Localization of PEPT1 and PEPT2 proton-coupled oligopeptide
transporter mRNA and protein in rat kidney
Hong
Shen1,
David E.
Smith1,
Tianxin
Yang2,
Yuning G.
Huang2,
Jürgen B.
Schnermann2, and
Frank C.
Brosius III3,4
1 College of Pharmacy and
Upjohn Center for Clinical Pharmacology,
2 Department of Physiology and
3 Division of Nephrology,
Department of Internal Medicine, University of Michigan, Ann Arbor
48109; and 4 Ann Arbor Veterans
Affairs Medical Center, Ann Arbor, Michigan 48105
 |
ABSTRACT |
To determine the
renal localization of oligopeptide transporters, Northern
blot analyses were performed and polyclonal antisera were generated
against PEPT1 and PEPT2, the two cloned rat
H+/peptide transporters. Under
high-stringency conditions, a 3.0-kb mRNA transcript of rat PEPT1 was
expressed primarily in superficial cortex, whereas a 3.5-kb mRNA
transcript of PEPT2 was expressed primarily in deep cortex/outer stripe
of outer medulla. PEPT1 antisera detected a specific band on
immunoblots of renal and intestinal brush-border membrane vesicles
(BBMV) with an apparent mobility of ~90 kDa. PEPT2 antisera detected
a specific broad band of ~85 kDa in renal but not in intestinal BBMV.
PEPT1 immunolocalization experiments showed detection of a brush border
antigen in S1 segments of the proximal tubule and in the brush border
of villi from all segments of the small intestine. In contrast, PEPT2
immunolocalization was primarily confined to the brush border of S3
segments of the proximal tubule. All other nephron segments in rat were
negative for PEPT1 and PEPT2 staining. Overall, our results
conclusively demonstrate that although PEPT1 is expressed in early
regions of the proximal tubule (pars convoluta), PEPT2 is specific for the latter regions of proximal tubule (pars recta).
Northern blot analysis; immunoblots; immunocytochemistry; proximal
tubules
 |
INTRODUCTION |
THE PROTON-COUPLED OLIGOPEPTIDE transporters, PEPT1 and
PEPT2, have been cloned in rabbit (4, 5, 11), rat (18, 20, 21), and
human (15, 16). Subsequent to this significant advance,
cloned peptide transporters have been functionally characterized using
Xenopus oocyte and HeLa cell
expression systems. In general, PEPT1- and PEPT2-mediated transport is
an active process in which translocation is energized by a
transmembrane electrochemical proton gradient (5, 10, 14). Whereas the
substrate affinity for PEPT1 is characterized as one of low
affinity/high capacity, the substrate affinity for PEPT2 is one of high
affinity/low capacity. However, there are also differences in pH
dependence, substrate specificity and tissue distribution. In
particular, it appears that PEPT2 is abundantly expressed in kidney
with minor contributions from PEPT1. The intestine, on the other hand,
is more uniform in expressing only PEPT1 (6, 12, 14). In this regard,
immunofluorescence has localized PEPT1 in the duodenum, jejunum, and
ileum and in the cortex but not medulla of kidney (19). To date,
immunolocalization studies have not been performed for PEPT1 and PEPT2
in kidney. However, it has recently been reported (23) that PEPT1 and
PEPT2 transcripts are differentially distributed along the proximal tubule, with PEPT1 being predominant in the convoluted segment and
PEPT2 being predominant in the straight segment. Since mRNA and protein
expression are not always coincident, the purpose of the present study
was to define the expression of oligopeptide transporter proteins
(i.e., PEPT1 and PEPT2) in rat kidney.
 |
METHODS |
Animals. All animal procedures
followed the guidelines of the University of Michigan Committee for the
Care and Use of Animals. Male Sprague-Dawley rats, weighing
150-200 g, were used as tissue donors for each experimental procedure.
Northern analysis. Total RNA was
prepared from different regions of rat kidney (i.e., superficial
cortex, middle cortex, deep cortex/outer stripe of outer medulla, inner
stripe of outer medulla, inner medulla, and papilla). These regions
were homogenized in Tri-Reagent (Molecular Research Center, Cincinnati,
OH), and RNA was obtained using a modification of the method of
Chomczynski and Sacchi (9), as described by the manufacturer (8).
