Transport mechanisms of carnosine in SKPT cells: contribution of apical and basolateral membrane transporters

Abstract

Purpose: The aim of this study was to investigate the transport properties of carnosine in kidney using SKPT cell cultures as a model of proximal tubular transport, and to isolate the functional activities of renal apical and basolateral transporters in this process.

Methods: The membrane transport kinetics of 10 microM [3H]carnosine was studied in SKPT cells as a function of time, pH, potential inhibitors and substrate concentration. A cellular compartment model was constructed in which the influx, efflux and transepithelial clearances of carnosine were determined. Peptide transporter expression was probed by RT-PCR.

Results: Carnosine uptake was 15-fold greater from the apical than basolateral surface of SKPT cells. However, the apical-to-basolateral transepithelial transport of carnosine was severely rate-limited by its cellular efflux across the basolateral membrane. The high-affinity, proton-dependence, concentration-dependence and inhibitor specificity of carnosine supports the contention that PEPT2 is responsible for its apical uptake. In contrast, the basolateral transporter is saturable, inhibited by PEPT2 substrates but non-concentrative, thereby, suggesting a facilitative carrier.

Conclusions: Carnosine is expected to have a substantial cellular accumulation in kidney but minimal tubular reabsorption in blood because of its high influx clearance across apical membranes by PEPT2 and very low efflux clearance across basolateral membranes.

Fig. 1

Schematic representation of the SKPT cellular model in which CL_AC and CL_BC represent the influx clearances from the apical and basolateral compartments, respectively, while CL_CA and CL_CB represent the respective efflux clearances to the apical and basolateral compartments (A); CL_AB represents the apical-to-basolateral transepithelial clearance and CL_BA represents the basolateral-to-apical transepithelial clearance (B). The clearance values are those determined experimentally for carnosine in this study (units, µl/mg/min).

Fig. 2

RT-PCR analysis of peptide transporter mRNA in SKPT cells, and in kidney, intestine and brain lysates (4 µg total RNA). Samples were separated on a 1.5% agarose gel, visualized with ethidium bromide, and screened for PEPT1 and PEPT2 transcripts (A) and for PHT1 and PHT2 transcripts (B). GAPDH controls for rat brain, intestine, kidney and SKPT cDNA samples are also displayed (C). In each gel, the right-hand lane is a 100 bp DNA ladder. The expected RT-PCR products are shown for each POT under the gel.

Fig. 3

Intracellular accumulation (A) and transcellular transport (B) of 10 µM [³H]carnosine as a function of time in SKPT cell monolayers at 37°C. The cellular efflux (C) of [³H]carnosine was determined after preloading the cells from the apical side with 10 µM carnosine for 2 hr at 37°C. For all experiments, the buffer pH was 6.0 in the apical compartment and 7.4 in the basolateral compartment. Data are expressed as mean ± SE (n=3–5).

Fig. 4

Effect of pH on the 15-min uptake of 10 µM [³H] carnosine in SKPT cell monolayers at 37°C from the apical (A) compartment (basolateral pH maintained at 7.4) and from the basolateral (B) compartment (apical pH maintained at 6.0). Data are expressed as mean ± SE (n=3–6). ** p < 0.01, as compared to pH 7.4. Relationship between pH and fractional ionization of carnosine (C).

Fig. 5

Effect of potential inhibitors on the 15-min apical (A) and basolateral (B) uptake of carnosine in SKPT cell monolayers at 37°C. For all experiments, the buffer pH was 6.0 in the apical compartment and 7.4 in the basolateral compartment. Data are expressed as mean ± SE (n=3–6). ** p < 0.01, as compared to control.

Fig. 6

Effect of concentration on the 15-min uptake of 1–500 µM [³H]carnosine from the apical (A) and basolateral (B) sides of SKPT cell monolayers at 37°C. For all experiments, the buffer pH was 6.0 in the apical compartment and 7.4 in the basolateral compartment. Data are presented as mean ± SE (n=3–6); the inset is a Woolf-Augustinsson-Hofstee plot of the transformed data (V, pmol/mg/15min versus V/S, µl/mg/15min).

Fig. 7

Stability of [³H]carnosine in the apical, basolateral, and intracellular compartments of SKPT cell monolayers as a function of time (pH 6.0 buffer in apical side and pH 7.4 buffer in basolateral side). Data are presented as mean ± SE (n=3).