Extracellular nucleotides inhibit oxalate transport by human intestinal Caco-2-BBe cells through PKC-δ activation

Nephrolithiasis remains a major health problem in Western countries. Seventy to 80% of kidney stones are composed of calcium oxalate, and small changes in urinary oxalate affect risk of kidney stone formation. Intestinal oxalate secretion mediated by the anion exchanger SLC26A6 plays an essential role in preventing hyperoxaluria and calcium oxalate nephrolithiasis, indicating that understanding the mechanisms regulating intestinal oxalate transport is critical for management of hyperoxaluria. Purinergic signaling modulates several intestinal processes through pathways including PKC activation, which we previously found to inhibit Slc26a6 activity in mouse duodenal tissue. We therefore examined whether purinergic stimulation with ATP and UTP affects oxalate transport by human intestinal Caco-2-BBe (C2) cells. We measured [14C]oxalate uptake in the presence of an outward Cl−gradient as an assay of Cl−/oxalate exchange activity, ≥50% of which is mediated by SLC26A6. We found that ATP and UTP significantly inhibited oxalate transport by C2 cells, an effect blocked by the PKC inhibitor Gö-6983. Utilizing pharmacological agonists and antagonists, as well as PKC-δ knockdown studies, we observed that ATP inhibits oxalate transport through the P2Y2receptor, PLC, and PKC-δ. Biotinylation studies showed that ATP inhibits oxalate transport by lowering SLC26A6 surface expression. These findings are of potential relevance to pathophysiology of inflammatory bowel disease-associated hyperoxaluria, where supraphysiological levels of ATP/UTP are expected and overexpression of the P2Y2receptor has been reported. We conclude that ATP and UTP inhibit oxalate transport by lowering SLC26A6 surface expression in C2 cells through signaling pathways including the P2Y2purinergic receptor, PLC, and PKC-δ.

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Cholinergic signaling inhibits oxalate transport by human intestinal T84 cells

AJP Cell Physiology ◽

10.1152/ajpcell.00075.2011 ◽

2012 ◽

Vol 302 (1) ◽

pp. C46-C58 ◽

Cited By ~ 15

Author(s):

Hatim A. Hassan ◽

Ming Cheng ◽

Peter S. Aronson

Keyword(s):

Calcium Oxalate ◽

Signaling Pathways ◽

Phospholipase C ◽

Muscarinic Receptor ◽

Surface Expression ◽

Intestinal Cell ◽

Common Disease ◽

T84 Cells ◽

Oxalate Secretion ◽

Oxalate Transport

Urolithiasis remains a very common disease in Western countries. Seventy to eighty percent of kidney stones are composed of calcium oxalate, and minor changes in urinary oxalate affect stone risk. Intestinal oxalate secretion mediated by anion exchanger SLC26A6 plays a major constitutive role in limiting net absorption of ingested oxalate, thereby preventing hyperoxaluria and calcium oxalate urolithiasis. Using the relatively selective PKC-δ inhibitor rottlerin, we had previously found that PKC-δ activation inhibits Slc26a6 activity in mouse duodenal tissue. To identify a model system to study physiologic agonists upstream of PKC-δ, we characterized the human intestinal cell line T84. Knockdown studies demonstrated that endogenous SLC26A6 mediates most of the oxalate transport by T84 cells. Cholinergic stimulation with carbachol modulates intestinal ion transport through signaling pathways including PKC activation. We therefore examined whether carbachol affects oxalate transport in T84 cells. We found that carbachol significantly inhibited oxalate transport by T84 cells, an effect blocked by rottlerin. Carbachol also led to significant translocation of PKC-δ from the cytosol to the membrane of T84 cells. Using pharmacological inhibitors, we observed that carbachol inhibits oxalate transport through the M3 muscarinic receptor and phospholipase C. Utilizing the Src inhibitor PP2 and phosphorylation studies, we found that the observed regulation downstream of PKC-δ is partially mediated by c-Src. Biotinylation studies revealed that carbachol inhibits oxalate transport by reducing SLC26A6 surface expression. We conclude that carbachol negatively regulates oxalate transport by reducing SLC26A6 surface expression in T84 cells through signaling pathways including the M3 muscarinic receptor, phospholipase C, PKC-δ, and c-Src.

