title: Coordinated regulation of TRPV5-mediated Ca²⁺ transport in primary distal convolution cultures.
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.
authors.affiliation: Department of Physiology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands.
authors.forename: Eline A E
authors.initials: EA
authors.lastname: van der Hagen
authors.forename: Marla
authors.initials: M
authors.lastname: Lavrijsen
authors.forename: Femke
authors.initials: F
authors.lastname: van Zeeland
authors.forename: Jeppe
authors.initials: J
authors.lastname: Praetorius
authors.forename: Olivier
authors.initials: O
authors.lastname: Bonny
authors.forename: René J M
authors.initials: RJ
authors.lastname: Bindels
authors.forename: Joost G J
authors.initials: JG
authors.lastname: Hoenderop
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D007686:Kidney Tubules, Distal / Q000378:metabolism*; D051379:Mice; D010281:Parathyroid Hormone / Q000378:metabolism; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology*; D050916:TRPV Cation Channels / Q000378:metabolism*; D014807:Vitamin D / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D000255:Adenosine Triphosphate; D002118:Calcium; D015220:Calcium Channels; D000252:Calcium-Transporting ATPases; D010281:Parathyroid Hormone; D053499:Plasma Membrane Calcium-Transporting ATPases; D050916:TRPV Cation Channels; D014807:Vitamin D; C000589038:ATP2B4 protein, mouse; C492240:Trpv5 protein, mouse; C118756:dihydroxy-vitamin D3; C506954:Atp2b1 protein, mouse