©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Characteristics of Na-coupled Glucose Transporters in Adult and Embryonic Rat Kidney (*)

(Received for publication, June 26, 1995; and in revised form, September 18, 1995)

Guofeng You Wen-Sen Lee (§) Elvino J. G. Barros (¶) Yoshikatsu Kanai (**) Teh-Li Huo Sadiqa Khawaja Rebecca G. Wells (§§) Sanjay K. Nigam Matthias A. Hediger (¶¶)

From the Renal Division, Department of Medicine, Brigham and Women's Hospital and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Two distinct Na-coupled glucose transporters (SGLTs) with either a high or a low affinity for glucose were shown to provide reabsorption of filtered glucose in the kidney. We have previously reported the characteristics of the high affinity Na/glucose cotransporter SGLT1 from rabbit, rat, and human kidney and the low affinity Na/glucose cotransporter SGLT2 from human kidney. Because the molecular identity of SGLT2 as the kidney cortical low affinity Na/glucose cotransporter has been recently challenged based on studies of the porcine low affinity Na/glucose cotransporter SAAT-pSGLT2 (Mackenzie, B., Panayotova-Heiermann, M., Loo, D. D. F., Lever, J. E., and Wright, E. M.(1994) J. Biol. Chem. 269, 22488-22491), we have reevaluated the properties of SGLT2 in greater detail. We furthermore report new data on the regulation of SGLT1 and SGLT2 during kidney development. To analyze and compare SGLT1 and SGLT2 in adult and embryonic kidney, we have cloned and characterized SGLT2 from rat kidney and determined its tissue distribution based on Northern analysis and in situ hybridization. When expressed in Xenopus oocytes, rat SGLT2 stimulated transport of alpha-methyl-D-glucopyranoside (2 mM) in oocytes up to 4.5-fold over controls with an apparent K of 3.0 mM. The transport properties (i.e. a Na to glucose coupling of 1:1 and lack of galactose transport) generally matched those of the kidney cortical low affinity system. We show that expression of rat SGLT2 mRNA is kidney specific and that it is strongly and exclusively expressed in proximal tubule S1 segments. Hybrid-depletion studies were performed to conclusively determine whether SGLT2 corresponds to the kidney cortical low affinity system. Injection of rat kidney superficial cortex mRNA into oocytes stimulated the uptake of alpha-methyl-D-glucopyranoside (2 mM) 2-3-fold. We show that hybrid depletion of this kidney RNA using an SGLT2 antisense oligonucleotide completely suppresses the uptake. These data strongly indicate that SGLT2 is the major kidney cortical low affinity glucose transporter. We therefore propose that SAAT-pSGLT2 be renamed SGLT3. Experiments addressing the expression of SGLT1 and SGLT2 mRNAs in embryonic rat kidneys reveal that the two Na/glucose cotransporters are developmentally regulated and that there may be a different splice variant for SGLT2 in embryonic kidney compared to the adult.


INTRODUCTION

Reabsorption of filtered glucose across epithelial cells of the kidney proceeds via Na-coupled glucose transporters (SGLTs) (^1)located in the brush-border membranes and facilitated glucose transporters (GLUTs) located in the basolateral membranes(1) . Studies of rabbit (2, 3, 4) and human (5) kidney brush border membrane vesicles and of rat kidney in vitro perfused tubules (6) revealed that efficient substrate transport in the mammalian kidney is provided by the concerted action of a low affinity high capacity and a high affinity low capacity Na/glucose cotransporter arranged in series along kidney proximal tubules.

