Coordinated Regulation of TRPV5-mediated Ca²⁺ Transport in Primary Distal Convolution Cultures

Pflugers Arch. 2014 Nov;466(11):2077-87. doi: 10.1007/s00424-014-1470-x. Epub 2014 Feb 21.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Animals
  • Calcium / metabolism*
  • Calcium Channels / metabolism*
  • Calcium-Transporting ATPases / metabolism
  • Kidney Tubules, Distal / metabolism*
  • Mice
  • Parathyroid Hormone / metabolism
  • Plasma Membrane Calcium-Transporting ATPases / metabolism
  • Protein Transport / physiology*
  • TRPV Cation Channels / metabolism*
  • Vitamin D / analogs & derivatives
  • Vitamin D / metabolism

Substances

  • Calcium Channels
  • PMCA4 protein, mouse
  • Parathyroid Hormone
  • TRPV Cation Channels
  • Trpv5 protein, mouse
  • dihydroxy-vitamin D3
  • Vitamin D
  • Adenosine Triphosphate
  • Plasma Membrane Calcium-Transporting ATPases
  • Atp2b1 protein, mouse
  • Calcium-Transporting ATPases
  • Calcium