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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 DH5alpha . 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).

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.

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.

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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., beta -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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Physiol 276(5):F658-F665
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