Coordinated regulation of TRPV5-mediated Ca²⁺ transport in primary distal convolution cultures.

Details

Serval ID
serval:BIB_11EA85DA130B
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Coordinated regulation of TRPV5-mediated Ca²⁺ transport in primary distal convolution cultures.
Journal
Pflugers Archiv : European Journal of Physiology
Author(s)
van der Hagen E.A., Lavrijsen M., van Zeeland F., Praetorius J., Bonny O., Bindels R.J., Hoenderop J.G.
ISSN
1432-2013 (Electronic)
ISSN-L
0031-6768
Publication state
Published
Issued date
2014
Peer-reviewed
Oui
Volume
466
Number
11
Pages
2077-2087
Language
english
Abstract
Fine-tuning of renal calcium ion (Ca(2+)) reabsorption takes place in the distal convoluted and connecting tubules (distal convolution) of the kidney via transcellular Ca(2+) transport, a process controlled by the epithelial Ca(2+) channel Transient Receptor Potential Vanilloid 5 (TRPV5). Studies to delineate the molecular mechanism of transcellular Ca(2+) transport are seriously hampered by the lack of a suitable cell model. The present study describes the establishment and validation of a primary murine cell model of the distal convolution. Viable kidney tubules were isolated from mice expressing enhanced Green Fluorescent Protein (eGFP) under the control of a TRPV5 promoter (pTRPV5-eGFP), using Complex Object Parametric Analyser and Sorting (COPAS) technology. Tubules were grown into tight monolayers on semi-permeable supports. Radioactive (45)Ca(2+) assays showed apical-to-basolateral transport rates of 13.5 ± 1.2 nmol/h/cm(2), which were enhanced by the calciotropic hormones parathyroid hormone and 1,25-dihydroxy vitamin D3. Cell cultures lacking TRPV5, generated by crossbreeding pTRPV5-eGFP with TRPV5 knockout mice (TRPV5(-/-)), showed significantly reduced transepithelial Ca(2+) transport (26 % of control), for the first time directly confirming the key role of TRPV5. Most importantly, using this cell model, a novel molecular player in transepithelial Ca(2+) transport was identified: mRNA analysis revealed that ATP-dependent Ca(2+)-ATPase 4 (PMCA4) instead of PMCA1 was enriched in isolated tubules and downregulated in TRPV5(-/-) material. Immunohistochemical stainings confirmed co-localization of PMCA4 with TRPV5 in the distal convolution. In conclusion, a novel primary cell model with TRPV5-dependent Ca(2+) transport characteristics was successfully established, enabling comprehensive studies of transcellular Ca(2+) transport.
Keywords
Adenosine Triphosphate/metabolism, Animals, Calcium/metabolism, Calcium Channels/metabolism, Calcium-Transporting ATPases/metabolism, Kidney Tubules, Distal/metabolism, Mice, Parathyroid Hormone/metabolism, Plasma Membrane Calcium-Transporting ATPases/metabolism, Protein Transport/physiology, TRPV Cation Channels/metabolism, Vitamin D/analogs & derivatives, Vitamin D/metabolism
Pubmed
Web of science
Funding(s)
Swiss National Science Foundation / Careers / PP00P3_133648
Create date
07/03/2014 2:44
Last modification date
24/01/2020 7:26
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