Several members of the SGLT family have been cloned and characterized. The Na/glucose transporter SGLT1 was isolated from rabbit(7) , rat(8) , pig(9) , and human (10) and has the characteristics of the high affinity transporter with K values for alpha-methyl-D-glucopyranoside (alphaMeGlc) of 110 µM for rabbit, 400 µM for rat, 800 µM for human and with a Na to glucose coupling ratio of 2:1 (for review, see (1) ). In kidney, SGLT1 is expressed in proximal tubule S3 segments (8) where it is expected to reabsorb the remainder of filtered glucose that was not reabsorbed in proximal tubule S1 segments. We previously isolated a second low affinity Na/glucose cotransporter, named SGLT2, from human kidney(11, 15) . The amino acid sequence of human SGLT2 is 59% identical to that of rat SGLT1. When expressed in Xenopus oocytes, SGLT2 mediated the phlorizin-inhibitable Na-coupled uptake of D-glucose or alphaMeGlc. D-Galactose, which is a substrate of SGLT1, was not recognized by the transporter. The apparent Kvalue for alphaMeGlc was 1.6 mM and that for Na was 250-300 mM. The Na to glucose coupling ratio of SGLT2 was 1:1. In situ hybridization, which involved probing rat kidney tissue with the human SGLT2 cRNA probe, indicated that SGLT2 is strongly expressed in proximal tubule S1 segments. Based on these data, we proposed that SGLT2 corresponds to the previously described kidney cortical low affinity Na/glucose cotransporter responsible for reabsorption of the bulk of filtered glucose in proximal tubule S1 segments.

Recent studies by Mackenzie et al.(12) led to the identification of a second low affinity Na/glucose transporter isoform from porcine kidney LLC-PK1 cells (SAAT-pSGLT2). This protein has 59% amino acid sequence identity to human SGLT2 and 75% identity to human SGLT1. SAAT-pSGLT2 was originally reported to be a Na-coupled neutral amino acid transporter (SAAT1) with the characteristics of system A(13) . However, subsequent oocyte expression studies revealed that its transport properties are representative of a low affinity Na/glucose cotransporter(12) . The apparent K value for alphaMeGlc was 2.0 mM and that for Na was 10 mM. Based on Hill equation analysis, the Na to glucose coupling ratio was 1:1, and D-galactose was not transported. Due to the much stronger stimulation of alphaMeGlc uptake by SAAT-pSGLT2 expressed in Xenopus oocytes (27-fold increase above controls for 50 µM alphaMeGlc) compared to human SGLT2 (only 2-3.5-fold increase above controls for 2 mM alphaMeGlc), these investigators suggested that SAAT-pSGLT2 corresponds to the cortical low affinity Na/glucose cotransporter. Since human SGLT2 is very similar (91% identity) to the SNST1 transporter from rabbit kidney reported to be a nucleoside transporter(14) , these investigators also proposed that human SGLT2 is the human version of SNST1.

We report the cDNA sequence, detailed functional characteristics, and localization in the kidney of the rat renal Na/glucose cotransporter SGLT2. Using hybrid-depletion studies we obtained strong evidence that SGLT2 corresponds to the rat renal cortical low affinity Na/glucose cotransporter. To evaluate the role of SGLT1 and SGLT2 during kidney development, we have studied the expression SGLT1 and SGLT2 in the rat embryonic kidney.


MATERIALS AND METHODS

Clone Isolation

A cDNA encoding the rat kidney low affinity Na/glucose cotransporter (SGLT2) was isolated by screening a rat kidney cDNA library prepared in ZAP (8) under low stringency conditions, using rabbit SGLT1 as a probe(7, 15) . The cDNA was sequenced using standard sequencing techniques(7, 15) .

In Vitro Transcription and Translation

SGLT2 cDNA ligated into the EcoRI-XhoI sites of pBluescript SK was used for in vitro transcription. Plasmid DNA was linearized with SalI and then in vitro transcribed using T3 RNA polymerase (Strategene), as described(8) .

Xenopus Oocyte Micro-injection and Transport Measurements

Xenopus ocyte expression was performed as described previously(7, 8, 11, 15) . Manually defolliculated oocytes were injected with about 40-50 ng of rat SGLT2 cRNA. 5 days after injection, the uptake of ^14C-labeled alphaMeGlc was determined. In all uptake experiments, oocytes were preincubated in choline solution (100 mM choline, 2 mM KCl, 1 mM MgCl(2), 1 mM CaCl(2), 10 mM HEPES, 5 mM Tris, pH 7.4) for 1 h. Then, 1-h uptakes were carried out in 0.75 ml of uptake solution (containing 3.0 µC(i) of ^14C-alphaMeGlc, 100 mM NaCl, 2 mM KCl, 1 mM MgCl(2), 1 mM CaCl(2), 10 mM HEPES, 5 mM Tris, pH 7.4). Unlabeled alphaMeGlc was added to the uptake solution to give a final concentration as indicated in the figure legends. Uptakes were terminated by washing each group of oocytes four to five times with ice-cold Na-free uptake solution. Individual oocytes were solubilized in 250 µl of 10% SDS and counted. Each uptake value represents the mean ± S.E. (n = 6-8 oocytes).

