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This Article Full Text Full Text (PDF) All Versions of this Article: 286/3/F483    most recent 00231.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (7) Citing Articles via Google Scholar Google Scholar Articles by Diepens, R. J. W. Articles by Hoenderop, J. G. J. Search for Related Content PubMed PubMed Citation Articles by Diepens, R. J. W. Articles by Hoenderop, J. G. J.

Characterization of a murine renal distal convoluted tubule cell line for the study of transcellular calcium transport

¹Department of Physiology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, NL-6500 HB Nijmegen, The Netherlands; and ²Institut National de la Santé et de la Recherche Médicale, U 478, Faculté de Médecine Xavier Bichat, 75870 Paris Cedex 18, France

Submitted 24 June 2003 ; accepted in final form 11 November 2003

To unravel the molecular regulation of renal transcellular Ca²⁺ transport, a murine distal convoluted tubule (mpkDCT) cell line derived from distal convoluted tubules (DCT) microdissected from a SV-PK/Tag transgenic mouse was characterized. This cell line originated from DCT only, as mRNA encoding for the DCT marker thiazide-sensitive Na⁺/Cl^- cotransporter was expressed, whereas mRNA encoding for the connecting tubule and collecting duct marker aquaporin-2 was not detected, as determined by reverse-transcriptase PCR. mpkDCT cells expressed mRNA encoding the Ca²⁺ channels TRPV5 and TRPV6 and other key players necessary for transcellular Ca²⁺ transport, i.e., calbindin-D_9k, calbindin-D_28k, plasma membrane Ca²⁺-ATPase isoform 1b, and Na⁺/Ca²⁺ exchanger 1. Primary cultures of DCT cells exhibited net transcellular Ca²⁺ transport of 0.4 ± 0.1 nmol·h^-1·cm^-2, whereas net transcellular Ca²⁺ transport across mpkDCT cells was significantly higher at 2.4 ± 0.4 nmol·h^-1·cm^-2. Transcellular Ca²⁺ transport across mpkDCT cells was completely inhibited by ruthenium red, an inhibitor of TRPV5 and TRPV6, but not by the voltage-operated Ca²⁺ channel inhibitors felodipine and verapamil. With the use of patch-clamp analysis, the IC₅₀ of ruthenium red on Na⁺ currents was between the values measured for TRPV5- and TRPV6-expressing HEK 293 cells, suggesting that TRPV5 and/or TRPV6 is possibly active in mpkDCT cells. Forskolin in combination with IBMX, 1,25-dihydroxyvitamin D₃, and 1-deamino-8-D-arginine vasopressin increased transcellular Ca²⁺ transport, whereas PMA and parathyroid hormone had no significant effect. In conclusion, the murine mpkDCT cell line provides a unique cell model in which to study the molecular regulation of transcellular Ca²⁺ transport in the kidney in vitro.

TRPV5; TRPV6; vitamin D₃; kidney; calcium reabsorption

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