OKP cells express the Na-dicarboxylate cotransporter NaDC-1

2004 ◽  
Vol 287 (1) ◽  
pp. C64-C72 ◽  
Author(s):  
Seiji Aruga ◽  
Ana M. Pajor ◽  
Kiyoshi Nakamura ◽  
Liping Liu ◽  
Orson W. Moe ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Xenopus Oocytes ◽  
Fluorescent Protein ◽  
Stone Formation ◽  
Chronic Metabolic Acidosis ◽  
Opossum Kidney ◽  
Citrate Transport ◽  
Increased Risk ◽  
Green Fluorescent ◽  
Urinary Citrate

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 ≥3 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 transport in rabbit nephron

1986 ◽  
Vol 251 (4) ◽  
pp. F683-F689 ◽  
Author(s):  
T. S. Brennan ◽  
S. Klahr ◽  
L. L. Hamm
Keyword(s):  
Proximal Tubule ◽  
Stone Formation ◽  
Absolute Magnitude ◽  
Thick Ascending Limb ◽  
Citrate Transport ◽  
Glucose Reabsorption ◽  
Collecting Tubule ◽  
Urinary Citrate ◽  
Layer Chromatography

Citrate is an important renal metabolic substrate and urinary inhibitor of calcium stone formation. Our purpose was to characterize citrate reabsorption in the rabbit nephron using isolated perfused tubules. Citrate reabsorption, measured by luminal disappearance of [14C]citrate, was found only in the proximal tubule, not in the cortical thick ascending limb or the cortical collecting tubule. In the proximal convoluted tubule, the collected fluid was also analyzed by thin-layer chromatography and by measurements of chemical citrate concentration using an ultramicroassay. Luminal disappearance of [14C]citrate was determined to accurately represent citrate reabsorption; no significant citrate secretion was found. The absolute magnitude of citrate reabsorption was approximately 3.4 pmol X mm-1 X min-1 using either 1 or 3 mM citrate in the perfusate. This rate of citrate reabsorption in the rabbit proximal tubule could account for all of renal citrate reabsorption but was severalfold lower than glucose reabsorption, which was studied for comparative purposes. In contrast, the magnitude of succinate transport (which probably occurs via the same transport system as citrate) was comparable with that of citrate. Citrate reabsorption was inhibited approximately 80% by 10(-5) M ouabain. This characterization of citrate transport in the intact proximal tubule should provide a useful model to study regulation of urinary citrate excretion.

scholarly journals Activation of dopamine D1-like receptors induces acute internalization of the renal Na+/phosphate cotransporter NaPi-IIa in mouse kidney and OK cells

2005 ◽  
Vol 288 (4) ◽  
pp. F740-F747 ◽  
Author(s):  
Desa Bacic ◽  
Paola Capuano ◽  
Michel Baum ◽  
Jianning Zhang ◽  
Gerti Stange ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Brush Border Membrane ◽  
Fluorescent Protein ◽  
Mouse Kidney ◽  
Proximal Tubules ◽  
Opossum Kidney ◽  
Kidney Slices ◽  
Opossum Kidney Cells ◽  
Green Fluorescent

