Dicarboxylate Cotransporter Research Articles

Molecular cloning and functional expression of a sodium-dicarboxylate cotransporter from human kidney.

The renal Na(+)-dicarboxylate cotransporter reabsorbs Krebs cycle intermediates, such as succinate and citrate, from the glomerular filtrate. The present study describes the cloning and characterization of the human renal Na(+)-dicarboxylate cotransporter, hNaDC-1. The amino acid sequence of hNaDC-1 is 78% identical to the rabbit renal Na(+)-dicarboxylate cotransporter, NaDC-1, and 42% identical to the rat renal Na(+)-sulfate transporter, NaSi-1. The carboxy terminus of hNaDC-1 protein contains two N-glycosylation sites that appear to be utilized. Xenopus oocytes injected with hNaDC-1 cRNA expressed a low-affinity Na(+)-dependent dicarboxylate transporter with Michaelis constant (Km) for succinate around 0.4 mM. The transport of succinate by hNaDC-1 was insensitive to the pH of the medium, whereas the transporter of citrate was stimulated by acidic pH. Northern blot analysis indicates that hNaDC-1 mRNA is found in both kidney and intestine. The gene for hNaDC-1 was localized to chromosome 17. This study provides the first demonstration that the human kidney contains a low-affinity Na(+)-dicarboxylate cotransporter with properties that resemble those of the Na(+)-dicarboxylate cotransporter on the apical membrane of the rabbit renal proximal tubule.

Expression of the renal Na + /dicarboxylate cotransporter, NaDC-1, in COS-7 cells

Expression of the renal Na + /dicarboxylate cotransporter, NaDC-1, in COS-7 cells

Expression of the renal Na+/dicarboxylate cotransporter, NaDC-1, in COS-7 cells.

The cloned rabbit renal Na+/dicarboxylate cotransporter, NaDC-1, was transiently expressed in the mammalian monkey kidney cell line, COS-7. Cells transfected with the plasmid pSV-201, containing the cDNA for the Na+/dicarboxylate cotransporter, expressed sodium-dependent succinate and citrate transport after 48 h. Cells transfected with control plasmid, pSV-SPORT, did not express sodium-dependent transport of succinate or citrate. The transport of succinate in cells expressing NaDC-1 was inhibited by di-and tricarboxylic acids, but not by monocarboxylic acids. Sodium-dependent transport of succinate was insensitive to changes in pH, while sodium-dependent citrate transport was stimulated by acidic pH. Succinate transport by NaDC-1 was saturable with an apparent Michaelis constant, Km, around 0.5 mM. The kinetics of sodium activation of succinate transport by NaDC-1 were sigmoidal, with an apparent Hill coefficient of 2.9, indicating that three sodium ions are involved in the transport of each succinate. Succinate transport by NaDC-1 was inhibited by lithium. The functional characteristics of NaDC-1 expressed in COS-7 cells correspond to those of the Na+/dicarboxylate cotransporter of the apical membrane of the renal proximal tubule.

Sequence and Functional Characterization of a Renal Sodium/Dicarboxylate Cotransporter

The cDNA coding for a rabbit renal Na+/dicarboxylate cotransporter, designated NaDC-1, was isolated by functional expression in Xenopus oocytes. NaDC-1 cDNA is approximately 2.3 kilobases in length and codes for a protein of 593 amino acids. NaDC-1 protein contains eight putative transmembrane domains, and the sequence and secondary structure are related to the renal Na+/sulfate transporter, NaSi-1. Northern analysis shows that the NaDC-1 message is abundant in kidney and small intestine, and related transporters may be found in liver, lung, and adrenal. The transport of succinate by NaDC-1 was sodium-dependent, sensitive to inhibition by lithium, and inhibited by a range of di- and tricarboxylic acids. This transporter also carries citrate, but it does not transport lactate. In kinetic experiments, the Km for succinate was around 0.4 mM and the Vmax was 15 nmol/oocyte/h, while the Hill coefficient of Na+ activation of succinate transport was 1.9. The transport of succinate by NaDC-1 was insensitive to changes in pH, whereas the transport of citrate increased with decreasing pH, in parallel with the concentration of divalent citrate in the medium. The results of the functional characterization indicate that NaDC-1 likely corresponds to the renal brush-border Na+/dicarboxylate cotransporter.

Hepatic Na(+)-dicarboxylate cotransport: identification, characterization, and acinar localization.