The Na to glucose coupling ratio was determined by combined ^14C-alphaMeGlc uptake and two electrode voltage clamp experiments(8, 11) . This method allowed a direct comparison of the initial rate of the alphaMeGlc uptake and the charge flux. Briefly, oocytes expressing either rat SGLT2 or rabbit SGLT1 (day 5 after injection) or water-injected control oocytes were used to measure the uptake of ^14C-alphaMeGlc (2 mM for SGLT2, 50 µM for SGLT1) during 1, 3, 5, and 10 min. After completion of the uptake measurements, electrophysiological experiments were performed using the same batch of cRNA- and water-injected oocytes. The conventional two-microelectrode voltage clamp method (Axoclamp-2A, Axon Instruments, CA) was used(16) . In rat SGLT2 cRNA- or water-injected oocytes, application of alphaMeGlc did not change the membrane potential significantly. Therefore, oocytes were clamped at their resting membrane potentials measured in the absence of alphaMeGlc. The inward currents evoked by bath-applied alphaMeGlc (3 mM) were recorded for each oocyte, and the rate of net charge flux was computed using Faraday's constant.

Northern Analysis

After electrophoresis of poly(A) RNA (3µg/lane) in a 1% formaldehyde/agarose gel, the RNA was blotted onto a nitrocellulose filter (Schleicher & Schuell). The filter was hybridized at 35 °C (low stringency) and 42 °C (high stringency) in 50% formamide, using P-labeled full-length rat SGLT2 cDNA as a probe, labeled using the T7 QuickPrime kit (Pharmacia). The filter was washed in 0.1 times SSC, 0.1% SDS at 40 °C (low stringency) or at 65 °C (high stringency).

In Situ Hybridization

In situ hybridization of rat kidney was performed as described previously (8) using 4% paraformaldehyde-fixed tissue sections (approximately 7-µm thickness). Briefly, S-labeled sense and antisense RNA probes were synthesized from the full-length clone (in pBluescript) after linearization of plasmid DNA with SalI or EcoRI using T3 or T7 RNA polymerase, respectively. RNA probes were hydrolyzed for 50 min to form probes of approximately 100 nucleotides. The probes were hybridized to tissue sections at 50 °C overnight in hybridization solution containing 50% formamide. Sections were washed in 5 times SSC for 30 min at 50 °C, in 50% formamide and 2 times SSC for 20 min at 50 °C, and then twice in 0.4 M NaCl for 20 min at 37 °C. After the sections were treated with RNase A and RNase T1 at 37 °C for 30 min and washed in 0.1 times SSC at 37 °C for 15 min, they were dipped into Kodak NTB2 emulsion and developed 6 days later. Counterstaining was performed with hematoxylin-eosin. Control experiments with S-labeled sense cRNA were performed to validate the specificity of the signal observed with the antisense probe.

Hybrid Depletion Studies

Rat superficial cortex poly(A) RNA or rat SGLT2 cRNA (1 µg/µl, heat-denatured) was incubated with 0.5 µg/µl of either rat SGLT1 or rat SGLT2 antisense oligonucleotides corresponding to regions near the start codons (nucleotide 50-72, TGCATCGGTGGCAGTAACAGCGG, for rat SGLT1 (8) and nucleotide 46-68, AGGATTATCAATCAGGACCTTCT, for rat SGLT2) in the presence of 50 mM NaCl at 42 °C for 20 min, cooled on ice, and injected into oocytes. SGLT1, SGLT2, and SAAT-pSGLT2 are minimally conserved for these regions(1) . 1-h uptakes of ^14C-alphaMeGlc (3 mM) were performed as described above.

