara, K. (1992) Nat. Genet. 2, 173-179[Medline] [Order article via Infotrieve]
6. Takenaka, M., Imai, E., Kaneko, T., Ito, T., Moriyama, T., Yamauchi, A., Hori, M., Kawamoto, S., and Okubo, K. (1998) Kidney Int. 53, 562-572[CrossRef][Medline] [Order article via Infotrieve]
7. Takenaka, M., Imai, E., Nagasawa, Y., Matsuoka, Y., Moriyama, T., Kaneko, T., Hori, M., Kawamoto, S., and Okubo, K. (2000) Kidney Int. 57, 19-24[Medline] [Order article via Infotrieve]
8. Nakajima, H., Takenaka, M., Kaimori, J., Nagasawa, Y., Kosugi, A., Kawamoto, S., Imai, E., Hori, M., and Okubo, K. (2002) Kidney Int. 61, 1577-1587[CrossRef][Medline] [Order article via Infotrieve]
9. Wright, E. M. (2001) Am. J. Physiol. 280, F10-F18
10. Hediger, M. A., Coady, M. J., Ikeda, T. S., and Wright, E. M. (1987) Nature 330, 379-381[CrossRef][Medline] [Order article via Infotrieve]
11. You, G., Lee, W. S., Barros, E. J., Kanai, Y., Huo, T. L., Khawaja, S., Wells, R. G., Nigam, S. K., and Hediger, M. A. (1995) J. Biol. Chem. 270, 29365-29371[Abstract/Free Full Text]
12. Kanai, Y., Lee, W. S., You, G., Brown, D., and Hediger, M. A. (1994) J. Clin. Invest. 93, 397-404[Medline] [Order article via Infotrieve]
13. Turk, E., Zabel, B., Mundlos, S., Dyer, J., and Wright, E. M. (1991) Nature 350, 354-356[CrossRef][Medline] [Order article via Infotrieve]
14. Santer, R., Schneppenheim, R., Dombrowski, A., Gotze, H., Steinmann, B., and Schaub, J. (1997) Nat. Genet. 17, 324-326[Medline] [Order article via Infotrieve]
15. Saito, H., Masuda, S., and Inui, K.-i. (1996) J. Biol. Chem. 271, 20719-20725[Abstract/Free Full Text]
16. Masuda, S., Saito, H., Nonoguchi, H., Tomita, K., and Inui, K.-i. (1997) FEBS Lett. 407, 127-131[CrossRef][Medline] [Order article via Infotrieve]
17. Uwai, Y., Okuda, M., Takami, K., Hashimoto, Y., and Inui, K.-i. (1998) FEBS Lett. 438, 321-324[CrossRef][Medline] [Order article via Infotrieve]
18. Ogihara, H., Saito, H., Shin, B. C., Terada, T., Takenoshita, S., Nagamichi, Y., Inui, K.-i., and Takata, K. (1996) Biochem. Biophys. Res. Commun. 220, 848-852[CrossRef][Medline] [Order article via Infotrieve]
19. Inui, K.-i., Okano, T., Takano, M., Kitazawa, S., and Hori, R. (1981) Biochim. Biophys. Acta 647, 150-154[Medline] [Order article via Infotrieve]
20. Takahashi, K., Nakamura, N., Terada, T., Okano, T., Futami, T., Saito, H., and Inui, K.-i. (1998) J. Pharmacol. Exp. Ther. 286, 1037-1042[Abstract/Free Full Text]
21. Motohashi, H., Sakurai, Y., Saito, H., Masuda, S., Urakami, Y., Goto, M., Fukatsu, A, Ogawa, O, and Inui, K.-i. (2002) J. Am. Soc. Nephrol. 13, 866-874[Abstract/Free Full Text]
22. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132[Medline] [Order article via Infotrieve]
23. Turner, R. J., and Moran, A. (1982) Am. J. Physiol. 242, F406-F414[Medline] [Order article via Infotrieve]
24. Takahashi, K., Masuda, S., Nakamura, N., Saito, H., Futami, T., Doi, T., and Inui, K.-i. (2001) Am. J. Physiol. 281, F1109-F1116
25. Cheung, P. T., and Hammerman, M. R. (1988) Am. J. Physiol. 254, F711-F718[Medline] [Order article via Infotrieve]
26. Rumsey, S. C., Kwon, O., Xu, G. W., Burant, C. F., Simpson, I., and Levine, M. (1997) J. Biol. Chem. 272, 18982-18989[Abstract/Free Full Text]
27. Kinne, R., Murer, H., Kinne-Saffran, E., Thees, M., and Sachs, G. (1975) J. Membr. Biol. 21, 375-395
28. Sridhar, R., Stroude, E. C., and Inch, W. R. (1979) In Vitro (Rockville) 15, 685-690
29. Lee, W. J., Peterson, D. R., Sukowski, E. J., and Hawkins, R. A. (1997) Am. J. Physiol. 272, C1552-C1557[Medline] [Order article via Infotrieve]
30. Oelberg, D. G., Xu, F., and Shabarek, F. (1994) Biochim. Biophys. Acta 1194, 92-98[Medline] [Order article via Infotrieve]
31. Hoffmann, K., and Stoffel, W. (1993) Biol. Chem. 374, 166
32. Hirokawa, T., Boon-Chieng, S., and Mitaku, S. (1998) Bioinformatics 14, 378-379[Abstract]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.