Interaction of α-KG with basolateral organic anion transporter in isolated rabbit renal S3 proximal tubules

To understand the basolateral p-aminohippurate (PAH) transporter in the S3 segment of rabbit proximal tubules and its relationship to the transporter in the S2 segment, we measured the 30-s uptake and efflux of PAH across the basolateral membrane of single isolated S3 segments at 37°C in bicarbonate-buffered media. Kinetic analysis of uptake data revealed a concentration of PAH at one-half Jmax of ∼107 μM (same as in the S2 segment) but a J_max of 600 fmol·min^-1·nl^-1 (one-tenth that of S2 segment). The coefficient for efflux across the basolateral membrane was also only one-sixth to one-tenth of that in the S2 segment. These data suggest that the basolateral PAH transporter is the same in both segments but that there are fewer transporters in the S3 than in the S2 segment. However, the apparent inhibitor constant values for cis-inhibition by probenecid (∼29 μM in S3, ∼15 μM in S2) and by α-ketoglutarate (α-KG) in the presence of LiCl (∼40 μM in S3, ∼160 μM in S2) suggest that the transporters may not be identical in the two segments. In bicarbonate-buffered medium, preloading the tubules with 100 μM α-KG did not trans-stimulate PAH uptake across the basolateral membrane, whereas preloading with 1.0 μM α-KG caused a significant stimulation of 43%. However, in N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid-buffered medium, preloading the tubules with 100 μM α-KG caused a twofold increase in PAH uptake. These data suggest that the initial intracellular concentration of α-KG (or metabolic state of the tubule) may influence PAH uptake and the trans-stimulatory effect of α-KG.