Expression of SGLT1 and SGLT2 in the Rat Developmental Kidney

Time-mated pregnant rats (day 10) were obtained commercially (Taconic Inc., Germantown, NY). They were anesthetized using methohexital and euthanized by cervical dislocation. The gravid uterus was removed from the rat after midline incision, and the individual embryos were dissected free of surrounding amnion. The embryos were placed in a 100-mm Petri dish with 10 ml of RNase free phosphate-buffered saline. The embryonic kidneys were dissected free of surrounding tissue under direct vision using a binocular dissecting microscope (6times magnification). The earliest dissectable kidneys were from days 11.5 to 12 embryos in which non-divided ureteric buds could be identified. Whole kidneys from embryonic rats (days 14, 16, 18, 20, newborn) were homogenized, RNA was prepared in 4 M guanidinium isothiocyanate, and mRNA was recovered after centrifugation through 5.7 M CsCl. It took 10-20 embryonic kidneys to recover 10 µg of RNA at embryonic day 14 and fewer kidneys for subsequent days.


RESULTS

Low stringency screening of a rat kidney cortex cDNA library using rabbit SGLT1 cDNA as a probe resulted in the isolation of a 2254 nucleotide cDNA (rat SGLT2 cDNA). When expressed in Xenopus oocytes, rat SGLT2 induced the Na-dependent alphaMeGlc uptake 3-5-fold above that of water-injected control oocytes (Fig. 3a). The cDNA sequence contains an open reading frame that encodes a 670-amino acid protein (rat SGLT2). The rat SGLT2 amino acid sequence is 60% identical to that of rat SGLT1(8) . An alignment of the two sequences is shown in Fig. 1. The rat and human (11, 15) SGLT2 amino acid sequences exhibit 85% identity. Like rat, rabbit, and human SGLT1 and human SGLT2, hydropathy analysis of rat SGLT2 predicts 12 distinct hydrophobic transmembrane segments interspersed with hydrophilic regions (see (8) ).


Figure 3: Transport characteristics of SGLT2 cRNA-injected Xenopus laevis oocytes. ^14C-alphaMeGlc (3 mM) uptakes were measured as described under ``Materials and Methods.'' Filled bars represent oocytes injected with rat SGLT2 cRNA, and open bars represent oocytes injected with H(2)O. Columns represent the mean ± S.E. (n = 5-8 oocytes). a, SGLT2-mediated alphaMeGlc uptake (left) and inhibition of uptake by sugar analogues (center and right). Gal, galactose; 3-O-MeGlc, 3-O-methyl-D-glucose, Glc, glucose. b, inhibition of SGLT2-mediated alphaMeGlc uptake by phlorizin.




Figure 1: Primary structure and topology of SGLT2. Alignment of the amino acid sequences of rat SGLT2 with SGLT1. Putative membrane-spanning regions (regions 1-12) and N-glycosylation sites (asterisks) are indicated.



In vitro translation of R12 cRNA using rabbit reticulocyte lysates followed by 15% SDS-polyacrylamide gel electrophoresis yielded a protein with an apparent molecular mass of 53 kDa in the absence and 59 kDa in the presence of dog pancreatic microsomes. The 6-kDa shift in molecular mass was reversed by treatment with endoglycosidase H (Fig. 2) and indicates that the protein is glycosylated at a single site, i.e. Asn-248 (see asterisks in Fig. 1).


Figure 2: In vitro translation of SGLT2. The figure shows an autoradiograph of an SDS-polyacrylamide gel (15%) used to analyze the in vitro translation products of rat SGLT2 cRNA obtained in the absence of pancreatic microsomes (first lane) and in the presence of microsomes after centrifugation (second lane). The third lane shows the product obtained in the presence of microsomes after deglycosylation with endoglycosidase H (Endo H).



Phlorizin, an inhibitor of both low and high affinity Na/glucose transporters, inhibited the rat SGLT2-mediated uptake of alphaMeGlc (3 mM) 95% at 5 µM phlorizin, and there was complete inhibition at 10 µM phlorizin (Fig. 3b). The substrate range of rat SGLT2 was tested by inhibition studies in which the inhibition of the uptake of ^14C-alphaMeGlc (3 mM, 1-h uptake) in the presence of various sugar analogues (30 mM inhibitor concentration) was determined. Fig. 3a shows that among the analogues tested, D-glucose effectively inhibited the uptake, whereas L-glucose, 3-O-methyl-D-glucose, D-galactose, and myo-inositol had little or no effect. This pattern of inhibition is consistent with the substrate range of human SGLT2.