Northern analyses for rat PEPT1 and PEPT2 were performed as described
previously (7). Briefly, 15 µg of RNA from each of the kidney
sections was electrophoresed on 6% formaldehyde plus 1% agarose gels
and transferred with 10× SSC (1× SSC is 150 mM NaCl and 15 mM sodium citrate, pH 7.0) to nylon membranes. The blots were
hybridized with uniformly
32P-labeled rat PEPT1 or PEPT2
cDNA probes in 50% formamide hybridization solution at 45°C
overnight, then washed several times at 45-46°C in 0.1×
SSC plus 0.1% SDS. PEPT1 and PEPT2 cDNA probes (547 and 561 bp,
respectively) were prepared by digestion of the pGEX-KT/PepT1 and
pGEX-KT/PepT2 recombinant plasmids using
BamH I and
EcoR I restriction enzymes, as
described subsequently.
Preparation of polyclonal antibodies.
Synthetic 12-amino acid peptides corresponding to the COOH-terminal
region of rat PEPT1 (YSSLEPVSQTNM, amino acids 699-710) and PEPT2
(NMINLETKNTRL, amino acids 718-729) were produced by the
University of Michigan Protein and Carbohydrate Structure Facility. An
NH2-terminal cysteine was added to
the peptides to facilitate coupling of the peptide to the carrier
protein, keyhole limpet hemocyanin (KLH). Purity was confirmed by
high-performance liquid chromatography and amino acids analysis.
For generation of fusion proteins encoding rat PEPT1 and PEPT2, cDNAs
encoding the large extracellular loop spanning putative transmembrane
domains 9 and 10 were used, because there is little sequence identity
between rat PEPT1 and PEPT2 in this region. Full-length rat PEPT1 (18)
and PEPT2 (21) cDNAs (gift of Dr. Matthias Hediger) were used as
templates for generation of the appropriate PEPT1 and PEPT2 PCR
products using the following primer pairs: PEPT1, 5'-TTT GGA TCC
CCC AGC GGA AAT CAA GTT CAA-3' (which corresponds to nucleotides
1219-1239 plus a BamH I enzyme
digestion site and three additional nucleotides to facilitate cleavage) and 5'-AGT CCT GTG ACA GAG AAG AC-3' (which corresponds to
the reverse complement of nucleotides 1846-1865); PEPT2,
5'-TTT GGA TCC CAG CCA GCT TCC CAA GAG ATA-3' (which
corresponds to nucleotides 1382-1402 plus a
BamH I enzyme digestion site and 3 additional nucleotides) and 5'-TTT GAA TTC CTG AAG ACC CTG ACT
GGT GA-3' (which corresponds to the reverse complement of
nucleotides 1923-1942 plus an
EcoR I enzyme digestion site and 3 additional nucleotides). The PEPT1 and PEPT2 cDNA fragments were
ligated into pGEX-KT vector (13) after
BamH I and
EcoR I digestion. Sequence of the cDNA inserts was confirmed by nucleotide sequencing by the University of
Michigan DNA Sequencing Core.
PEPT1- and PEPT2-glutathione S-transferase fusion proteins
fusion proteins were induced in the
Escherichia coli strain DH5
. Bacterial lysates were analyzed by SDS-PAGE, and fusion proteins were
purified by glutathione-agarose affinity chromatography from those
which expressed a predominant polypeptide of the predicted molecular
weight (13).
Antisera were generated against the KLH-conjugated synthetic peptides
and fusion proteins in New Zealand White rabbits (Lampire Biological
Laboratories, Pipersville, PA). Rabbits were injected (subcutaneous and
intradermal) with 0.5 mg of synthetic peptide or 0.25 mg of fusion
protein in complete Freund's adjuvant. Subsequent injections of
antigen in incomplete Freund's adjuvant suspension were performed in
rabbits 2 wk later and every 4 wk thereafter. Hyperimmune serum was
collected 2 wk after each injection, and the specificity and titer of
antisera were assessed. The specificity of immunoblot and
immunolocalization experiments was assured by preincubation of the
antisera with an appropriate immunizing peptide (10-20 µg/ml) or
fusion protein (5-10 µg/ml) compared with preincubation with a
nonspecific antigen. Incubations with preimmune sera were also
assessed. Peptide and fusion-protein antisera were prepared in
parallel, because it was uncertain at the outset which approach would
work better. Although the antisera against fusion proteins showed
patterns similar to those of synthetic peptides, staining was much
weaker when using the fusion-protein antisera. As a result, only the
peptide antisera were further processed for immunolocalization studies
(see below).