The Na+/phosphate cotransporter NaPi-IIa (SLC34A1) is the major transporter mediating the reabsorption of Pi in the proximal tubule. Expression and activity of NaPi-IIa is regulated by several factors, including parathyroid hormone, dopamine, metabolic acidosis, and dietary Pi intake. Dopamine induces natriuresis and phosphaturia in vivo, and its actions on several Na+-transporting systems such as NHE3 and Na+-K+-ATPase have been investigated in detail. Using freshly isolated mouse kidney slices, perfused proximal tubules, and cultured renal epithelial cells, we examined the acute effects of dopamine on NaPi-IIa expression and localization. Incubation of isolated kidney slices with the selective D1-like receptor agonists fenoldopam (10 μM) and SKF-38393 (10 μM) for 1 h induced NaPi-IIa internalization and reduced expression of NaPi-IIa in the brush border membrane (BBM). The D2-like selective agonist quinpirole (1 μM) had no effect. The D1 and D2 agonists did not affect the renal Na+/sulfate cotransporter NaSi in the BBM of the proximal tubule. Studies with isolated perfused proximal tubules demonstrated that activation of luminal, but not basolateral, D1-like receptors caused NaPi-IIa internalization. In kidney slices, inhibition of PKC (1 μM chelerythrine) or ERK1/2 (20 μM PD-098089) pathways did not prevent the fenoldopam-induced internalization. Inhibition with the PKA blocker H-89 (10 μM) abolished the effect of fenoldopam. Immunoblot demonstrated a reduction of NaPi-IIa protein in BBMs from kidney slices treated with fenoldopam. Incubation of opossum kidney cells transfected with NaPi-IIa-green fluorescent protein chimera shifted fluorescence from the apical membrane to an intracellular pool. In summary, dopamine induces internalization of NaPi-IIa by activation of luminal D1-like receptors, an effect that is mediated by PKA.

Asymmetrical targeting of type II Na-Picotransporters in renal and intestinal epithelial cell lines

2000 ◽  
Vol 278 (3) ◽  
pp. F361-F368 ◽  
Author(s):  
N. Hernando ◽  
S. Sheikh ◽  
Z. Karim-Jimenez ◽  
H. Galliker ◽  
J. Forgo ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Intestinal Epithelial ◽  
Type Ii ◽  
Opossum Kidney ◽  
Epithelial Cell Lines ◽  
Molecular Determinants ◽  
Cellular Context ◽  
Green Fluorescent

Targeting of newly synthesized transporters to either the apical or basolateral domains of polarized cells is crucial for the function of epithelia, such as in the renal proximal tubule or in the small intestine. Recently, different sodium-phosphate cotransporters have been identified. Type II cotransporters can be subdivided into two groups: type IIa and type IIb. Type IIa is predominantly expressed in renal proximal tubules, whereas type IIb is located on the intestinal and lung epithelia. To gain some insights into the polarized targeting of the type II cotransporters, we have transiently expressed type IIa and type IIb cotransporters in several epithelial cell lines: two lines derived from renal proximal cells (opossum kidney and LLC-PK1), one from renal distal cells (Madin-Darby canine kidney), and one from colonic epithelium (CaCo-2). We studied the expression of the transporters fused to the enhanced green fluorescent protein. Our data indicate that the polarized targeting is dependent on molecular determinants most probably located at the COOH terminus of the cotransporters as well as on the cellular context.

scholarly journals Calcium sensitivity of dicarboxylate transport in cultured proximal tubule cells

2011 ◽  
Vol 300 (2) ◽  
pp. F425-F432 ◽  
Author(s):  
Kathleen S. Hering-Smith ◽  
Faith R. Schiro ◽  
Ana M. Pajor ◽  
L. Lee Hamm
Keyword(s):  
Proximal Tubule ◽  
Transport Process ◽  
Urinary Calcium ◽  
Extracellular Calcium ◽  
Calcium Excretion ◽  
Proximal Tubule Cells ◽  
Citrate Transport ◽  
Dicarboxylate Transport ◽  
Tubule Cells ◽  
Urinary Citrate

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.

scholarly journals Citrate and succinate transport in proximal tubule cells

2000 ◽  
Vol 278 (3) ◽  
pp. F492-F498 ◽  
Author(s):  
Kathleen S. Hering-Smith ◽  
Cecilia T. Gambala ◽  
L. Lee Hamm
Keyword(s):  
Proximal Tubule ◽  
Apical Membrane ◽  
Low Ph ◽  
Extracellular Ph ◽  
Kidney Cells ◽  
Proximal Tubule Cells ◽  
Opossum Kidney ◽  
Opossum Kidney Cells ◽  
Tubule Cells ◽  
Urinary Citrate