Saturation of uptake mediated by rat SGLT2 expressed in oocytes with increasing alphaMeGlc concentration is shown in Fig. 4. SGLT2 induced saturable alphaMeGlc uptake with and apparent K(m) value of 3.0 mM. This value is significantly higher than that of human SGLT2, which is 1.6 mM.


Figure 4: Concentration dependence of alphaMeGlc uptakes. The uptake of ^14C-alphaMeGlc was measured between final alphaMeGlc concentrations of 0.5 and 6.0 mM in rat SGLT2-cRNA-injected oocytes. The inset represents the Eadie-Hofstee plot for the uptakes.



We determined the stoichiometry of rat SGLT2-induced transport by combining ^14C-alphaMeGlc uptake and two-electrode voltage clamp experiments (Fig. 5). According to this approach, the Na-influx is measured as a current. As Fig. 5illustrates, the calculated initial rate of the net Na-flux in rat SGLT2 cRNA-injected oocytes is 0.91 pmol/min. This value is similar to the initial rate of alphaMeGlc uptake, which is 0.96 pmol/min. We therefore conclude that the Na to glucose coupling ratio for SGLT2 is 1:1, meaning that one Na ion is cotransported by rat SGLT2 with each glucose molecule.


Figure 5: Stoichiometry of SGLT2: comparison of the initial rate of the ^14C-alphaMeGlc uptake (left) and the Na influx (right) calculated based on the alphaMeGlc-evoked inward current. The alphaMeGlc concentration was 3 mM. The mean influx values obtained from water-injected control oocytes have been subtracted.



Northern blot analysis was used to study the tissue distribution of SGLT2. A Northern blot containing poly(A) RNA from different rat tissues, including kidney superficial cortex, remaining cortex, outer and inner stripe of outer medulla, and inner medulla was probed with P-labeled full-length SGLT2 cDNA under either low (Fig. 6, top) or high (Fig. 6, bottom) stringency condition. Fig. 6shows that expression of the 2.2-kb band corresponding to rat SGLT2 is restricted to kidney cortex. This specific expression in kidney is consistent with its predicted involvement in the reabsorption of filtered glucose. Under low stringency conditions, the rat SGLT2 probe hybridized to a 4.0-kb band in kidney, duodenum, jejunum, and ileum that corresponds to rat SGLT1(8) , and to an additional band of size 2.5 kb in brain, liver, kidney, duodenum, jejunum, ileum, and colon, which may correspond to an as yet uncharacterized transporter with similarity to SGLT2.


Figure 6: Tissue distribution of SGLT2 in rat based on Northern analysis. Autoradiographs obtained by probing Northern blots of poly(A) RNA (3 µg) from rat tissues with P-labeled rat SGLT2 cDNA at low (top) and high (bottom) stringency are shown.



The exact site of expression of SGLT2 mRNA along the kidney nephron was determined by in situ hybridization using S-labeled antisense cRNA probes and paraformaldehyde-fixed rat kidney frozen sections (Fig. 7a). Sections adjacent to those used for in situ hybridization were analyzed by immunocytochemistry using antibodies that are specific for proximal tubule S1, S2, or S3 segments (Fig. 8). The following segment-specific marker antibodies were used: Anti-GLUT2 antibodies, which react with proximal tubule S1 segments(17) , anti-carbonic anhydrase type IV antibodies, which react densely with S2 segments and only lightly with S1 and S3 segment(18) , and anti-ecto-ATPase antibodies, which specifically react with S3 segments(19) . Fig. 8a shows that the hybridization pattern obtained using rat SGLT2 cRNA probe matches the immunostaining obtained using the GLUT2 antibody (Fig. 8b) but not the immunostaining patterns using the anti-carbonic anhydrase type IV antibody (Fig. 8c) or the anti-ecto-ATPase antibody (not shown). Based on these data, it can be concluded that low affinity SGLT2 is strongly and specifically expressed in proximal tubule S1 segments. By contrast, Fig. 7b shows that the in situ hybridization signal, which corresponds to the rat SGLT1 message, is present in the outer stripe of the outer medulla and in medullary rays. This distribution is consistent with our previous localization of SGLT1 in proximal tubule S3 segments(8) .


