(Received for publication, June 26, 1995; and in revised form, September 18, 1995)
From the
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
-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
-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.
Reabsorption of filtered glucose across epithelial cells of the
kidney proceeds via Na-coupled glucose transporters
(SGLTs) (
)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
-methyl-D-glucopyranoside (
MeGlc) 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
MeGlc. D-Galactose, which is a substrate of SGLT1, was not recognized
by the transporter. The apparent K
value
for
MeGlc 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
MeGlc 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
MeGlc uptake by SAAT-pSGLT2 expressed
in Xenopus oocytes (27-fold increase above controls for 50
µM
MeGlc) compared to human SGLT2 (only
2-3.5-fold increase above controls for 2 mM
MeGlc),
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.
The Na to glucose coupling ratio was determined by combined
C-
MeGlc uptake and two electrode voltage clamp
experiments(8, 11) . This method allowed a direct
comparison of the initial rate of the
MeGlc 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
C-
MeGlc (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
MeGlc did not
change the membrane potential significantly. Therefore, oocytes were
clamped at their resting membrane potentials measured in the absence of
MeGlc. The inward currents evoked by bath-applied
MeGlc (3
mM) were recorded for each oocyte, and the rate of net charge
flux was computed using Faraday's constant.
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
MeGlc 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. C-
MeGlc (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
O. Columns represent the mean ± S.E. (n = 5-8
oocytes). a, SGLT2-mediated
MeGlc 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
MeGlc 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
MeGlc (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
C-
MeGlc (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 MeGlc concentration is shown in Fig. 4. SGLT2
induced saturable
MeGlc uptake with and apparent K
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 MeGlc
uptakes. The uptake of
C-
MeGlc was measured between
final
MeGlc 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 C-
MeGlc 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
MeGlc
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 C-
MeGlc uptake (left)
and the Na
influx (right) calculated based on
the
MeGlc-evoked inward current. The
MeGlc 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
C-
MeGlc (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
MeGlc induced by rat SGLT2 cRNA. In
conclusion, our data strongly indicate that the
MeGlc uptake
stimulated by rat kidney superficial cortex is entirely due to the
expression of rat SGLT2. It is unlikely that the
MeGlc 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 MeGlc uptake. Open bar represents oocytes injected with H
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.
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
>
MeGlc
3-O-methyl-D-glucose, whereas D-galactose, L-glucose, and inositol were not
recognized by the transporter.
The apparent
K of rat SGLT2 of 3.0 mM is
significantly higher than that of human SGLT2, which is 1.6
mM. A K
of 3.0 mM is consistent
with the values reported for the rabbit kidney low affinity glucose
transporter: the reported K
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
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
for
MeGlc(20) , and the effect was enhanced by
membrane depolarization.
As indicated above, the apparent low V of SGLT2 expressed in oocytes may be in part
related to the high apparent K
for Na
of 250-300 mM(11) . At a
Na
concentration of 100 mM, the extracellular
Na
concentration is significantly below the apparent
K
(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
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
for
MeGlc 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
MeGlc 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
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
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U29881[GenBank].