Palmitate transport by rat renal basolateral membrane vesicles in the presence of albumin

Abstract

Long chain fatty acids are an important metabolic fuel for renal proximal tubules. Because of their insolubility, they are carried in the blood primarily bound to albumin. Processes involved in the cellular uptake of albumin-bound ligands are incompletely understood. To determine how fatty acids enter renal tubular cells when substrate was presented primarily bound to albumin (molar ratio albumin:palmitate = 1.0), (3H)palmitate uptake by basolateral membrane vesicles (BLMV) isolated from rat renal cortex was measured by a rapid filtration technique. The pattern of uptake was strikingly affected by buffer composition. In low-ionic-strength buffer (300 mM mannitol, 20 mM Tris-N-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)), (3H)palmitate uptake plateaued in less than 1 min. The calculated volume of distribution of (3H)palmitate indicated extensive binding. In high-ionic-strength buffer with an inwardly directed NaCl, sodium gluconate, or KCl concentration gradient (100 mM) or equal intravesicular and extravesicular NaCl concentration (100 mM), uptake was biphasic with peak values 1.4- to 3-fold higher than plateau values. To test whether uptake could be specifically inhibited, phloretin (200 μM), probenecid (500 μM), or 4,4′-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS; 500 μM) was added to NaCl uptake buffer. These agents were without effect on initial uptake but inhibited the normal biphasic pattern. The biphasic uptake pattern suggested binding followed by translocation to the interior of BLMV with subsequent mediated efflux. Mediated palmitate efflux was demonstrated in BLMV preloaded with (3H)-palmitate in NaCl buffer by (1) phloretin inhibition of efflux and (2) trans-stimulation of efflux by p-aminohippurate. Results suggest that the translocation of fatty acids derived from albumin:fatty acid complexes occurs only in the presence of high external ion concentration. Subsequent equilibration of fatty acids between albumin, BLMV, and vesicle interior is mediated by the organic anion transport system.