Urinary citrate, which inhibits calcium nephrolithiasis, is determined by proximal reabsorption via an apical dicarboxylate transporter. Citrate is predominantly trivalent at physiological pH, but citrate− 2 is transported at the apical membrane. We now demonstrate that low-Ca solutions induce transport of citrate− 2 and succinate in opossum kidney cells. With 1.2 mM extracellular Ca, citrate uptake was pH insensitive and not competed by succinate− 2. In contrast, with low extracellular Ca, citrate uptake increased twofold, was inhibited by succinate (and other dicarboxylates), was stimulated by lowering extracellular pH (consistent with citrate− 2 transport), and increased further by lowering extracellular Mg. The effect of Ca was incrementally concentration dependent, between 0 and 1.2 mM. The effect of Ca was not simply complexation with citrate because succinate (which is complexed significantly less) was affected by Ca similarly. Incubation of cells for 48 h in a low-pH media increased citrate transport (studied at control pH) more than twofold, suggesting induction of transporters.

Citrate transport in proximal cell line

1992 ◽  
Vol 263 (1) ◽  
pp. C220-C225 ◽  
Author(s):  
D. Law ◽  
K. S. Hering-Smith ◽  
L. L. Hamm
Keyword(s):  
Proximal Tubule ◽  
Cell Line ◽  
Transport Process ◽  
Competitive Inhibition ◽  
Basolateral Membrane ◽  
Opossum Kidney ◽  
Cell Monolayers ◽  
Citrate Transport ◽  
Sodium Dependent ◽  
A Cell

Citrate uptake into kidney proximal tubules occurs via an apical dicarboxylate transporter and a poorly characterized process in the basolateral membrane. We used OK cells, a cell line derived from opossum kidney, to study citrate transport in proximal tubule-like cells. Citrate uptake into cell monolayers was studied using [14C]citrate with [3H]mannitol as a volume marker. Citrate uptake into these cells was sodium dependent and saturable with increasing concentrations of citrate. In contrast to previous models, citrate transport was altered minimally by changes in pH from 6.2 to 7.0 and increased at pH 7.4 to 7.8. A variety of di- and tricarboxylates were tested for interaction with citrate transport. The dicarboxylates succinate, malate, and oxaloacetate at 1 mM concentration inhibited citrate uptake minimally (uptake at least 80% of control); one dicarboxylate, alpha-ketoglutarate, did inhibit citrate uptake significantly. In contrast, the tricarboxylates isocitrate and tricarballylate inhibited citrate uptake significantly, indicating probable competitive inhibition with the transport process. These characteristics are distinctly different from those of the apical membrane dicarboxylate transporter. 1,2,3-Benzenetricarboxylic acid, an inhibitor of the mitochondrial tricarboxylate transporter, did not alter citrate uptake. In conclusion, the OK proximal cell line exhibits a novel citrate transport process compared with the apical transport of citrate described in most proximal systems. This transport process probably involves the trivalent species of citrate in contrast to the usual predominant transport of divalent citrate. This transport process may represent a process similar to that in the basolateral membrane of the proximal tubule.

scholarly journals Overexpression of truncated γ-tubulins disrupts mitotic aster formation in Xenopus oocyte extracts

2005 ◽  
Vol 389 (3) ◽  
pp. 611-617 ◽  
Author(s):  
Tomoya Kotani ◽  
Masakane Yamashita
Keyword(s):  
Xenopus Oocyte ◽  
Xenopus Oocytes ◽  
Fluorescent Protein ◽  
Cytoplasmic Dynein ◽  
Spindle Pole ◽  
Middle Part ◽  
Microtubule Organization ◽  
Activity Inhibition ◽  
Green Fluorescent