Rat liver perfusion studies suggest that the transport of alpha-ketoglutarate (KG) and related dicarboxylates exhibits acinar heterogeneity, in that the uptake and subsequent metabolism of these organic anions appears to occur predominantly in the perivenous region. However, the isolated perfused liver as an experimental model cannot distinguish intra-acinar differences in either the rate of solute uptake and/or efflux or intracellular binding and/or metabolism. Therefore, the driving forces and acinar localization of KG transport were examined using rat basolateral liver plasma membrane vesicles (blLPMV) isolated from control animals and animals treated 24 h before with selective perivenous and periportal toxins [carbon tetrachloride (CCl4) and allyl alcohol (AA), respectively]. In control blLPMV, [14C]KG uptake into an osmotically sensitive space was markedly stimulated by an inwardly directed Na+ gradient but not by inwardly directed gradients of other monovalent cations. The Na+ ionophore, gramicidin, had a small but significant inhibitory effect on Na(+)-dependent KG uptake, demonstrating that KG uptake was not the result of an intravesicular positive Na+ diffusion potential. The protonophore, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, had no effect on Na+ gradient-driven KG uptake, indicating that KG uptake was not the indirect result of coordinated activities of Na-H and KG-OH exchange. Na+ gradient-driven KG uptake was electrogenic (occurring with the net transfer of positive charge), and cis-inhibited by other tricarboxylic acid cycle intermediates, including succinate, fumarate, and malate and by citrate, but not by the dicarboxylates oxalate and malonate nor by glutamate and taurocholate (TC).(ABSTRACT TRUNCATED AT 250 WORDS)

PAH/alpha-KG countertransport stimulates PAH uptake and net secretion in isolated rabbit renal tubules.

Possible stimulation of both basolateral uptake and net transepithelial secretion of p-aminohippurate (PAH) by PAH/alpha-ketoglutarate (alpha-KG) countertransport was examined in intact perfused and nonperfused rabbit proximal S2 renal tubules. Preloading tubules with alpha-KG (100 microM) for 30 min increased [14C]-PAH rate of uptake by nonperfused tubules and rate of net secretion by perfused tubules approximately three- to sixfold. During stimulation of net secretion, intracellular [14C]PAH concentration increased to about the same extent as net secretion. Presence of Li+ (2 mM) or absence of Na+ (inhibitors of Na(+)-dicarboxylate cotransport) in bathing medium during alpha-KG preloading eliminated stimulation of PAH transport. Addition of unlabeled alpha-KG (1 mM) to bathing medium stimulated efflux of [14C]PAH across the basolateral membrane of tubules with oil-filled lumina, further supporting the concept of PAH/alpha-KG countertransport. Preloading with succinate (100 microM) also stimulated rates of [14C]PAH uptake by nonperfused tubules and net secretion by perfused tubules, but stimulation was only approximately 1.5-fold. Moreover, preloading with methyl succinate, a slowly metabolized derivative of succinate, did not stimulate [14C]PAH uptake by nonperfused tubules or net secretion by perfused tubules. Thus it seems most likely that succinate preloading stimulates PAH transport via metabolism, possibly by conversion to alpha-KG, not by direct countertransport for PAH. This study indicates for the first time in intact mammalian proximal S2 renal tubules that PAH/alpha-KG countertransport can stimulate net PAH secretion by generating an increased intracellular PAH concentration.

特集 細胞膜輸送 薬物の生体膜輸送

The present review is mainly concerned with the transport mechanisms of drugs in the plasma membranes of the renal epithelial cells. Organic anions and cations are actively secreted from plasma to filtrate in the proximal tubules. Studies with isolated membrane vesicles have generated a great deal of information about the transport mechanisms of organic ions in basolateral and brush-border membranes. p-Aminohippurate (PAH ; an organic anion) is actively transported in basolateral membranes by two functionally linked transport systems, Na+/dicarboxylate cotransporter and dicarboxylate/PA H exchanger. In rat brush-border membranes, PAH is probably transported by two distinct transport systems, anion exchanger and potential-sensitive transport system. On the other hand, tetraethylammonium (TEA ; an organic cation) is transported by a potential-sensitive, carrier-mediated system in basolateral membranes. In brush-border membranes, TEA is actively transported by H+/TEA antiport system. Thus, the vesicle studies have provided new insights into our understanding of renal tubular transport mechanisms of drugs.

PAH-alpha-KG countertransport stimulates PAH uptake and net secretion in isolated snake renal tubules

The possibility that p-aminohippurate (PAH)-alpha-ketoglutarate (alpha-KG) countertransport could stimulate basolateral uptake and net transepithelial secretion of PAH was examined in intact isolated nonperfused and perfused snake (Thamnophis spp.) distal proximal renal tubules. Preloading tubules with alpha-KG (100 microM) for 30 min increased [14C]PAH rate of uptake by nonperfused tubules and rate of net secretion by perfused tubules approximately three- to fivefold without increasing cellular ATP content. During stimulation of net secretion, intracellular [14C]PAH concentration increased to same extent as net secretion. Presence of Li+ (2 mM) or absence of Na+ (inhibitors of Na(+)-dicarboxylate cotransport) in bath during alpha-KG preloading eliminated stimulation of PAH transport. Addition of unlabeled alpha-KG to bathing medium stimulated efflux of [14C]PAH across basolateral membrane of tubules with oil-filled lumina, further supporting the concept of PAH-alpha-KG countertransport. Preloading with succinate (100 microM) also stimulated [14C]PAH uptake by nonperfused tubules and net secretion by perfused tubules, but stimulation was only 1.5- to 2-fold and cellular ATP content increased. Moreover, preloading with methyl succinate, a slowly metabolized derivative of succinate, did not stimulate PAH uptake by nonperfused tubules or secretion by perfused tubules. Thus succinate appears to stimulate PAH transport via metabolism, possibly by conversion to alpha-KG, not by direct countertransport. This study indicates for the first time in intact renal tubules that PAH-alpha-KG countertransport can stimulate net PAH secretion by generating an increased intracellular PAH concentration.

