title: Regulation of vitamin D-1alpha-hydroxylase in a human cortical collecting duct cell line.
abstract: BACKGROUND
  Recent studies have shown that renal expression of 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) is not restricted to proximal tubules. To investigate the significance of this expression, we characterized the regulation of 1alpha-OHase expression and activity in a human cortical collecting duct cell line (HCD).
  
  
  METHODS
  Expression of 1alpha-OHase mRNA and protein was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses. Enzyme activity was quantified using 25-hydroxyvitamin D3 as the substrate; conversion to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and 24,25-dihydroxyvitamin D3 was then determined by thin-layer chromatography.
  
  
  RESULTS
  HCD cells expressed mRNA and protein for 1alpha-OHase. However, basal 1,25(OH)2D3 production was lower than that observed in proximal tubule HKC-8 cells. In both cell lines, synthesis of 1,25(OH)2D3 was increased by forskolin, parathyroid hormone, and low calcium medium. Conversely, treatment with 1,25(OH)2D3 itself decreased 1alpha-OHase activity. This effect was more pronounced in HCD cells, which also demonstrated significantly higher levels of 24-hydroxylase activity. The most striking induction of 1alpha-OHase activity was observed in the HCD cells following incubation with lipopolysaccharide, which was coincident with the expression of mRNA for both CD14 and Toll-like receptor 4.
  
  
  CONCLUSIONS
  These results highlight the capacity for synthesis of 1,25(OH)2D3 in cells from more distal areas of the nephron. However, more sensitive feedback regulation and immune induction of 1alpha-OHase in the HCD cells suggest a more localized role for 1,25(OH)2D3 production in the distal nephron.
authors.affiliation: Division of Medical Sciences, The University of Birmingham, Queen Elizabeth Hospital, Birmingham, England, United Kingdom.
authors.forename: R
authors.initials: R
authors.lastname: Bland
authors.forename: D
authors.initials: D
authors.lastname: Zehnder
authors.forename: S V
authors.initials: SV
authors.lastname: Hughes
authors.forename: P M
authors.initials: PM
authors.lastname: Ronco
authors.forename: P M
authors.initials: PM
authors.lastname: Stewart
authors.forename: M
authors.initials: M
authors.lastname: Hewison
mesh_terms: D002117:Calcitriol / Q000378:metabolism; D002118:Calcium / Q000008:administration & dosage / Q000494:pharmacology; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D029721:Drosophila Proteins; D006801:Humans; D007672:Kidney Cortex; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism; D010281:Parathyroid Hormone / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D065668:Vitamin D3 24-Hydroxylase
chemical_list: D003470:Culture Media; D029721:Drosophila Proteins; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D010281:Parathyroid Hormone; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D005576:Colforsin; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C406322:vitamin D 1-alpha hydroxylase; D065668:Vitamin D3 24-Hydroxylase; D002117:Calcitriol; D002118:Calcium