Figure 7: Comparison of the localization of SGLT2 and SGLT1 message to rat kidney. Bright field micrographs showing the pattern of in situ hybridization of S-labeled rat SGLT2 and SGLT1 antisense-cRNA probes to cryosections of paraformaldehyde-fixed rat kidney. a, the hybridization signal of SGLT2 is predominantly present over the tubules in the cortex (Co), whereas the signal is absent in outer stripe of outer medulla (OSOM) and inner stripe of outer medulla (ISOM). b, the major hybridization signal of SGLT1 is detected in the outer stripe of outer medulla (OSOM), including the medullary rays (MR), and only at a lower level in the cortex (Co). Bar, 70 µm.




Figure 8: Localization of SGLT2 mRNA in rat kidney tubules. Bright field micrographs of adjacent rat kidney cortex sections showing the pattern of in situ hybridization of S-labeled rat SGLT2 antisense-cRNA probe (a) or the immunostaining with kidney tubule segment-specific marker antibodies against GLUT2 (S1 segments) (b) or carbonic anhydrase (S2 segments) (c). G, glomerulus. Bar, 10 µm.



In the present study, we also studied the contribution of rat SGLT2 to rat kidney low affinity Na/glucose cotransport based on oocyte expression studies. As indicated in the Introduction, this evaluation is of particular importance since Mackenzie et al.(12) reported the molecular characterization of a second low affinity Na/glucose cotransporter (SAAT-pSGLT2), which is expressed in a variety of pig tissues including intestine, spleen, muscle, and also kidney. Micro-injection of rat kidney superficial cortex mRNA into oocytes resulted in a 2-fold increase in the uptake of ^14C-alphaMeGlc (2 mM, 1-h uptake) (Fig. 9). Incubation before injection of superficial cortex mRNA with an antisense oligonucleotide corresponding to the 5`-portion of the rat SGLT2 coding region completely suppressed this uptake, whereas incubation with a rat SGLT1 antisense oligonucleotide had no effect on the uptake. These findings were verified in a second independent experiment (data not shown). Control experiments furthermore showed that antisense SGLT2 but not SGLT1 oligonucleotide completely suppressed the uptake of alphaMeGlc induced by rat SGLT2 cRNA. In conclusion, our data strongly indicate that the alphaMeGlc uptake stimulated by rat kidney superficial cortex is entirely due to the expression of rat SGLT2. It is unlikely that the alphaMeGlc uptake produced by kidney superficial cortex mRNA is the result of SAAT-pSGLT2 activity since the rat SGLT2 antisense oligonucleotide would not be expected to deplete SAAT-pSGLT2 mRNA due to its low homology to rat SGLT2 (only 60% amino acid sequence identity).


Figure 9: Hybrid depletion of alphaMeGlc uptake. Open bar represents oocytes injected with H(2)O. Cross-hatched bars represent oocytes injected with rat kidney superficial cortex poly(A) RNA incubated with antisense SGLT1 or antisense SGLT2. Filled bars represent oocytes injected with SGLT2 cRNA incubated with antisense SGLT1 or antisense SGLT2. Columns represent the mean ± S.E. (n = 5-8 oocytes).



To study the expression of SGLT1 and SGLT2 during kidney development, we prepared Northern blots of RNA from embryonic rat kidneys (embryonic days 14-21) (Fig. 10). In control experiments, Northern blots were probed with P-labeled glycerophosphate dehydrogenase cDNA to verify that the RNA loading is similar for each lane. Expression of the 4-kb SGLT1 mRNA appeared on embryonic day 18 and gradually increased until birth (Fig. 10). Expression of SGLT2 mRNA appeared on embryonic day 17 and gradually increased until day 19. Interestingly, the message level decreased between day 19 and birth, and the same result was obtained using a second Northern blot (not shown). A striking observation was also that the size of rat SGLT2 mRNA was 2.6 kb before birth and 2.2 kb after birth. The shift in molecular weight is evident in Fig. 10a and is further illustrated on a different Northern blot shown in Fig. 10b, which shows embryonic day 19 and adult kidney cortex.


