Characterization of a murine renal distal convoluted tubule cell line for the study of transcellular calcium transport.
To unravel the molecular regulation of renal transcellular Ca(2+) 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 Ca(2+) channels TRPV5 and TRPV6 and other key players necessary for transcellular Ca(2+) transport, i.e., calbindin-D(9k), calbindin-D(28k), plasma membrane Ca(2+)-ATPase isoform 1b, and Na(+)/Ca(2+) exchanger 1. Primary cultures of DCT cells exhibited net transcellular Ca(2+) transport of 0.4 +/- 0.1 nmol.h(-1).cm(-2), whereas net transcellular Ca(2+) transport across mpkDCT cells was significantly higher at 2.4 +/- 0.4 nmol.h(-1).cm(-2). Transcellular Ca(2+) transport across mpkDCT cells was completely inhibited by ruthenium red, an inhibitor of TRPV5 and TRPV6, but not by the voltage-operated Ca(2+) channel inhibitors felodipine and verapamil. With the use of patch-clamp analysis, the IC(50) 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 D(3), and 1-deamino-8-d-arginine vasopressin increased transcellular Ca(2+) 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 Ca(2+) transport in the kidney in vitro.
Diepens
Robin J W
RJ
Department of Physiology, Nijmegen Center for Molecualr Life Sciences, University Medical Center, The Netherlands.
den Dekker
Els
E
Bens
Marcelle
M
Weidema
A Freek
AF
Vandewalle
Alain
A
Bindels
René J M
RJ
Hoenderop
Joost G J
JG
D000818:Animals; D002117:Calcitriol / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004553:Electric Conductivity; D005260:Female; D017136:Ion Transport; D007686:Kidney Tubules, Distal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D012333:RNA, Messenger / Q000378:metabolism; D012430:Ruthenium Red / Q000494:pharmacology
D012333:RNA, Messenger; D012430:Ruthenium Red; D000242:Cyclic AMP; D002117:Calcitriol; D002118:Calcium