Characterization of rPEPT2-Mediated Gly-Sar Transport Parameters in the Rat Kidney Proximal Tubule Cell Line SKPT-0193 cl.2 Cultured in Basic Growth Media

Abstract

The rat proximal kidney tubule cell line SKPT-0193 cl.2 (SKPT) expresses the di-/tripeptide transporter PEPT2 (rPEPT2) and has been used to study PEPT2-mediated transport. Traditionally, SKPT cells have been cultured in growth media supplemented with epidermal growth factor (EGF), apotransferrin, dexamethasone, and insulin. It was recently demonstrated that omission of EGF from the culture media caused a drastic increase in the expression of rPEPT2. The hypothesis was therefore that the SKPT cell line might be able to differentiate and express rPEPT2 in the absence of the four agonists traditionally added. The aim of the study was thus to characterize Gly-Sar transport parameters in SKPT cells cultured in basic growth media (conventional media without added agonists). Morphology was studied using confocal laser scanning microscopy (CLSM) and immunohistochemistry. Monolayer integrity was evaluated using transepithelial electrical resistance (TEER) measurements and [(3)H]-mannitol permeabilities. Di-/tripeptide transporter activity was studied using [(14)C]-glycylsarcosine ([(14)C]-Gly-Sar). SKPT cells grown in basic media for 4 days formed confluent monolayers with a TEER of 5.03 +/- 0.33 kOmega.cm(2) (n = 5). Apical Gly-Sar uptake peaked after 3-6 days in culture. Uptake at day 4 was 5.89 +/- 0.30 pmol.cm(-2).min(-1) (n = 3). Di-/tripeptide uptake displayed an optimum at approximately pH 6. Affinity values for cephalexin, kyotorphin, and delta-aminolevulinic acid were comparable to those obtained in other PEPT2-expressing model systems. It can be concluded that SKPT cells grown in the absence of the agonists traditionally added to the culture media retain all necessary properties for PEPT2-mediated peptide uptake studies. Furthermore, the absence of the agonists might facilitate studies of hormonal regulation of PEPT2 expression and transport activity.

hPEPT1 is responsible for uptake and transport of Gly-Sar in the human bronchial airway epithelial cell-line Calu-3

“…In Caco-2 cells (hPEPT1), the K i value for Gly-Pro is 0.3 mM [5], whereas in SKPT cells (rPEPT2), the K i value is 0.027 mM [3]. For δ-aminolevulinic acid, a K i value of 1.5 mM has been found in Caco-2 cells [21], and we have found a K i value of 0.23 mM in SKPT cells [7]. Although affinity data may vary depending on apical pH, transporter origin, etc., K i values obtained for Gly-Pro and δ-aminolevulinic acid in this study are more representative of hPEPT1-mediated kinetics than hPEPT2-mediated kinetics.…”

“…Based on available inhibition data, this indicates that Gly-Sar transport is mediated by hPEPT1 rather than hPEPT2. In Caco-2 cells, affinity of cephalexin for hPEPT1 is 9-14 mM [8,10,22], whereas we have determined the affinity for PEPT2 in SKPT cells to 0.04-0.05 mM [7,38]. In our transport studies, this implicates that, at an apical concentration of 2.5 mM cephalexin, the transport of Gly-Sar should be inhibited by approximately 97% if the transporter responsible for Gly-Sar transport was hPEPT2 and 15% if hPEPT1.…”

Transport Mechanisms of Carnosine in SKPT Cells: Contribution of Apical and Basolateral Membrane Transporters

“…In particular, Bravo et al (37) reported similar apical-to-basolateral and basolateral-to-apical fluxes of GlySar even though the apical uptake of dipeptide was about 5× greater than its basolateral uptake in SKPT cells. In the study by Neumann et al (38) the transepithelial apical-to-basolateral flux of GlySar was only 28% higher than its reverse flux (i.e., basolateral to apical direction) in SKPT cells despite the apical uptake of GlySar being about 3.5× greater than its basolateral uptake.…”

“…3A) of carnosine. To account for the anomaly between transcellular transport and apical uptake, Bravo et al (37) speculated that a low basolateral transport activity may limit the carrier-mediated transepithelial flux of GlySar in SKPT cells. Our kinetic analysis agrees with this assessment and has demonstrated that carnosine is effluxed at a much slower rate across the basolateral versus apical membrane of SKPT cells (Fig.…”

Kyotorphin transport and metabolism in rat and mouse neonatal astrocytes

“…Several pieces of evidence indicate that the high affinity system is mediated by Pept2 : 1) Pept2 KO astrocytes only had the low affinity system; 2) cefadroxil, a PEPT2 substrate (Shen et al, 2005; Shen et al, 2007), blocked the high affinity system; 3) L-KTP is a substrate for PEPT2 (Thakkar et al, 2008) which is a high affinity oligopeptide transporter; and 4) L-KTP blocks PEPT2-mediated GlySar uptake with IC 50 values of 5-30 μM in choroid plexus epithelial cells, synaptosomes and kidney cells, suggesting a high affinity for PEPT2 (Bravo et al, 2005; Fujita et al, 2004; Teuscher et al, 2001). In rat, the concentration of L-KTP in different brain regions varies between 0.14 – 2.1 μmol/kg tissue (Ueda et al, 1980).…”