Figure 10: Developmental expression of SGLT2 and SGLT1 in rat embryonic kidneys. a, high stringency Northern analysis of mRNA from rat total embryonic kidneys (embryonic days 14-21) or rat adult kidney cortex (C) probed with P-labeled SGLT2 or SGLT1 cDNA. Stronger signals in adult kidney compared to embryonic kidneys are observed because of the enrichment of SGLT mRNAs in the cortex. b) A different Northern blot is shown to further illustrate the shift in size of SGLT2 mRNA between embryonic day 19 and adult kidney cortex. To verify equal loading, the blots were probed with glycerophosphate dehydrogenase (GAPDH) cDNA.




DISCUSSION

The functional characterization of the kidney low affinity Na/glucose cotransporter SGLT2 has been complicated by the apparent low level of expression of this protein in Xenopus oocytes. This issue was evident, regardless of whether kidney superficial cortex mRNA or SGLT2 cRNA was expressed. One reason for this behavior may be the low affinity of this transporter for Na (see below). Nevertheless, rat kidney SGLT2 exhibited a considerably higher level of expression in oocytes compared to human SGLT2. Rat SGLT2 induced uptakes up to 4.5-fold above controls, making its functional characterization more reliable. In general, its transport properties match well those of the low affinity Na/glucose cotransporter previously reported for rabbit and human kidney cortex(2, 3, 4, 5, 6) .

The Na to glucose coupling of rat SGLT2 was found to be 1:1, in analogy to human SGLT2. Also analogous to human SGLT2, the substrate specificity decreases in the order D-glucose > alphaMeGlc 3-O-methyl-D-glucose, whereas D-galactose, L-glucose, and inositol were not recognized by the transporter.

The apparent K(m) of rat SGLT2 of 3.0 mM is significantly higher than that of human SGLT2, which is 1.6 mM. A K(m) of 3.0 mM is consistent with the values reported for the rabbit kidney low affinity glucose transporter: the reported K(m) values for rabbit kidney cortex are 6 mM based on brush border membrane vesicle studies (2) and 1.64 mM based on in vitro perfused tubule studies(6) . It should be noted that the K(m) value of 6 mM is probably an overestimation since the vesicle studies were performed at an extracellular Na concentration of only 40 mM. In the case of SGLT1, reduction of extracellular Na resulted in an increase in the apparent K(m) for alphaMeGlc(20) , and the effect was enhanced by membrane depolarization.

As indicated above, the apparent low V(max) of SGLT2 expressed in oocytes may be in part related to the high apparent K(m) for Na of 250-300 mM(11) . At a Na concentration of 100 mM, the extracellular Na concentration is significantly below the apparent K(m) (oocytes do not tolerate Na concentration significantly above this concentration). Na binding to SGLT2 may therefore become rate limiting at this Na concentration. This would be in analogy to studies of Parent et al.(20) on SGLT1. Rabbit SGLT1 has an apparent K(m) of 30 mM, and the studies revealed that Na binding becomes rate limiting at Na concentrations below 10 mM.

Using Northern analysis and in situ hybridization combined with immunocytochemistry, we show that rat SGLT2 expression is kidney specific and that it is strongly and specifically expressed in proximal tubule S1 segments. This demonstration is important since our previous localization study was based on the use of a human sequence to probe rat tissue(11) . We also directly compare its expression with SGLT1, which is present in proximal tubule S3 segments (Fig. 7). The data presented in this figure illustrate that the low and high affinity Na/glucose cotransporters are arranged in series in kidney tubules, consistent with earlier studies of kidney brush border membrane vesicles (2, 3, 4) and rabbit kidney in vitro perfused tubules(6) . Our data therefore agree with the view that the bulk of filtered glucose in proximal tubule S1 segments is absorbed by a low affinity Na/glucose cotransporter with a Na to glucose coupling ratio of 1:1, whereas reabsorption of the remainder of filtered glucose in S3 segments occurs by a high affinity Na/glucose cotransporter which is energized by the cotransport of 2 Na ions.

