a-Ketoglutarate Transport in Rat Renal Brush-Border and Basolateral Membrane Vesicles

The dicarboxylate, a-ketoglutarate (aKG), has been identified as the most likely physiological anion involved in renal proximal tubule basolateral membrane (BLM) dicarboxylate/organic anion exchange. In the present study, we characterized the uptake of aKG in BLM and brush-border membrane (BBM) vesicles isolated from rat kidney. In both membrane preparations, aKG uptake was Na-dependent, saturable, electrogenic and inhibited by Li. The initial rate of aKG (5 mM) uptake in BLM vesicles was twice that in BBM vesicles (258 6 8.2 vs. 126 6 3.9 pmol/mg/5 sec). The BLM transporter had a high affinity for aKG (apparent Km 5 15.2 mM), but a relatively low transport capacity (Vmax 5 386 pmol/mg/5 sec). In contrast, the BBM transporter had characteristics of a low-affinity (Km 5 158 mM), high-capacity (Vmax 5 1106 pmol/mg/5 sec) system. Other dicarboxylates such as succinate, malate, fumarate and glutarate at a concentration of 1 mM inhibited aKG uptake into BLM and BBM vesicles to the same extent (.90%). The tricarboxylate, citrate, also inhibited aKG uptake (70–80%). However, of these Krebs’ cycle intermediates, only aKG and glutarate were able to affect p-aminohippurate (PAH) uptake into BLM vesicles. These results lend further support for a BLM PAH/aKG exchanger. Furthermore, if extracellular aKG plays a role in the operation of the PAH/aKG exchanger, the highaffinity Na-dependent aKG transporter located in the BLM is the likely source of the organic anion. Renal organic anion transport plays an important role in the secretion and clearance of a wide range of endogenous metabolites and xenobiotics (Moller and Sheikh, 1983). Transepithelial secretion of organic anions such as PAH occurs exclusively in the proximal tubule and involves the transport of the anion across the BLM into the tubule cell against a steep electrochemical gradient. Movement of PAH out of the cell across the BBM into the tubule lumen occurs down its electrochemical gradient but likely involves a mediated process (Pritchard and Miller, 1993). The events involved in the concentrative uptake of PAH and other molecules that share this transport system have been the focus of much research, but only recently have studies shown that PAH uptake across the BLM is a tertiary active transport process. According to this model (Shimada et al., 1987; Pritchard, 1988), uptake of PAH into the cell across the BLM occurs in exchange for a dicarboxylate moving out of the cell down its concentration gradient. The outwardly directed gradient for the dicarboxylate is maintained by intracellular synthesis as well as by Na-coupled entry of the dicarboxylate into the cell which depends on the inwardly directed Na gradient produced and maintained by Na-K-ATPase. This model was originally proposed based on studies on BLM vesicles (Shimada et al., 1987; Pritchard, 1988) but has subsequently been shown to function in isolated proximal tubules (Chatsudthipong and Dantzler, 1992; Sullivan and Grantham, 1992; Shpun et al., 1995) and renal cortical slices (Pritchard, 1990). aKG has been identified as the most likely physiological anion involved in dicarboxylate-PAH exchange. This is based primarily on its intracellular abundance in the proximal tubule (Pritchard, 1995) and its potency compared with other dicarboxylates to stimulate PAH uptake in the proximal tubule (Pritchard, 1988; Sullivan and Grantham, 1992). In view of its importance in the transport of organic anions, the purpose of this study was to characterize the uptake of aKG in BLM and BBM vesicles isolated from rat kidney. Materials and Methods Membrane vesicle preparation. BBM vesicles were isolated from the renal cortex of male Sprague-Dawley rats (250–300 g, Taconic Farms, Germantown, NY) by the Mg precipitation method as described previously (Edwards et al., 1996). Renal cortices were homogenized at 4°C in 50 mM mannitol and 10 mM HEPES-Tris (pH 7.5) with a Polytron (3 3 30 sec at speed 6). MgCl2 was added to a final concentration of 10 mM, and the homogenate was stirred on ice for 20 min. The homogenate was centrifuged for 10 min at 2,000 3 g, Received for publication November 20, 1996. ABBREVIATIONS: BBM, brush border membrane; BLM, basolateral membrane; PAH, p-aminohippurate; aKG, a-ketoglutarate; HEPES, N-2hydroxyethylpiperazine-N9-ethanesulfonic acid. 0022-3565/97/2813-1059$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 281, No. 3 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 281:1059–1064, 1997 1059 at A PE T Jornals on Sptem er 3, 2017 jpet.asjournals.org D ow nladed from and the resulting supernatant was centrifuged for 20 min at 35,000 3 g. The pellet was resuspended in intravesicular buffer (see below) with use of a glass/Teflon homogenizer and centrifuged for 15 min at 35,000 3 g. The loosely packed BBM layer was gently washed off the pellet and recentrifuged an additional three times as described above. The final BBM pellet was resuspended in intravesicular buffer at a protein concentration of 10 to 15 mg/ml and stored at room temperature until uptake studies were performed on the same