Characterization of vitamin D-deficient klotho(-/-) mice: do increased levels of serum 1,25(OH)2D3 cause disturbed calcium and phosphate homeostasis in klotho(-/-) mice?
BACKGROUND
Klotho(-/-) mice display disturbed Ca(2+) and vitamin D homeostasis. Renal cytochrome p450 27b1 (Cyp27b1), the enzyme that catalyzes the hydrolysis to 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), is increased in klotho(-/-) mice, and a 1,25(OH)(2)D(3)-deficient diet partially normalized Ca(2+) homeostasis in these klotho(-/-) mice. The aim of the present study was to further delineate the interplay between 1,25(OH)(2)D(3) and klotho and their relative contribution to the Ca(2+) homeostasis of klotho(-/-) mice.


METHODS
Double-klotho(-/-)/Cyp27b1(-/-) mice were generated and mice aged 8-12 weeks were housed in metabolic cages to collect 24-h urine. Blood samples were taken and the animals were sacrificed, and the kidney and duodenum tissues were sampled for RNA extraction. The bone was fixed in 10% v/v formalin and analysed by microcomputed tomography (μCT) scans.


RESULTS
Klotho(-/-)/Cyp27b1(-/-) mice, like Cyp27b1(-/-) mice, displayed significantly decreased serum total calcium concentrations compared with wild-type mice (1.44 ± 0.03 and 2.25 ± 0.02 mM) along with normal urinary total calcium excretion. Hyperphosphataemia of klotho(-/-) mice normalized to wild-type levels in klotho(-/-)/Cyp27b1(-/-) mice. The mRNA levels of duodenal transient receptor potential vanilloid subtype 6 (TRPV6) and calcium-binding protein-D(9K), and renal calbindin-D(28K) and NCX1 were significantly reduced in the double knockouts compared with wild-type or klotho(-/-) mice. Elevated TRPV5 protein levels in klotho(-/-) mice normalized to wild type in klotho(-/-)/Cyp27b1(-/-) mice, but were decreased in Cyp27b1(-/-) mice. μCT scans showed that klotho(-/-)/Cyp27b1(-/-) mice, as Cyp27b1(-/-) mice, display significant bone hypomineralization and severely decreased bone mass. Klotho(-/-) mice show a reduced bone mass and increased trabecular numbers.


CONCLUSIONS
Klotho(-/-)/Cyp27b1(-/-) mice resemble Cyp27b1(-/-) mice. Since 1,25(OH)(2)D(3) is absent in these mice, our results imply that Ca(2+) homeostasis in klotho(-/-) mice is affected by their excessive 1,25(OH)(2)D(3) levels.
Department of Physiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
Titia E
TE
Woudenberg-Vrenken
Bram C J
BC
van der Eerden
AnneMiete W C M
AW
van der Kemp
Johannes P T M
JP
van Leeuwen
René J M
RJ
Bindels
Joost G J
JG
Hoenderop
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D001842:Bone and Bones / Q000378:metabolism*; D002118:Calcium / Q000097:blood* / Q000652:urine; D004386:Duodenum / Q000378:metabolism; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism*; D006706:Homeostasis; D007150:Immunohistochemistry; D000090265:Klotho Proteins; D051379:Mice; D018345:Mice, Knockout; D010710:Phosphates / Q000097:blood* / Q000652:urine; D060888:Real-Time Polymerase Chain Reaction; D050916:TRPV Cation Channels / Q000378:metabolism; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000097:blood; D014808:Vitamin D Deficiency / Q000097:blood* / Q000652:urine
D010710:Phosphates; D050916:TRPV Cation Channels; C118756:dihydroxy-vitamin D3; D014807:Vitamin D; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D005966:Glucuronidase; D000090265:Klotho Proteins; D002118:Calcium