Coordinated regulation of TRPV5-mediated Ca2+ transport in primary distal convolution cultures
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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 45Ca2+ assays showed apical-to-basolateral transport rates of 13.5 ± 1.2 nmol/h/cm2, 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 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.
KeywordsKidney TRPV5 PMCA4 COPAS Calcium Primary culture
We are grateful to the ‘COPAS sorting team’: Hans Meijer, Thomas van der Velden, AnneMiete van der Kemp and Laura Klein for maintenance of mice and/or subsequent sorting of the tubule material. We thank Sjoerd Verkaart for critical reading of the manuscript. We thank Nicolas Markadieu for help with setting up the primary cultures. We thank Rico Bongaarts from Union Biometrica for technical assistance with the COPAS. We thank Candice Stoudmann for technical help.
This work was financially supported by the Netherlands Organization for Scientific Research (NWO 819.02.012), the European Science Foundation (EURYI 2006) and EURenOmics funding from the European Union Seventh Framework Programme (FP7/2007–2013, agreement no. 305608).
The authors declare no conflict of interest.
- 5.Brini M, Carafoli E (2011) The plasma membrane Ca(2) + ATPase and the plasma membrane sodium calcium exchanger cooperate in the regulation of cell calcium. Cold Spring Harb Perspect Biol 3Google Scholar
- 18.Hoenderop JG, van Leeuwen JP, van der Eerden BC, Kersten FF, van der Kemp AW, Merillat AM, Waarsing JH, Rossier BC, Vallon V, Hummler E, Bindels RJ (2003) Renal Ca2+ wasting, hyperabsorption, and reduced bone thickness in mice lacking TRPV5. J Clin Invest 112:1906–1914PubMedCrossRefPubMedCentralGoogle Scholar
- 27.Markadieu N, San-Cristobal P, Nair AV, Verkaart S, Lenssen E, Tudpor K, van Zeeland F, Loffing J, Bindels RJ, Hoenderop JG (2012) A primary culture of distal convoluted tubules expressing functional thiazide-sensitive NaCl transport. Am J Physiol Renal Physiol 303:F886–892PubMedCrossRefGoogle Scholar
- 28.Okunade GW, Miller ML, Pyne GJ, Sutliff RL, O'Connor KT, Neumann JC, Andringa A, Miller DA, Prasad V, Doetschman T, Paul RJ, Shull GE (2004) Targeted ablation of plasma membrane Ca2 + −ATPase (PMCA) 1 and 4 indicates a major housekeeping function for PMCA1 and a critical role in hyperactivated sperm motility and male fertility for PMCA4. J Biol Chem 279:33742–33750PubMedCrossRefGoogle Scholar