Mechanisms of spindle pole formation rely on minus-end-directed motor proteins. γ-Tubulin is present at the centre of poles, but its function during pole formation is completely unknown. To address the role of γ-tubulin in spindle pole formation, we overexpressed GFP (green fluorescent protein)-fused γ-tubulin (γ-Tu-GFP) in Xenopus oocytes and produced self-assembled mitotic asters in the oocyte extracts. γ-Tu-GFP associated with endogenous α-, β- and γ-tubulin, suggesting that it acts in the same manner as that of endogenous γ-tubulin. During the process of aster formation, γ-Tu-GFP aggregated as dots on microtubules, and then the dots were translocated to the centre of the aster along microtubules in a manner dependent on cytoplasmic dynein activity. Inhibition of the function of γ-tubulin by an anti-γ-tubulin antibody resulted in failure of microtubule organization into asters. This defect was restored by overexpression of γ-Tu-GFP, confirming the necessity of γ-tubulin in microtubule recruitment for aster formation. We also examined the effects of truncated γ-tubulin mutants, which are difficult to solubly express in other systems, on aster formation. The middle part of γ-tubulin caused abnormal organization of microtubules in which minus ends of microtubules were not tethered, but dispersed. An N-terminus-deleted mutant prevented recruitment of microtubules into asters, similar to the effect of the anti-γ-tubulin antibody. The results indicate possible roles of γ-tubulin in spindle pole formation and show that the system developed in the present study could be useful for analysing roles of many proteins that are difficult to solubly express.

PKC-α-mediated remodeling of the actin cytoskeleton is involved in constitutive albumin uptake by proximal tubule cells

2005 ◽  
Vol 288 (6) ◽  
pp. F1227-F1235 ◽  
Author(s):  
Deanne H. Hryciw ◽  
Carol A. Pollock ◽  
Philip Poronnik
Keyword(s):  
Proximal Tubule ◽  
Fluorescent Protein ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cell ◽  
Apical Surface ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Albumin Endocytosis ◽  
In Cells

One key role of the renal proximal tubule is the reabsorption of proteins from the glomerular filtrate by constitutive receptor-mediated endocytosis. In the opossum kidney (OK) renal proximal tubule cell line, inhibition of protein kinase C (PKC) reduces albumin uptake, although the isoforms involved and mechanisms by which this occurs have not been identified. We used pharmacological and molecular approaches to investigate the role of PKC-α in albumin endocytosis. We found that albumin uptake in OK cells was inhibited by the pan-PKC blocker bisindolylmaleimide-1 and the isoform-specific PKC blockers Gö-6976 and 2′,3,3′,4,4′-hexahydroxy-1,1′-biphenyl-6,6′-dimethanol dimethyl ether, indicating a role for PKC-α. Overexpression of a kinase deficient PKC-α(K368R) but not wild-type PKC-α significantly reduced albumin endocytosis. Western blot analysis of fractionated cells showed an increased association of PKC-α-green fluorescent protein with the membrane fraction within 10–20 min of exposure to albumin. We used phalloidin to demonstrate that albumin induces the formation of clusters of actin at the apical surface of OK cells and that these clusters correspond to the location of albumin uptake. These clusters were not present in cells grown in the absence of albumin. In cells treated either with PKC inhibitors or overexpressing kinase-deficient PKC-α(K368R) this actin cluster formation was significantly reduced. This study identifies a role for PKC-α in constitutive albumin uptake in OK cells by mediating assembly of actin microfilaments at the apical membrane.

scholarly journals Specific connectivity between photoreceptors and horizontal cells in the zebrafish retina

2016 ◽  
Vol 116 (6) ◽  
pp. 2799-2814 ◽  
Author(s):  
Lauw J. Klaassen ◽  
Wim de Graaff ◽  
Jorrit B. van Asselt ◽  
Jan Klooster ◽  
Maarten Kamermans
Keyword(s):  
Promoter Activity ◽  
Xenopus Oocytes ◽  
Fluorescent Protein ◽  
Horizontal Cell ◽  
Feedback Signal ◽  
Kinetic Properties ◽  
Strongly Coupled ◽  
H1 Cells ◽  
Green Fluorescent ◽  
Biphasic Responses

