title: 25-Hydroxyvitamin D3 1-α-hydroxylase-dependent stimulation of renal klotho expression by spironolactone.
abstract: BACKGROUND
  Klotho, a transmembrane protein, protease and hormone mainly expressed in kidney, is required for the suppression of 1,25(OH)2D3-generating 25-hydroxyvitamin D3 1-alpha-hydroxylase (Cyp27b1) by FGF23. Conversely, 1,25(OH)2D3 stimulates, by activating the vitamin D3 receptor (Vdr), the expression of klotho, thus establishing a negative feedback loop. Klotho protects against renal and vascular injury. Klotho deficiency accelerates aging and early death, effects at least partially due to excessive formation of 1,25(OH)2D3 and subsequent hyperphosphatemia. Klotho expression is inhibited by aldosterone. The present study explored the interaction of aldosterone and DOCA as well as the moderately selective mineralocorticoid receptor antagonist spironolactone on klotho expression.
  
  
  METHODS
  mRNA levels were determined utilizing quantitative RT-PCR in human embryonic kidney cells (HEK293) or in renal tissues from mice without or with prior mineralocorticoid (aldosterone or DOCA) and/or spironolactone treatment. In HEK293 cells, protein levels were determined by western blotting. The experiments in HEK293 cells were performed without or with silencing of CYP27B1, of vitamin D3 receptor (VDR) or of mineralocorticoid receptor (NR3C2).
  
  
  RESULTS
  In HEK293 cells aldosterone and in mice DOCA significantly decreased KLOTHO gene expression, effects opposed by spironolactone treatment. Spironolactone treatment alone significantly increased KLOTHO and CYP27B1 transcript levels in HEK293 cells (24 hours) and mice (8 hours or 5 days). Moreover, spironolactone significantly increased klotho and CYP27B1 protein levels in HEK293 cells (48 hours). Reduced NR3C2 expression following silencing did not significantly affect KLOTHO and CYP27B1 transcript levels in presence or absence of spironolactone. Silencing of CYP27B1 and VDR significantly blunted the stimulating effect of spironolactone on KLOTHO mRNA levels in HEK293 cells.
  
  
  CONCLUSIONS
  Besides blocking the effects of aldosterone, spironolactone upregulates KLOTHO gene expression by upregulation of 25-hydroxyvitamin D3 1-alpha-hydroxylase with subsequent activation of the vitamin D3 receptor by 1,25(OH)2D3, an effect possibly independent from the mineralocorticoid receptor.
authors.affiliation: Department of Physiology, University of Tübingen, Tübingen, Germany.
authors.forename: Ioana
authors.initials: I
authors.lastname: Alesutan
authors.forename: Martina
authors.initials: M
authors.lastname: Feger
authors.forename: Tatsiana
authors.initials: T
authors.lastname: Pakladok
authors.forename: Sobuj
authors.initials: S
authors.lastname: Mia
authors.forename: Mohamed Siyabeldin E
authors.initials: MS
authors.lastname: Ahmed
authors.forename: Jakob
authors.initials: J
authors.lastname: Voelkl
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000502:physiology*; D000818:Animals; D005260:Female; D000089703:Fibroblast Growth Factor-23; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005966:Glucuronidase / Q000096:biosynthesis* / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D000090265:Klotho Proteins; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D013148:Spironolactone / Q000494:pharmacology*
chemical_list: C000717427:FGF23 protein, human; C000717428:Fgf23 protein, mouse; D013148:Spironolactone; D000089703:Fibroblast Growth Factor-23; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D005966:Glucuronidase; D000090265:Klotho Proteins