The presence of a proton-coupled electrogenic high-affinity peptide transporter in the apical membrane of tubular cells has been demonstrated by microperfusion studies and by use of brush border membrane vesicles. The transporter mediates tubular uptake of filtered di-and tripeptides and aminocephalosporin antibiotics. We have used expression cloning in Xenopus laevis oocytes for identification and characterization of the renal high-affinity peptide transporter. Injection of poly(A)+ RNA isolated from rabbit kidney cortex into oocytes resulted in expression of a pH-dependent transport activity for the aminocephalosporin antibiotic cefadroxil. After size fractionation of poly(A)+ RNA the transport activity was identified in the 3.0-to 5.0-kb fractions, which were used for construction of a cDNA library. The library was screened for expression of cefadroxil transport after injection of complementary RNA synthesized in vitro from different pools of clones. A single clone (rPepT2) was isolated that stimulated cefadroxil uptake into oocytes "70-fold at a pH of 6.0. Kinetic analysis of cefadroxil uptake expressed by the transporter's complementary RNA showed a single saturable high-affinity transport system shared by dipeptides, tripeptides, and selected amino-f3-lactam antibiotics. Electrophysiological studies established that the transport activity is electrogenic and affected by membrane potential. Sequencing of the cDNA predicts a protein of 729 amino acids with 12 membrane-spanning domains. Although there is a significant amino acid sequence identity (47%) to the recently cloned peptide transporters from rabbit and human small intestine, the renal transporter shows distinct structural and functional differences.

Renal epithelial cells express membrane transport proteins capable of cellular uptake of a large variety of di- and tripeptides. These transporters contribute to renal amino acid homeostasis and the efficiency of conservation of amino acid nitrogen. In addition, these transporters appear to play a role in the renal handling of xenobiotics that possess a peptide backbone. Peptide carriers specialized in transport of di- and tripeptides have been identified in bacteria, fungi, plants, and epithelial cells of mammalian intestine and kidney. They appear to represent an archaic transporter family conserved throughout evolution. As a unique feature, these peptide carriers utilize a transmembrane-electrochemical proton gradient as the driving force that enables them to transport peptides against a concentration gradient. Renal peptide transporters have been characterized in terms of mechanism of transport function and substrate specificity in a number of model systems. Within the last two years, kidney peptide transporters of a variety of species have been identified by cloning techniques. In this review we discuss the physiological importance of renal peptide carriers and the transport mechanisms at the cellular level. We also present the recent advancements in functional expression of the cloned proteins that provide first insights into their molecular architecture and mode of operation.

l. Recently two genes have been identified by expression cloning that encode mammalian epithelial peptide transporters capable of translocating di- and tripeptides and selected peptidomimetics by stereoselective and rheogenic substrate-H+ cotransport. PepT1 from rabbit or human small intestine induces a transport activity with high transport capacity but rather low substrate affinity when expressed in Xenopus oocytes. In contrast, the renal carrier PepT2 is a high affinity-type transporter with a lower maximal transport capacity. In addition, both transporters show differences in pH dependence and substrate specificity. 2. As a first approach to identify structural components of the transport proteins that determine their phenotypical characteristics, we constructed a recombinant chimeric peptide transporter (CH1Pep) in which the aminoterminal region (residues 1-401) is derived from PepT2 whereas the carboxyterminal region (residues 402-707) starting at the end of transmembrane domain 9 is derived from PepT1. Expression of PepT1, PepT2 and CH1Pep in Xenopus oocytes allowed the characteristics of the transporters to be determined by flux studies employing a radiolabelled dipeptide and by the two-electrode voltage clamp technique. 3. Our studies indicate that CH1Pep conserves the characteristics of PepT2 including the high affinity for dipeptides and peptidomimetics, the substrate specificity, the pH dependence of transport activation and the electrophysiological parameters. We conclude that the phenotypical characteristics of the renal peptide transporter are determined by its amino-terminal region.

Di- and tripeptides and peptide mimetics such as beta-lactam antibiotics are efficiently reabsorbed from the tubular lumen by a high-affinity peptide transporter. We have recently identified and characterized this H+-coupled high-affinity peptide transport system in the porcine proximal tubular cell line LLC-PK1. Here we describe for the first time the regulation of the renal high-affinity peptide cotransporter at the cellular level. Uptake of 5 microM 3H-D-Phe-L-Ala into LLC-PK1 cells was significantly increased by lowering [Ca2+]in and decreased by increasing [Ca2+] in. Moreover, it was shown that the [Ca2+]in effects on peptide transport activity were dependent on Ca2+ entry from the extracellular site (e.g., via a store-regulated capacitative Ca2+ influx). Protein kinase C (PKC) was found to transmit the effects of [Ca2+]in on peptide transport. Although we demonstrate by pHin measurements that the PKC inhibitor staurosporine did decrease the transmembrane H+ gradient and consequently should have reduced the driving force for peptide uptake, the only effect on transport kinetics of 3H-D-Phe-L-Ala observed was a significant decrease in Km from 22.7+/-2.5 microM to 10.2+/-1.9 microM with no change in maximal velocity.

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.