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

作者：van der Hagen Eline A E; Lavrijsen Marla; van Zeeland Femke; Praetorius Jeppe; Bonny Olivier; Bindels Rene J M; Hoenderop Joost G J^*

来源：Pflugers Archiv-European Journal of Physiology, 2014, 466(11): 2077-2087.

DOI：10.1007/s00424-014-1470-x

• Kidney
• TRPV5
• PMCA4
• COPAS
• Calcium
• Primary culture

Fine-tuning of renal calcium ion (Ca2+) reabsorption takes place in the distal convoluted and connecting tubules (distal convolution) of the kidney via transcellular Ca2+ transport, a process controlled by the epithelial Ca2+ channel Transient Receptor Potential Vanilloid 5 (TRPV5). Studies to delineate the molecular mechanism of transcellular Ca2+ 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 Ca-45(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 D-3. Cell cultures lacking TRPV5, generated by crossbreeding pTRPV5-eGFP with TRPV5 knockout mice (TRPV5(-/-)), showed significantly reduced transepithelial Ca2+ 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 Ca2+ transport was identified: mRNA analysis revealed that ATP-dependent Ca2+-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 Ca2+ transport characteristics was successfully established, enabling comprehensive studies of transcellular Ca2+ transport.

• 出版日期2014-11