Citrate transport in proximal cell line

Law, David S.; Hering-Smith, Kathleen S.; Hamm, L. Lee

doi:10.1152/ajpcell.1992.263.1.c220

Cited by 14 publications

(19 citation statements)

References 9 publications

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“…It is unfortunate that NaDC-1 expression is low, making it necessary to transfect the cells with NaDC-1 to study regulation. Our finding of acid regulation is slightly different from what Hamm and colleagues (6,8) found. In their studies using OK cells, acute decreases in pH had no effect on citrate transport, whereas acute increases raised transporter activity.…”

Section: Discussioncontrasting

confidence: 99%

“…In their studies using OK cells, acute decreases in pH had no effect on citrate transport, whereas acute increases raised transporter activity. As these authors (8) noted, this response to changes in pH is not typical of the proximal tubule apical membrane citrate transporter. In further studies (6), they did find that both acute and chronic (48 h) decreases in media pH increased transporter activity.…”

Section: Discussionmentioning

confidence: 91%

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OKP cells express the Na-dicarboxylate cotransporter NaDC-1

Aruga¹,

Pajor²,

Nakamura³

et al. 2004

American Journal of Physiology-Cell Physiology

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Urinary citrate concentration, a major factor in the formation of kidney stones, is primarily determined by its rate of reabsorption in the proximal tubule. Citrate reabsorption is mediated by the Na-dicarboxylate cotransporter-1 (NaDC-1). The opossum kidney (OKP) cell line possesses many characteristics of the renal proximal tubule. The OKP NaDC-1 (oNaDC-1) cDNA was cloned and encodes a 2.4-kb mRNA. When injected into Xenopus oocytes, the cotransporter is expressed and demonstrates Na-coupled citrate transport with a stoichiometry of Ն3 Na:1 citrate, specificity for di-and tricarboxylates, pH-dependent citrate transport, and pH-independent succinate transport, all characteristics of the other NaDC-1 orthologs. Chronic metabolic acidosis increases proximal tubule citrate reabsorption, leading to profound hypocitraturia and an increased risk for stone formation. Under the conditions studied, endogenous OKP NaDC-1 mRNA abundance is not regulated by changes in media pH. In OKP cells transfected with a green fluorescent protein-oNaDC-1 construct, however, media acidification increases Na-dependent citrate uptake, demonstrating posttranscriptional acid regulation of NaDC-1 activity.citrate; acid base; nephrocalcinosis; nephrolithiasis; opossum kidney cells INGESTED CITRATE IS ABSORBED in the intestine. When citrate is metabolized to neutral end products, base equivalents are produced. Citrate is present in the circulation at low concentrations and is freely filtered by the renal glomerulus. Its concentration in urine is important because it plays a key role in preventing nephrocalcinosis and nephrolithiasis. Citrate's urinary excretion rate is regulated primarily by its rate of reabsorption in the proximal tubule, a process mediated by an apical membrane electrogenic 3Na ϩ -citrate 2Ϫ cotransporter (16). Substantial evidence supports the idea that this apical membrane 3Na ϩ -citrate 2Ϫ cotransporter is encoded by the Nadicarboxylate cotransporter-1 (NaDC-1) (14). NaDC-1 expressed in Xenopus oocytes exhibits many characteristics of the apical membrane 3Na ϩ -citrate 2Ϫ transporter, including Nacoupled electrogenic transport, specificity for di-and tricarboxylates, pH-dependent citrate transport, and pH-independent succinate transport (14). NaDC-1 protein has been localized to the apical membrane of the proximal tubule (2). Chronic metabolic acidosis, which is known to increase proximal tubule citrate absorption as well as the activity of the apical membrane 3Na ϩ -citrate 2Ϫ cotransporter, also increases renal cortical NaDC-1 mRNA and apical membrane protein abundance (2, 5, 7). OKP cells are an opossum kidney cell line that possesses many characteristics of the renal proximal tubule. These cells have proved extremely useful in studying the mechanisms of regulation of proximal tubule H ϩ , Na ϩ , and phosphate transport. Recently, OK cells, another opossum kidney cell line, were demonstrated to exhibit Na-dependent citrate transport with many of the characteristics of NaDC-1 (6). The purpose of our study was to clone ...

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Section: Discussioncontrasting

confidence: 99%

Section: Discussionmentioning

confidence: 91%

OKP cells express the Na-dicarboxylate cotransporter NaDC-1

Aruga¹,

Pajor²,

Nakamura³

et al. 2004

American Journal of Physiology-Cell Physiology

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“…In previous studies to investigate proximal tubule citrate transport, our laboratory has used the OK (opossum kidney) proximal tubule cell line (13,21). Our initial cell culture studies demonstrated that citrate uptake in normal calciumcontaining solutions (calcium concentration 1.2 mM) occurred via an apparent tricarboxylate transporter resembling the basolateral uptake of citrate in isolated basolateral membrane vesicles (21,35).…”