Immunoblotting. Immunoblot analyses
were performed with rat PEPT1 and PEPT2 antisera prepared against both
synthetic peptides and fusion proteins. Brush-border membrane vesicles
(BBMV) were prepared from rat kidney and small intestine using a method
described previously (3). Membrane vesicles were solubilized in sample loading buffer (1% SDS, 50 mM Tris · HCl, pH 7.0, 20% glycerol, and 5% mercaptoethanol) and heated at 100°C for 3 min. Proteins (50 µg/lane) were separated on 7.5% SDS-PAGE gels and
transferred to nitrocellulose membranes as described previously (7).
Membranes were blocked with 6% nonfat dry milk in TBS-T (20 mM
Tris · HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20)
for 2 h at room temperature and probed with rat PEPT1 or PEPT2 antisera
(1:1,000 dilution in blocking buffer). The filters were washed three
times in TBS-T, blocked again, and incubated with goat anti-rabbit IgG
conjugated to horseradish peroxidase (Vector Laboratories, Burlingame,
CA). The filters were then washed several times in TBS-T, and the bound antibody was detected on X-ray film by an enhanced chemiluminescence method (Amersham, Arlington Heights, IL).
Immunocytochemistry. For
immunolocalization studies, the antisera directed against rat PEPT1 and
PEPT2 synthetic peptides were affinity purified using a SulfoLink kit,
as described by the manufacturer (Pierce, Rockford, IL). Rat kidneys
were perfusion fixed, via the descending aorta, with cold PBS followed
by periodate-lysine-paraformaldehyde (PLP) for PEPT1 or Carnoy's
fixative for PEPT2 studies. After initial fixation, the kidneys were
kept in fixation solution overnight at 4°C. The kidneys were then
dehydrated and embedded in paraffin blocks. Serial 5-µm sections were
subsequently dewaxed, rehydrated, and immunostained, as described
previously (17). In brief, tissue sections were incubated in PBS
containing 10% normal goat serum for 30 min at room temperature,
followed by a 3-h incubation with affinity-purified rat PEPT1 or PEPT2
antisera (30 µg/ml). Sections were washed for three periods, 5 min
each period, in PBS containing 2.7% NaCl and were incubated with
FITC-conjugated goat anti-rabbit IgG (Vector Laboratories) at a 1:100
dilution in PBS-10% normal goat serum for 1 h. Tissue sections were
mounted in Vectashield mounting medium (Vector Laboratories) and were
examined and photographed on a Zeiss Axioskop 50 microscope equipped
for epifluorescence, using Kodak Color Elite 400 film.
 |
RESULTS |
Distribution of PEPT1 and PEPT2 mRNA in
kidney. Northern blot analysis was used to assess the
overall distribution of oligopeptide transporters in the kidney. PEPT1
and PEPT2 cDNA probes, corresponding to the large extracellular loop
between membrane domains 9 and 10, were hybridized with total RNA
isolated from different regions of rat kidney. As shown in Fig.
1, under high-stringency conditions, a
3.0-kb mRNA transcript of rat PEPT1 was strongly expressed in superficial cortex and at much lower levels in middle cortex. No PEPT1
transcripts were found in deep cortex, outer or inner medulla, or
papilla. In contrast, a 3.5-kb mRNA transcript of PEPT2 was strongly
and predominantly expressed in deep cortex/outer stripe of outer
medulla. A very faint band was detected in middle cortex, and PEPT2 was
undetectable in superficial cortex, inner stripe of outer medulla,
inner medulla, or papilla. These results suggest that PEPT1 and PEPT2
transcripts are confined to and differentially expressed in the
proximal tubule of rat kidney.

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Fig. 1.
Renal distribution of PEPT1 and PEPT2 mRNAs. Total RNAs (15 µg/lane)
from different regions of rat kidney were sequentially analyzed by
high-stringency Northern analysis with specific PEPT1
(bottom) and PEPT2 cDNA probes
(top).
|
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Specificity of polyclonal PEPT1 and PEPT2
antibodies. Polyclonal antisera against rat PEPT1 and
PEPT2 were raised by immunizing rabbits with synthetic peptides
corresponding to their respective carboxy-terminal amino acids. The
carboxy termini were selected given the complete lack of homology
between PEPT1 and PEPT2 in this region. Western blot analyses were then
performed to determine whether PEPT1 and PEPT2 proteins could be
detected in BBMV prepared from rat kidney and small intestine. In this
regard, PEPT1 antisera recognized a major polypeptide fragment with an
apparent molecular mass of ~90 kDa (Fig.