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Adenosinergic signaling inhibits oxalate transport by human intestinal Caco2-BBE cells through the A2B adenosine receptor

AJP Cell Physiology ◽

10.1152/ajpcell.00024.2017 ◽

2018 ◽

Vol 315 (5) ◽

pp. C687-C698 ◽

Cited By ~ 1

Author(s):

Daniel Jung ◽

Altayeb Alshaikh ◽

Sireesha Ratakonda ◽

Mohamed Bashir ◽

Ruhul Amin ◽

...

Keyword(s):

Phospholipase C ◽

Adenosine Receptor ◽

Surface Expression ◽

Oxalate Secretion ◽

Urine Oxalate ◽

Inflammatory Bowel ◽

Sirna Knockdown ◽

Oxalate Transport ◽

Exchange Activity

Most kidney stones (KS) are composed of calcium oxalate, and small increases in urine oxalate affect the stone risk. Intestinal oxalate secretion mediated by anion exchanger SLC26A6 (PAT1) plays a crucial role in limiting net absorption of ingested oxalate, thereby preventing hyperoxaluria and related KS, reflecting the importance of understanding regulation of intestinal oxalate transport. We previously showed that ATP and UTP inhibit oxalate transport by human intestinal Caco2-BBE cells (C2). Since ATP is rapidly degraded to adenosine (ADO), we examined whether intestinal oxalate transport is regulated by ADO. We measured [14C]oxalate uptake in the presence of an outward Cl gradient as an assay of Cl-oxalate exchange activity, ≥49% of which is PAT1-mediated in C2 cells. We found that ADO significantly inhibited oxalate transport by C2 cells, an effect completely blocked by the nonselective ADO receptor antagonist 8- p-sulfophenyltheophylline. ADO also significantly inhibited oxalate efflux by C2 cells, which is important since PAT1 mediates oxalate efflux in vivo. Using pharmacological antagonists and A2B adenosine receptor (A2B AR) siRNA knockdown studies, we observed that ADO inhibits oxalate transport through the A2B AR, phospholipase C, and PKC. ADO inhibits oxalate transport by reducing PAT1 surface expression as shown by biotinylation studies. We conclude that ADO inhibits oxalate transport by lowering PAT1 surface expression in C2 cells through signaling pathways including the A2B AR, PKC, and phospholipase C. Given higher ADO levels and overexpression of the A2B AR in inflammatory bowel disease (IBD), our findings have potential relevance to pathophysiology of IBD-associated hyperoxaluria and related KS.

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Sat1 is dispensable for active oxalate secretion in mouse duodenum

AJP Cell Physiology ◽

10.1152/ajpcell.00385.2011 ◽

2012 ◽

Vol 303 (1) ◽

pp. C52-C57 ◽

Cited By ~ 12

Author(s):

Narae Ko ◽

Felix Knauf ◽

Zhirong Jiang ◽

Daniel Markovich ◽

Peter S. Aronson

Keyword(s):

