Identification of a renal cell line that constitutively expresses the kidney-specific high-affinity H⁺/peptide cotransporter

In this study we describe for the first time the identification of a renal cell line that expresses the kidney-specific high-affinity H⁺/peptide cotransport system. The kidney cell line SKPT-0193 Cl.2 was obtained by SV40 transformation of rat proximal tubular cells. The transport of the dipeptide glycylsarcosine (Gly-Sar) was studied in this cell line grown as a confluent monolayer on impermeable plastic supports. Uptake of the dipeptide was rapid and was stimulated sixfold by an inwardly directed H⁺ gradient, with optimal uptake occurring at an extracellular pH of 6.0. The uptake was markedly reduced by the protonophore carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone whether measured at pH 7.5 or 6.0. Intracellular acidification of the cells by NH₄Cl prepulse also reduced the uptake of glycylsarcosine. The dipeptide uptake was found to he mediated by a high- affinity transport system with a Michaelis-Menten constant (K(t)) of 67 ± 2 μM and a maximal transport velocity of 1.20 ± 0.02 nmol · 10 min^-1 · mg protein^-1. Studied over a concentration range of 5 μM to 5 mM, there was no evidence for a second saturable transport component. Di- and tripeptides, but not glycine, were strong inhibitors of glycylsarcosine uptake, indicating that these peptides also interact with the transport system with high affinity. Northern blot analysis of poly(A)⁺RNA from these cells using cDNA probes specific for the human intestinal peptide transporter (PEPT 1) or the human kidney-specific peptide transporter (PEPT 2) revealed that the transport system expressed in these cells is PEPT 2. It is concluded that the SKPT-0193 Cl.2 cell line constitutively expresses the kidney-specific high- affinity H⁺/peptide cotransporter described in the proximal tubular epithelial cells of the normal kidney.