Dicarboxylate transport in human placental brush-border membrane vesicles

Pathways for transport of dicarboxylic acid metabolites by human placental epithelia were investigated using apical membrane vesicles isolated by divalent cation precipitation. The presence of Na +/dicarboxylate cotransport was assessed directly by [ 14C]succinate tracer flux measurements and indirectly by fluorescence determinations of voltage sensitive dye responses. The imposition of an inwardly directed Na + gradient stimulated vesicle uptake of succinate achieving levels approximately 5-fold greater than those observed at equilibrium. The increased succinate uptake was specific for Na + as no stimulation was observed in the presence of Li +, K + or choline + gradients. In addition to concentrative accumulation of succinate, a direct coupling of Na +/succinate cotransport was suggested by the absence of a sizeable conductive pathway for succinate uptake and decreased succinate uptake levels associated with a more rapid decay of an imposed Na + gradient. Na + gradient-driven succinate uptake was not the result of parallel Na +/H + and succinate/OH − exchange activities but was reduced by the Na +-coupled transport inhibitor harmaline. The voltage sensitivity of Na + gradient-driven succinate uptake suggests Na +/succinate cotransport is electrogenic occurring with net transfer of positive charge. Substrate-specificity studies suggest the tricarboxylic acid cycle intermediates as candidates for transport by the Na +-coupled pathway. Decreasing pH increased the citrate-induced inhibition of succinate uptake suggesting divalent citrate as the preferred substrate for transport. Initial rate determinations of succinate uptake indicate succinate interacts with a single saturable site ( K m 33 μM) with a maximal transport rate of 0.5 nmol/mg per min.

Indirect coupling to Na+ of p-aminohippuric acid uptake into rat renal basolateral membrane vesicles.

Experiments with basolateral membrane vesicles prepared from rat kidney cortex were performed to study the mechanism by which p-aminohippuric acid (PAH) is taken up across the contraluminal membrane and is concentrated in proximal tubule cells. An inward Na+ gradient failed to stimulate [3H]PAH uptake compared with K+ or Li+ and did not cause intravesicular PAH accumulation above equilibrium distribution. In the absence of Na+, the dicarboxylates glutarate and suberate cis-inhibited and trans-stimulated [3H]PAH uptake, indicating a common transport system. In the presence of Na+, 10 microM glutarate in the incubation medium did not cis-inhibit, but rather stimulated [3H]PAH uptake and caused PAH accumulation above equilibrium distribution ("overshoot"). Li+ diminished this stimulation, but was without effect on [3H]PAH/PAH- and [3H]PAH/glutarate exchange. The data indicate the coexistence of a Na+ -coupled, Li+-sensitive transport system for dicarboxylates and a Li+ -insensitive PAH/dicarboxylate exchanger in the basolateral membrane. We propose that dicarboxylates are cotransported with Na+ into the cell and subsequently exchange for extracellular PAH at the basolateral membrane. PAH uptake is thereby indirectly coupled to Na+ via the Na+/dicarboxylate cotransporter.

Coupling between sodium and succinate transport across renal brush border membrane vesicles.

The coupling between Na+ and succinate transport through the renal brush border Na+-dicarboxylate cotransporter was examined in a vesicle preparation. Initial rates of Na+ and succinate uptake were measured simultaneously using radioactive isotopes under zero-trans (Na+ and succinate-free intravesicular solutions) and voltage-clamped conditions (p.d. = 0 mV). The coupling coefficient is defined as the ratio of the succinate-dependent Na+ flux to the Na+-dependent succinate flux. The cis Na+ and succinate concentrations were varied from 0.05-5 mM succinate and 25-150 mM Na+. The coupling coefficient was approximately 3 and was independent of the succinate and Na+ concentrations tested. These results suggest that only the free carrier and the fully loaded carrier (carrier + 3 Na+ + succinate2-) are able to cross the brush border membrane; i.e., there is little evidence of slippage in the coupling between Na+ and succinate fluxes through the membrane via this carrier. A coupling coefficient of 3 is consistent with the electrogenic nature of the Na-dicarboxylate cotransporter.