Calcium Oxalate ◽

Basolateral Membrane ◽

Complete Removal ◽

Calcium Oxalate Stones ◽

Anion Transporter ◽

Oxalate Secretion ◽

In Series ◽

Disulfonic Stilbene ◽

Oxalate Transport

Mice deficient for the apical membrane oxalate transporter SLC26A6 develop hyperoxalemia, hyperoxaluria, and calcium oxalate stones due to a defect in intestinal oxalate secretion. However, the nature of the basolateral membrane oxalate transport process that operates in series with SLC26A6 to mediate active oxalate secretion in the intestine remains unknown. Sulfate anion transporter-1 (Sat1 or SLC26A1) is a basolateral membrane anion exchanger that mediates intestinal oxalate transport. Moreover, Sat1-deficient mice also have a phenotype of hyperoxalemia, hyperoxaluria, and calcium oxalate stones. We, therefore, tested the role of Sat1 in mouse duodenum, a tissue with Sat1 expression and SLC26A6-dependent oxalate secretion. Although the active secretory flux of oxalate across mouse duodenum was strongly inhibited (>90%) by addition of the disulfonic stilbene DIDS to the basolateral solution, secretion was unaffected by changes in medium concentrations of sulfate and bicarbonate, key substrates for Sat1-mediated anion exchange. Inhibition of intracellular bicarbonate production by acetazolamide and complete removal of bicarbonate from the buffer also produced no change in oxalate secretion. Finally, active oxalate secretion was not reduced in Sat1-null mice. We conclude that a DIDS-sensitive basolateral transporter is involved in mediating oxalate secretion across mouse duodenum, but Sat1 itself is dispensable for this process.

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Absence of the sulfate transporter SAT-1 has no impact on oxalate handling by mouse intestine and does not cause hyperoxaluria or hyperoxalemia

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00299.2018 ◽

2019 ◽

Vol 316 (1) ◽

pp. G82-G94 ◽

Cited By ~ 5

Author(s):

Jonathan M. Whittamore ◽

Christine E. Stephens ◽

Marguerite Hatch

Keyword(s):

Calcium Oxalate ◽

Stone Formation ◽

Distal Colon ◽

Toxic Waste ◽

Oxalate Excretion ◽

Anion Transporter ◽

Oxalate Secretion ◽

Mechanistic Basis ◽

Urinary Oxalate Excretion ◽

Sat 1

The anion exchanger SAT-1 [sulfate anion transporter 1 (Slc26a1)] is considered an important regulator of oxalate and sulfate homeostasis, but the mechanistic basis of these critical roles remain undetermined. Previously, characterization of the SAT-1-knockout (KO) mouse suggested that the loss of SAT-1-mediated oxalate secretion by the intestine was responsible for the hyperoxaluria, hyperoxalemia, and calcium oxalate urolithiasis reportedly displayed by this model. To test this hypothesis, we compared the transepithelial fluxes of 14C-oxalate, 35[Formula: see text], and 36Cl− across isolated, short-circuited segments of the distal ileum, cecum, and distal colon from wild-type (WT) and SAT-1-KO mice. The absence of SAT-1 did not impact the transport of these anions by any part of the intestine examined. Additionally, SAT-1-KO mice were neither hyperoxaluric nor hyperoxalemic. Instead, 24-h urinary oxalate excretion was almost 50% lower than in WT mice. With no contribution from the intestine, we suggest that this may reflect the loss of SAT-1-mediated oxalate efflux from the liver. SAT-1-KO mice were, however, profoundly hyposulfatemic, even though there were no changes to intestinal sulfate handling, and the renal clearances of sulfate and creatinine indicated diminished rates of sulfate reabsorption by the proximal tubule. Aside from this distinct sulfate phenotype, we were unable to reproduce the hyperoxaluria, hyperoxalemia, and urolithiasis of the original SAT-1-KO model. In conclusion, oxalate and sulfate transport by the intestine were not dependent on SAT-1, and we found no evidence supporting the long-standing hypothesis that intestinal SAT-1 contributes to oxalate and sulfate homeostasis. NEW & NOTEWORTHY SAT-1 is a membrane-bound transport protein expressed in the intestine, liver, and kidney, where it is widely considered essential for the excretion of oxalate, a potentially toxic waste metabolite. Previously, calcium oxalate kidney stone formation by the SAT-1-knockout mouse generated the hypothesis that SAT-1 has a major role in oxalate excretion via the intestine. We definitively tested this proposal and found no evidence for SAT-1 as an intestinal anion transporter contributing to oxalate homeostasis.