2), and PEPT2 antisera detected a broad band at ~85 kDa in renal BBMV (Fig. 3).
Polyclonal antibodies were also generated against fusion proteins
containing the large extracellular loop of PEPT1 or PEPT2, since
there is little amino acid identity between the two
transporters in this region. As shown in Figs. 2 and 3,
similar results were observed with antisera prepared from fusion
proteins, although the signals were much weaker. With respect to
intestinal BBMV, a similar pattern of positive staining was observed
for rat PEPT1, whereas rat PEPT2 was undetectable (data not shown).
Most importantly, all immunoblot (Figs. 2 and 3) and immunolocalization
(Figs. 4E,
5E, and
6B) detection was prevented by
preincubation of antisera with the appropriate immunizing synthetic
peptide or fusion protein, but not by preincubation with a nonspecific
antigen. Thus specificity of the antisera was confirmed.

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Fig. 2.
Specificity of antisera generated against PEPT1. Polyclonal antibodies
raised against a PEPT1 synthetic peptide (residues 699-710; PEPT1
anti-peptide Ab) and a PEPT1 fusion protein (PEPT1 anti-FP Ab) both
recognized a polypeptide of ~90 kDa in rat renal brush-border
membrane vesicle (BBMV). Immunoreactivity was completely blocked by
preincubation of antiserum with the synthetic peptide or fusion protein
(P1) used to generate the antibodies but not by PEPT2 synthetic peptide
or PEPT2 fusion protein (P2).
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Fig. 3.
Specificity of antisera generated against PEPT2. Polyclonal antibodies
raised against a PEPT2 synthetic peptide (residues 718-729; PEPT2
anti-peptide Ab) and a PEPT2 fusion protein (PEPT2 anti-FP Ab) both
recognized a polypeptide of ~85 kDa in rat renal BBMV.
Immunoreactivity was completely blocked by preincubation of antiserum
with the synthetic peptide or fusion protein (P2) used to generate the
antibodies but not by PEPT1 synthetic peptide or PEPT1 fusion protein
(P1).
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Fig. 4.
Immunolocalization of PEPT1 in rat kidney proximal convoluted tubule
segments. Low magnification
(A-C)
showed strong PEPT1-specific immunostaining in outer cortex
(A), progressively weaker staining
in deeper cortical regions (B), and
no staining in outer medulla (C).
Higher magnification
(D-F)
showed bright immunofluorescent staining of the brush border for PEPT1,
and no staining for the basolateral membrane
(D). All staining completely
disappeared when the section was probed with antiserum preincubated
with PEPT1 immunizing peptide (E).
S1 segment of proximal convoluted tubule emanating from the glomerulus
was intensely stained for PEPT1 (F).
Sections containing the inner medulla and papilla were negative (data
not shown). Thick white marker bar at bottom
right in C is
equivalent in length to 95.2 µm for
A-C,
47.6 µm for D and
E, and 23.8 µm for
F. Thin white line on
left in
C separates the outer and inner
stripes of outer medulla.
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The finding of a specific immunoreactive protein of ~90 kDa for rat
PEPT1 is consistent with the molecular mass of glycosylated protein
predicted from the cDNA clone (18, 20) and the molecular mass
previously reported in rat intestinal BBMV subjected to 8% SDS-PAGE
(24). Likewise, the apparent molecular mass of rat PEPT2 (~85 kDa) is
consistent with its predicted weight based on the cDNA clone and
putative glycosylation sites (21). Although a molecular mass of
75-85 kDa was observed for PEPT1 in rat small intestine and kidney
cortex brush-border membranes (20, 24), the difference is small
and probably reflects the researchers' use of 10%
polyacrylamide gels to separate the samples.
Localization of PEPT1 and PEPT2 proteins in rat
kidney. The cellular localization of rat PEPT1 and
PEPT2 proteins was further investigated by indirect immunofluorescence
using affinity-purified antibodies against the synthetic peptide. Using
freshly removed kidneys fixed in PLP (for PEPT1) or Carnoy's solution
(for PEPT2), we incubated 5-µm sections with PEPT1 or PEPT2 antisera.