Low stringency Northern analysis of rat tissues (Fig. 6a) shows that, in addition to the 2.2-kb SGLT2 band, there is strong hybridization to the 4-kb SGLT1 band in both kidney and intestine (see (8) ). In addition, there is also a band at 2.5 kb in brain, liver, and colon. Its distribution appears to be distinct from that reported for other members of the SGLT family such as the myo-inositol transporter, which is primarily expressed in the renal medulla, and the SAAT-pSGLT2 transporter, which is expressed in intestine, spleen, liver, skeletal muscle, and kidney.

Wright and colleagues (12) recently reported that SAAT/pSGLT2 is a low affinity Na/glucose cotransporter. The apparent K(m) for alphaMeGlc of SAAT-pSGLT2 is 2 mM and that for Na is 10 mM. In analogy to rat and human SGLT2, the Na to glucose coupling ratio for SAAT-pSGLT2 was found to be 1:1. At the amino acid sequence level, however, SAAT-pSGLT2 has only 60% identity to rat SGLT2, and it is more similar to rat SGLT1 with 78% identity. Because SAAT-pSGLT2 expression in Xenopus oocytes induced a much higher (27-fold) increase of the uptake of alphaMeGlc than human SGLT2, the investigators suggested that SAAT-pSGLT2 corresponds to the cortical low affinity Na/glucose transporter(12) . They also proposed that human SGLT2 is the human version of the previously reported rabbit nucleotide transporter SNST1. Our hybrid-depletion studies (Fig. 9), however, strongly indicate that SGLT2 is the major kidney cortical low affinity Na/glucose cotransporter. Also consistent with SGLT2 being the cortical low affinity Na/glucose cotransporter is the apparent K(m) for Na of human SGLT2, which is between 250 and 300 mM(11) , a value which is close to that of 228 mM reported by Turner & Moran (4) for rabbit kidney cortex. In contrast, the K(m) for Na of 10 mM of SAAT/pSGLT2 is substantially below that value. Our data on human SGLT2 also indicate that the substrate binding site of SGLT2 does not recognize uridine. It is therefore unlikely that rat and human SGLT2 correspond to a nucleoside transporter. In summary, we conclude that SGLT2 is the major kidney cortical low affinity Na/glucose cotransporter.

The reported tissue distribution of SAAT-pSGLT2 furthermore suggests that it is not a kidney-specific low affinity glucose transporter. Expression of SAAT-pSGLT2 mRNA was reported to be strong in intestine, spleen, liver, and muscle and only at a lower level in kidney(13) . Thus, SAAT1-pSGLT2 may therefore represent an additional low affinity Na/glucose cotransporter isoform that is expressed also in non-renal tissues. Consequently, we propose that SAAT-pSGLT2 be renamed SGLT3.

At present, there is little information available about the expression of transporters in the embryonic kidney. The data shown in Fig. 10reveal that SGLT1 and SGLT2 mRNA expression starts on embryonic days 18 and 17, respectively. This time frame appears to be consistent with the expression of brush border membrane proteins in the developing nephrons (see (21) ). The decrease of the SGLT2 message between days 19 and 21 is an interesting observation as well as the finding that the size of rat SGLT2 mRNA changes from 2.6 kb before birth to 2.2 kb after birth. Our data suggest that a different splice variant of SGLT2 may be expressed in the embryo compared to the adult kidney.


FOOTNOTES

*
This work was supported by National Institute of Health Grants DK 43632 and 43171 (to M. A. H.), an American Heart Association Fellowship (to G. Y.), a Juvenile Diabetes Foundation International Fellowship (to W-S. L.), and an International Human Frontier Science Program Organization Long Term Research Fellowship (to Y. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank(TM)/EMBL Data Bank with accession number(s) U29881[GenBank].

§
Present address: Cardiovascular Biology Laboratory, Harvard School of Public Health, 677 Huntington Ave., Boston, MA 02115.

Present address: Division of Nephrology, UFRGS, Hospital de Clinicas de Porto Alegre, Brazil.

**
Present address: Dept. of Pharmacology, Kyorin University, School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181, Japan.

§§
Present address: The Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142.

¶¶
To whom all correspondence should be addressed: Renal Division, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. Tel.: 617-732-5850; Fax: 617-732-6392.

(^1)
The abbreviations used are: SGLT, Na-coupled glucose transporter; kb, kilobase(s); GLUT, facilitated glucose transporter; alphaMeGlc, alpha-methyl-D-glucopyranoside.


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