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Extracellular Nucleotides Regulate Arterial Calcification by Activating Both Independent and Dependent Purinergic Receptor Signaling Pathways

International Journal of Molecular Sciences ◽

10.3390/ijms21207636 ◽

2020 ◽

Vol 21 (20) ◽

pp. 7636 ◽

Cited By ~ 1

Author(s):

Britt Opdebeeck ◽

Isabel R. Orriss ◽

Ellen Neven ◽

Patrick C. D’Haese ◽

Anja Verhulst

Keyword(s):

Purinergic Receptors ◽

Molecular Mechanisms ◽

Purinergic Receptor ◽

Bone Mineralization ◽

Purinergic Signaling ◽

Extracellular Nucleotides ◽

Arterial Calcification ◽

Nucleoside Triphosphate ◽

Vascular Cells ◽

Bone Homeostasis

Arterial calcification, the deposition of calcium-phosphate crystals in the extracellular matrix, resembles physiological bone mineralization. It is well-known that extracellular nucleotides regulate bone homeostasis raising an emerging interest in the role of these molecules on arterial calcification. The purinergic independent pathway involves the enzymes ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs), ecto-nucleoside triphosphate diphosphohydrolases (NTPDases), 5′-nucleotidase and alkaline phosphatase. These regulate the production and breakdown of the calcification inhibitor—pyrophosphate and the calcification stimulator—inorganic phosphate, from extracellular nucleotides. Maintaining ecto-nucleotidase activities in a well-defined range is indispensable as enzymatic hyper- and hypo-expression has been linked to arterial calcification. The purinergic signaling dependent pathway focusses on the activation of purinergic receptors (P1, P2X and P2Y) by extracellular nucleotides. These receptors influence arterial calcification by interfering with the key molecular mechanisms underlying this pathology, including the osteogenic switch and apoptosis of vascular cells and possibly, by favoring the phenotypic switch of vascular cells towards an adipogenic phenotype, a recent, novel hypothesis explaining the systemic prevention of arterial calcification. Selective compounds influencing the activity of ecto-nucleotidases and purinergic receptors, have recently been developed to treat arterial calcification. However, adverse side-effects on bone mineralization are possible as these compounds reasonably could interfere with physiological bone mineralization.

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Modulation of epithelial sodium channel activity by lipopolysaccharide in alveolar type II cells: involvement of purinergic signaling

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00170.2009 ◽

2010 ◽

Vol 298 (3) ◽

pp. L417-L426 ◽

Cited By ~ 18

Author(s):

Émilie Boncoeur ◽

Valérie Tardif ◽

Marie-Claude Tessier ◽

Frédéric Morneau ◽

Jacynthe Lavoie ◽

...

Keyword(s):

Epithelial Cells ◽

Purinergic Receptor ◽

Purinergic Signaling ◽

Surface Protein ◽

Extracellular Atp ◽

Alveolar Epithelial Cells ◽

Extracellular Nucleotides ◽

Alveolar Type ◽

Alveolar Epithelial ◽

Atp Signaling

Pseudomonas aeruginosa is a gram-negative bacterium that causes chronic infection in cystic fibrosis patients. We reported recently that P. aeruginosa modulates epithelial Na+ channel (ENaC) expression in experimental chronic pneumonia models. For this reason, we tested whether LPS from P. aeruginosa alters ENaC expression and activity in alveolar epithelial cells. We found that LPS induces a ∼60% decrease of ENaC apical current without significant changes in intracellular ENaC or surface protein expression. Because a growing body of evidence reports a key role for extracellular nucleotides in regulation of ion channels, we evaluated the possibility that modulation of ENaC activity by LPS involves extracellular ATP signaling. We found that alveolar epithelial cells release ATP upon LPS stimulation and that pretreatment with suramin, a P2Y2 purinergic receptor antagonist, inhibited the effect of LPS on ENaC. Furthermore, ET-18-OCH3, a PLC inhibitor, and Go-6976, a PKC inhibitor, were able to partially prevent ENaC inhibition by LPS, suggesting that the actions of LPS on ENaC current were mediated, in part, by the PKC and PLC pathways. Together, these findings demonstrate an important role of extracellular ATP signaling in the response of epithelial cells to LPS.

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