The purpose of this study was to evaluate the role, relevance, and regional dependence of peptide transporter (PEPT) 1 expression and function in mouse intestines using the model dipeptide glycylsarcosine (GlySar). After isolating specific intestinal segments, in situ single-pass perfusions were performed in wild-type and Pept1 knockout mice. The permeability of [³H]GlySar was measured as a function of perfusate pH, dipeptide concentration, potential inhibitors, and intestinal segment, along with PEPT1 mRNA and protein. We found the permeability of GlySar to be saturable (K_m = 5.7 mM), pH-dependent (maximal value at pH 5.5), and specific for PEPT1; other peptide transporters, such as PHT1 and PHT2, were not involved, as judged by the lack of GlySar inhibition by excess concentrations of histidine. GlySar permeabilities were comparable in the duodenum and jejunum of wild-type mice but were much larger than that in ileum (approximately 2-fold). A PEPT1-mediated permeability was not observed for GlySar in the colon of wild-type mice (<10% residual uptake compared to proximal small intestine). Moreover, GlySar permeabilities were very low and not different in the duodenum, jejunum, ileum, and colon of Pept1 knockout mice. Functional activity of intestinal PEPT1 was confirmed by real-time polymerase chain reaction and immunoblot analyses. Our findings suggest that a loss of PEPT1 activity (e.g., due to polymorphisms, disease, or drug interactions) should have a major effect in reducing the intestinal absorption of di-/tripeptides, peptidomimetics, and peptide-like drugs.

The strategic localization of peptide transporter 2 (PEPT2), a proton-coupled oligopeptide transporter, to the apical membrane of epithelial cells in the kidney and choroid plexus suggests that it plays an important role in the disposition of peptides/mimetics in the body. Therefore, the in vivo significance of PEPT2 was investigated in wild-type and PEPT2 null mice following an i.v. bolus dose (0.05 μmol/g body weight) of [¹⁴C]glycylsarcosine (GlySar). In PEPT2 null mice, the clearance (total and renal) of GlySar was markedly increased (2-fold), resulting in concomitantly lower systemic concentrations. In addition, renal reabsorption was almost abolished, and GlySar was eliminated by glomerular filtration. Of the 46% of GlySar reabsorbed in wild-type mice, PEPT2 accounted for 86% and PEPT1 accounted for 14% of reabsorbed substrate. Analysis of GlySar uptake in kidney sections revealed that PEPT2 was primarily localized in the outer medullary region. Wild-type mice also had greater choroid plexus concentrations of GlySar and a 5-fold greater choroid plexus/cerebrospinal fluid (CSF) ratio as compared with null mice at 60 min. Null mice exhibited a greater CSF/blood ratio at 60 min (0.9 versus 0.2) and area under the curve (AUC)_CSF/AUC_blood ratio over 60 min (0.45 versus 0.12), indicating that PEPT2 significantly reduces the exposure of GlySar in CSF. Our in vivo results demonstrate that PEPT2 is the predominant peptide transporter in kidney and that it acts as an efflux transporter in choroid plexus. Thus, PEPT2 may have profound effects on the sensitivity and/or toxicity of peptides and peptide-like drugs.

Pept2 knockout mice are an important tool to evaluate the evolving role and relevance of this proton-coupled oligopeptide transporter beyond drug disposition, where the transporter also modulates the pharmacodynamic and toxicodynamic effects of drug substrates. Our in vivo studies with glycylsarcosine in Pept2 knockout mice have established "proof of concept" that PEPT2 can have a significant effect on dipeptide disposition. Subsequent studies with the aminocephalosporin antibiotic cefadroxil have shown relevance to pharmacology and infectious disease. Finally, studies with the endogenous peptidomimetic 5-aminolevulinic acid have demonstrated relevance to toxicology in the framework of porphyria- and lead-induced neurotoxicity. These studies have consistently demonstrated the dual action of PEPT2 with respect to its apical localization in choroid plexus epithelium and kidney in: 1) effluxing substrates from CSF into choroid plexus, thereby affecting regional pharmacokinetics in brain; and 2) reabsorbing substrates from renal tubular fluid into proximal tubules, thereby affecting systemic pharmacokinetics and exposure. Moreover, these studies have shown that the regional effect of PEPT2 in limiting substrate concentrations in the CSF is more dramatic than its effect in increasing systemic exposure. In the case of 5-aminolevulinic acid, such regional modulation of drug disposition translates directly into significant changes in neurotoxicity.

Purpose. To define the tubular localization and tissue distribution of PEPT1 (low-affinity, high-capacity transporter) and PEPT2 (high-affinity, low-capacity transporter) in rat kidney. Methods. mRNA...