In this regard, the general distribution of PEPT1-specific fluorescence in rat cortex and outer medulla is shown in Fig. 4
(A-C).
Immunostaining was the strongest in outer cortex with lower levels
being observed in deeper cortical regions. Staining was absent in inner
cortex, outer and inner medulla, and papilla. As observed under
high-power magnification (Fig. 4, D
and F), PEPT1 staining was greatest
in proximal tubule S1 segments, although some PEPT1-specific
fluorescence was seen in other proximal convoluted segments. It is also
clear that the PEPT1 transporter is specifically localized in the
apical border of proximal tubules. No specific fluorescence was
observed in glomeruli or other tubular segments.
The immunolocalization pattern of PEPT2 was very different in rat
kidney, compared with PEPT1. In this regard, PEPT2-specific staining
was restricted to the outer stripe of outer medulla, which includes the
medullary rays protruding into the deeper cortical regions (Fig.
5,
A-C).
Specific staining was absent in outer cortex, inner stripe of outer
medulla, inner medulla, and papilla. High-power magnification (Fig. 5,
D and
F) shows PEPT2-specific
immunostaining to be localized in the apical border of proximal
tubules. Cortical staining was also observed in proximal tubule
segments within a medullary ray (i.e., corresponds to distal straight
part of S2 and S3 segments) but not in those in S1 segments (Fig. 5,
B and
F). Specific immunofluorescent
staining was not detected in glomeruli or other tubular segments.

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Fig. 5.
Immunolocalization of PEPT2 in rat kidney proximal straight tubule
segments. Low magnification
(A-C)
showed strong PEPT2-specific immunostaining in outer but not inner
stripe of outer medulla (C), some
staining in deeper cortical regions
(B), and no staining in outer cortex
(A). Higher magnification
(D-F)
showed bright immunofluorescent staining of the brush border for PEPT2
and no staining for the basolateral membrane
(D). All staining completely
disappeared when the section was probed with antiserum preincubated
with PEPT2 immunizing peptide (E).
In the deeper cortical regions, S1 segments showed negative
fluorescence (B and
F), whereas other segments of
proximal tubule within the medullary ray (i.e., corresponding to distal
straight part of S2 and S3 segments) were heavily fluorescent. Sections
containing the inner medulla and papilla were negative (data not
shown). In F, "g" is glomerulus.
Thick white marker bar at bottom right
in C is equivalent in length to 95.2 µm for
A-C,
47.6 µm for D and
E, and 23.8 µm for
F. Thin white line on
left in
C separates outer and inner stripes of
outer medulla.
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Further studies were performed on the immunolocalization of PEPT1 and
PEPT2 in rat intestine. Although the PEPT1 studies served as a positive
control, there are no studies in which PEPT2-specific antibodies have
been tested in intestine. As shown in Fig.
6 (A and
C), strong immunofluorescent
staining was seen for PEPT1 in the brush border of villi in the
jejunum. The intensity of staining was weaker in crypt cells, and no
staining was detected in goblet cells. In agreement with our immunoblot
experiments, PEPT2 could not be detected in the brush border of
intestinal villi (data not shown).

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Fig. 6.
Immunolocalization of PEPT1 in rat small intestine. Strong
immunostaining was detected for PEPT1 in the brush border of jejunum,
whereas crypt cells were weakly fluorescent and goblet cells were
negative (A and
C). Immunostaining was blocked by
preincubation of the antisera with synthetic peptide used to raise
antibody (B). White marker bar in
C is equivalent in length to 95.2 µm
for A and
B and to 47.6 µm for
C.
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 |
DISCUSSION |
Peptide transporters have been studied extensively because of their
physiological, pharmacological, and clinical importance and because of
potential applications in drug delivery and targeting (1, 2, 14). In
this regard, the intestine and kidney are pivotal for the conservation
and regulation of peptide-bound amino acids. In addition to this
physiological attribute, oligopeptide transporters have a significant
role in the absorption, disposition, and efficacy of a variety of
peptidomimetic drugs used for the treatment of infection (e.g.,
-lactam antibiotics), hypertension (e.g., angiotensin converting
enzyme inhibitors), and cancer (e.g., bestatin). Furthermore, there are
advantages of dipeptide mixtures over free amino acid mixtures and,
when coupled to peptide transporter function, dipeptides may have
therapeutic value as a nitrogen source for enteral and parenteral nutrition.
