Characterization of peptide transport mediated by PEPT2 in renal LLC-PK1 cells

Reabsorption of filtered di- and tripeptides and peptide mimetics from the tubular lumen into renal epithelial cells is mediated by a H⁺-coupled high affinity transport process. Here we describe the characteristics of uptake of the radiolabeled dipeptide ³H-D-Phe-L-Ala into porcine proximal tubule LLC-PK1 cells. Saturable dipeptide influx occurs with a pH-optimum of 6.0-6.5, a Km of 61.5 ± 5 μM and with app. Ki-values of 20-80 μM for inhibition by a variety of differently charged dipeptides or β-lactam antibiotics. H⁺/dipeptide cotransport was also assessed by pH_in-measurements. Peptide transport in LLC-PK1 cells therefore shows all the characteristics of the high-affinity PepT2 transporter cloned from kidney cDNA libraries of various species. Further studies were performed with regard to regulation of the transporter expressed in LLC-PK1 cells. It was shown that transport is affected by preincubation of cells with compounds altering PKC activity or modulating Ca²⁺in. Moreover, Ca²⁺ was required to enter the cytosol from the extracellular side (e.g. by capacitative influx) in order to induce effects on peptide transport. Kinetic analysis revealed that the regulatory components exert their effects via a change in Km and not in Vmax.