The functional and morphological connectivity between various horizontal cell (HC) types (H1, H2, H3, and H4) and photoreceptors was studied in zebrafish retina. Since HCs are strongly coupled by gap junctions and feedback from HCs to photoreceptors depends strongly on connexin (Cx) hemichannels, we characterized the various HC Cxs (Cx52.6, Cx52.7, Cx52.9, and Cx55.5) in Xenopus oocytes. All Cxs formed hemichannels that were conducting at physiological membrane potentials. The Cx hemichannels differed in kinetic properties and voltage dependence, allowing for specific tuning of the coupling of HCs and the feedback signal from HCs to cones. The morphological connectivity between HC layers and cones was determined next. We used zebrafish expressing green fluorescent protein under the control of Cx promoters. We found that all HCs showed Cx55.5 promoter activity. Cx52.7 promoter activity was exclusively present in H4 cells, while Cx52.9 promoter activity occurred only in H1 cells. Cx52.6 promoter activity was present in H4 cells and in the ventral quadrant of the retina also in H1 cells. Finally, we determined the spectral sensitivities of the HC layers. Three response types were found. Monophasic responses were generated by HCs that contacted all cones (H1 cells), biphasic responses were generated by HCs that contacted M, S, and UV cones (H2 cells), and triphasic responses were generated by HCs that contacted either S and UV cones (H3 cells) or rods and UV cones (H4 cells). Electron microscopy confirms that H4 cells innervate cones. This indicates that rod-driven HCs process spectral information during photopic and luminance information during scotopic conditions.

scholarly journals Effect of renal lipid accumulation on proximal tubule Na+/H+ exchange and ammonium secretion

2008 ◽  
Vol 294 (6) ◽  
pp. F1315-F1322 ◽  
Author(s):  
I. Alexandru Bobulescu ◽  
Michele Dubree ◽  
Jianning Zhang ◽  
Paul McLeroy ◽  
Orson W. Moe
Keyword(s):  
Fatty Acids ◽  
Proximal Tubule ◽  
Lipid Accumulation ◽  
Ammonium Excretion ◽  
Sprague Dawley ◽  
Opossum Kidney ◽  
Intracellular Lipids ◽  
Increased Risk ◽  
Zucker Diabetic Fatty Rats ◽  
Stimulation Of

Patients with metabolic syndrome have increased risk of uric acid nephrolithiasis due to lower urinary pH and impaired ammonium excretion. The pathophysiology underlying these urinary changes is unknown. We used two animal models and a cell culture model to study whether the alteration in renal acidification is associated with renal fat infiltration (steatosis). Compared with pair-fed lean control rats, Zucker diabetic fatty rats have higher renal triglyceride content, decreased urinary ammonium and pH, and lower levels of brush border membrane Na+/H+ exchanger-3 (NHE3), a major mediator of ammonium excretion. High-fat feeding in Sprague-Dawley rats results in transient lowering of urinary ammonium and pH, with all parameters returning to normal when the animals resumed eating normal chow. This is consistent with an absence of diet-induced renal steatosis in these animals. To examine the direct effect of fat accumulation, we incubated opossum kidney (OKP) cells with a mixture of long-chain fatty acids and found accumulation of intracellular lipids with concomitant dose-dependent decrease in NHE3 activity, surface biotin-accessible NHE3 protein, and ammonium secretion. A lower dose of fatty acids that leads to intracellular lipid accumulation but does not change baseline NHE3 is sufficient to abolish the stimulation of NHE3 by insulin and to partially block the stimulation of NHE3 by glucocorticoid hormones; acid regulation of NHE3 in lipid-loaded OKP cells is not affected. These findings suggest that renal steatosis decreases ammonium secretion in the proximal tubule, in part by reducing NHE3 activity and by impairing the regulation of NHE3 by specific agonists.

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