Abstract
Luminal nucleotide stimulation is known to reduce Na+ transport in the distal nephron. Previous studies suggest that this mechanism may involve the thiazide-sensitive Na+-Cl− cotransporter (NCC), which plays an essential role in NaCl reabsorption in the cells lining the distal convoluted tubule (DCT). Here we show that stimulation of mouse DCT (mDCT) cells with ATP or UTP promoted Ca2+ transients and decreased the expression of NCC at both mRNA and protein levels. Specific siRNA-mediated silencing of P2Y2 receptors almost completely abolished ATP/UTP-induced Ca2+ transients and significantly reduced ATP/UTP-induced decrease of NCC expression. To test whether local variations in the intracellular Ca2+ concentration ([Ca2+]i) may control NCC transcription, we overexpressed the Ca2+-binding protein parvalbumin selectively in the cytosol or in the nucleus of mDCT cells. The decrease in NCC mRNA upon nucleotide stimulation was abolished in cells overexpressing cytosolic PV but not in cells overexpressing either a nuclear-targeted PV or a mutated PV unable to bind Ca2+. Using a firefly luciferase reporter gene strategy, we observed that the activity of NCC promoter region from −1 to −2,200 bp was not regulated by changes in [Ca2+]i. In contrast, high cytosolic calcium level induced instability of NCC mRNA. We conclude that in mDCT cells: (1) P2Y2 receptor is essential for the intracellular Ca2+ signaling induced by ATP/UTP stimulation; (2) P2Y2-mediated increase of cytoplasmic Ca2+ concentration down-regulates the expression of NCC; (3) the decrease of NCC expression occurs, at least in part, via destabilization of its mRNA.
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Abbreviations
- ATP/UTP:
-
Adenosine-5′-triphosphate/uridine-5′-triphosphate
- [Ca2+]e :
-
Extracellular Ca2+ concentration
- [Ca2+]i :
-
Intracellular Ca2+ concentration
- DCT:
-
Distal convoluted tubule
- mDCT:
-
Mouse DCT
- EGTA-AM:
-
Ethylene glycol tetra (acetoxymethyl ester)
- FITC:
-
Fluorescein isothiocyanate
- GAPDH:
-
Glyceraldehyde 3-phosphate dehydrogenase
- NCC:
-
Na+-Cl− cotransporter
- nCaRE:
-
Negative calcium response element
- PLC:
-
Phospholipase C
- PV:
-
Parvalbumin
- TRPM6:
-
Transient receptor potential cation channel, subfamily M, member 6
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Acknowledgments
The authors thank Prof. J. Loffing and Mrs. M. Carrel (Dept. of Anatomy, University of Zurich) for help and fruitful discussions. This study was supported by the Belgian agencies "Fonds National de la Recherche Scientifique" and "Fonds de la Recherche Scientifique Médicale", Concerted Research Action (10-15/029) and Inter-University Attraction Poles (IUAP P7/13) from the Belgian Federal Government and by the NCCR Kidney.CH program (Swiss National Science Foundation); the Gebert Rüf Stiftung (Project GRS-038/12); and the Swiss National Science Foundation 310030-146490 (to OD).
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The authors have declared that no conflict of interest exists.
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P. Gailly and M. Szutkowska contributed equally to this work.
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Gailly, P., Szutkowska, M., Olinger, E. et al. P2Y2 receptor activation inhibits the expression of the sodium-chloride cotransporter NCC in distal convoluted tubule cells. Pflugers Arch - Eur J Physiol 466, 2035–2047 (2014). https://doi.org/10.1007/s00424-013-1438-2
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DOI: https://doi.org/10.1007/s00424-013-1438-2