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confidence: 99%

Calcium sensitivity of dicarboxylate transport in cultured proximal tubule cells

Hering-Smith

Schiro

Pajor

et al. 2011

American Journal of Physiology-Renal Physiology

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Urinary citrate is an important inhibitor of calcium nephrolithiasis and is primarily determined by proximal tubule reabsorption. The major transporter to reabsorb citrate is Na(+)-dicarboxylate cotransporter (NaDC1), which transports dicarboxylates, including the divalent form of citrate. We previously found that opossum kidney (OK) proximal tubule cells variably express either divalent or trivalent citrate transport, depending on extracellular calcium. The present studies were performed to delineate the mechanism of the effect of calcium on citrate and succinate transport in these cells. Transport was measured using isotope uptake assays. In some studies, NaDC1 transport was studied in Xenopus oocytes, expressing either the rabbit or opossum ortholog. In the OK cell culture model, lowering extracellular calcium increased both citrate and succinate transport by more than twofold; the effect was specific in that glucose transport was not altered. Citrate and succinate were found to reciprocally inhibit transport at low extracellular calcium (<60 μM), but not at normal calcium (1.2 mM); this mutual inhibition is consistent with dicarboxylate transport. The inhibition varied progressively at intermediate levels of extracellular calcium. In addition to changing the relative magnitude and interaction of citrate and succinate transport, decreasing calcium also increased the affinity of the transport process for various other dicarboxylates. Also, the affinity for succinate, at low concentrations of substrate, was increased by calcium removal. In contrast, in oocytes expressing NaDC1, calcium did not have a similar effect on transport, indicating that NaDC1 could not likely account for the findings in OK cells. In summary, extracellular calcium regulates constitutive citrate and succinate transport in OK proximal tubule cells, probably via a novel transport process that is not NaDC1. The calcium effect on citrate transport parallels in vivo studies that demonstrate the regulation of urinary citrate excretion with urinary calcium excretion, a process that may be important in decreasing urinary calcium stone formation.

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“…In kidney proximal tubules, reabsorption of Krebs cycle intermediates such as citrate, succinate, ␣-ketoglutarate, malate, and fumarate has been shown to be accomplished by Na ϩ -coupled transporters (1)(2)(3)(4)(5)(6)(7)(8)(9). Numerous studies have been performed in intact proximal tubules (10), isolated brush border membrane vesicles (BBMV) 1 (1-5, 11, 12), and basolateral membrane vesicles (BLMV) (12)(13)(14), mostly using citrate or succinate as substrates.…”

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confidence: 99%

“…The functional differences between BBMV and BLMV transport suggest that there exist different transporter isoforms on the apical and basolateral sides. Also, a possible presence of a trivalent citrate transport system in the basolateral membrane of the proximal tubule cell line from opossum kidney was proposed (9).…”

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Characterization of a Rat Na+-Dicarboxylate Cotransporter

Chen

Shayakul

Berger

et al. 1998

Journal of Biological Chemistry

106

128

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The metabolism of Krebs cycle intermediates is of fundamental importance for eukaryotic cells. In the kidney, these intermediates are transported actively into epithelial cells. Because citrate is a potent inhibitor for calcium stone formation, excessive uptake results in nephrolithiasis due to hypocitraturia. We report the cloning and characterization of a rat kidney dicarboxylate transporter (SDCT1). In situ hybridization revealed that SDCT1 mRNA is localized in S3 segments of kidney proximal tubules and in enterocytes lining the intestinal villi. Signals were also detected in lung bronchioli, the epididymis, and liver. When expressed in Xenopus oocytes, SDCT1 mediated electrogenic, sodium-dependent transport of most Krebs cycle intermediates (K m ‫؍‬ 20-60 M), including citrate, succinate, ␣-ketoglutarate, and oxaloacetate. Of note, the acidic amino acids L-and D-glutamate and aspartate were also transported, although with lower affinity (K m ‫؍‬ 2-18 mM). Transport of citrate was pH-sensitive. At pH 7.5, the K m for citrate was high (0.64 mM), whereas at pH 5.5, the K m was low (57 M). This is consistent with the concept that the ؊2 form of citrate is the transported species. In addition, maximal currents at pH 5.5 were 70% higher than those at pH 7.5, and our data show that the ؊3 form acts as a competitive inhibitor. Simultaneous measurements of substrate-evoked currents and tracer uptakes under voltage-clamp condition, as well as a thermodynamic approach, gave a Na ؉ to citrate or a Na ؉ to succinate stoichiometry of 3 to 1. SDCT1-mediated currents were inhibited by phloretin. This plant glycoside also inhibited the SDCT1-specific sodium leak in the absence of substrate, indicating that at least one Na ؉ binds to the transporter before the substrate. The data presented provide new insights into the biophysical characteristics and physiological implications of a cloned dicarboxylate transporter.In kidney proximal tubules, reabsorption of Krebs cycle intermediates such as citrate, succinate, ␣-ketoglutarate, malate, and fumarate has been shown to be accomplished by Na ϩ -coupled transporters (1-9). Numerous studies have been performed in intact proximal tubules (10), isolated brush border membrane vesicles (BBMV) 1 (1-5, 11, 12), and basolateral membrane vesicles (BLMV) (12-14), mostly using citrate or succinate as substrates. In BBMV, succinate uptake was found to be mediated with low affinity (K m ϭ ϳ1 mM) (5,8,12,15). Studies on the pH dependence suggested that citrate is transported in its protonated divalent form (Cit Ϫ2 ) (1, 2, 12), whereas succinate is transported either in its deprotonated (Ϫ2) or protonated (Ϫ1) form (11). In addition, it was shown that the Ϫ3 form of citrate (trivalent form, Cit Ϫ3 ) inhibits transport of Cit Ϫ2 (11). Radiotracer studies revealed that the cotransport process exhibits a stoichiometry of 2-3 sodium ions/dicarboxylate molecule (2,8,16). On the other hand, experiments with a voltage-sensitive dye showed that the cotransport was electrogenic (14, 17), which favors a 3...

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Citrate transport in proximal cell line

Cited by 14 publications

References 9 publications

OKP cells express the Na-dicarboxylate cotransporter NaDC-1

OKP cells express the Na-dicarboxylate cotransporter NaDC-1

Calcium sensitivity of dicarboxylate transport in cultured proximal tubule cells

Characterization of a Rat Na+-Dicarboxylate Cotransporter

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