Despite the significance of peptide transporter processes, few studies
have addressed their tissue distribution and localization, especially
at the level of protein expression. For example, Western blot analyses
have detected PEPT1 protein in brush-border membranes prepared from rat
small intestine and kidney cortex (20). Using an anti-PEPT1 antibody
(19), immunofluorescence localization of PEPT1 protein was further
established along the rat digestive tract and villus-crypt axis. Thus
positive staining for PEPT1 was observed in the small intestine
(duodenum, jejunum, and ileum), but not in the esophagus, stomach,
colon or rectum. Absorptive epithelial cells in the villi were highly
enriched for PEPT1 protein; however, crypt and goblet cells showed
little or no staining. Our immunolocalization studies with PEPT1 were
consistent with that of others (19) and further demonstrate that PEPT2
was not present in the brush border of intestinal villi.
No studies have, as yet, been reported with respect to the
immunolocalization of PEPT1 and PEPT2 in kidney. Some investigators (6,
14) have proposed that the distribution of peptide transporters is
heterogeneous in kidney such that PEPT2 is responsible for peptide
reabsorption in more proximal parts and PEPT1 in more distal parts of
the nephron. Such conjecture was based on a scenario in which filtered
di- and tripeptides are immediately reabsorbed in proximal regions by a
high-affinity/low-capacity transporter. On the other hand, larger
peptides and proteins would be hydrolyzed by various peptidases as they
pass down the tubule and, as a result, an advantageous reabsorption of
increasing amounts of small peptides could occur in distal regions by a
low-affinity/high-capacity transporter. In conflict with this scenario
is a recent study in which the mRNA expression of rat PEPT1 and PEPT2
was evaluated using RT-PCR of microdissected rat nephron segments and
in situ hybridization of rat kidney sections (23). In that study, PEPT1 mRNA was found to be specifically expressed in early parts of the
proximal tubule (pars convoluta), whereas PEPT2 was expressed preferentially (but not exclusively) in latter parts of the proximal tubule (pars recta). All other segments along the nephron were negative
for PEPT1 or PEPT2 transcripts.
In the present study, we demonstrate for the first time that the
oligopeptide transport proteins of PEPT1 and PEPT2 are differentially expressed in rat kidney. Using polyclonal antisera against rat PEPT1
and PEPT2, immunolocalization experiments revealed that PEPT1 proteins
were found in S1 and other convoluted segments of the proximal tubule
with stronger signals being detected in earlier regions. In contrast,
PEPT2 proteins were restricted primarily to S3 segments. All other
nephron segments were negative for PEPT1 or PEPT2. As further observed,
both peptide transporters were expressed in the brush border as opposed
to basolateral membranes of rat kidney. This finding was consistent
with our high-stringency Northern analyses in which rat PEPT1 mRNA was
expressed primarily in superficial cortex, with weak signals in the
middle cortex and no signals in deeper sections of kidney. PEPT2 mRNA,
on the other hand, was abundantly expressed in deep cortex/outer
stripe, with little expression in middle cortex and no expression in
other kidney sections.
In conclusion, definitive evidence is provided for the heterogeneous
distribution of PEPT1 and PEPT2 proteins in rat kidney, a finding that
corroborates our previous results using RT-PCR and in situ
hybridization techniques (23). Overall, the data suggest that peptides
and peptidomimetics are processed sequentially in proximal regions of
the nephron, first by a high-capacity/low-affinity transporter (PEPT1)
and second by a low-capacity/high-affinity transporter (PEPT2). The
physiological significance of a differential localization of
oligopeptide transporters in proximal tubule is uncertain at the
present time. However, on the basis of the greater abundance of PEPT2
over PEPT1 in kidney (14, 23), it is likely that peptides are
predominantly reabsorbed in kidney by the high-affinity transporter,
PEPT2. Such speculation is supported by free-flow microinfusion studies
in rats (22) in which glycylsarcosine reabsorption was shown to occur
from late as opposed to early proximal tubules.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by National Institutes of Health
Grant R01-GM-35498.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. E. Smith,
Upjohn Center for Clinical Pharmacology, 1310 E. Catherine St., Univ.
of Michigan, Ann Arbor, MI 48109-0504 (E-mail:
smithb{at}umich.edu).
Received 31 August 1998; accepted in final form 22 December 1998.
 |
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