Endogenous expression of the renal high-affinity H+-peptide cotransporter in LLC-PK1 cells

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Abstract
The reabsorption of filtered di- and tripeptides as well as certain peptide mimetics from the tubular lumen into renal epithelial cells is mediated by an H+-coupled high-affinity transport process. Here we demonstrate for the first time H+-coupled uptake of dipeptides into the renal proximal tubule cell line LLC-PK1. Transport was assessed 1) by uptake studies using the radiolabeled dipeptided-[3H]Phe-l-Ala, 2) by cellular accumulation of the fluorescent dipeptided-Ala-Lys-AMCA, and 3) by measurement of intracellular pH (pHi) changes as a consequence of H+-coupled dipeptide transport. Uptake ofd-Phe-l-Ala increased linearly over 11 days postconfluency and showed all the characteristics of the kidney cortex high-affinity peptide transporter, e.g., a pH optimum for transport ofd-Phe-l-Ala of 6.0, an apparent K m value for influx of 25.8 ± 3.6 μM, and affinities of differently charged dipeptides or the β-lactam antibiotic cefadroxil to the binding site in the range of 20–80 μM. pHi measurements established the peptide transporter to induce pronounced intracellular acidification in LLC-PK1 cells and confirm its postulated role as a cellular acid loader.