Pseudovitamin D-deficiency rickets (PDDR) is an autosomal disease, characterized by growth retardation, hyperpara-thyroidism, rickets and undetectable levels of 1,25-dihydroxy-vitamin D₃ (1,25(OH)₂D₃). Mice homozygous for the 25-hydroxyvitamin D₃-1α-hydroxylase (1α-OHase) gene presented the same clinical phenotype as patients with PDDR. To determine whether the severe hypocalcaemia in PDDR is secondary to disturbed 1,25(OH)₂D₃-stimulated Ca²⁺ reabsorption, the effect of 1α-OHase gene disruption was examined on serum Ca²⁺ and renal expression of the epithelial Ca²⁺ channel (ECaC1), the calbindins, Na⁺-Ca²⁺ exchanger (NCX1) and Ca²⁺-ATPase (PMCA1b) in 1α-OHase knockout mice. Animals were humanely killed according to the guidelines of the Animal Welfare committee. Both serum Ca²⁺ (1α-OHase^-/- mice: 1.20 ± 0.05 versus 1α-OHase^+/- mice: 2.19 ± 0.01 mM) and mRNA/protein expression of ECaC1 (41 ± 3 %, mean ± S.E.M., n = 6), calbindin-D_28K (31 ± 2 %), calbindin-D_9K (58 ± 7 %), NCX1 (10 ± 2 %), PMCA1b (69 ± 3 %) were significantly decreased in 1α-OHase^-/- mice. Feeding the 1α-OHase^-/- mice a Ca²⁺-enriched diet normalized expression of these Ca²⁺ proteins except for the calbindin-D9K expression that was not significantly altered. In contrast to 1α-OHase^+/- mice in which dietary Ca²⁺ reduced (ECaC1, calbindin-D28K, calbindin-D9K), or did not change (NCX1, PMCA1b) the expression levels of the Ca²⁺ transport proteins. Elevation of the Ca²⁺ transport proteins by dietary Ca²⁺ was accompanied by normalization of the serum Ca²⁺ concentration in 1α-OHase^-/- mice from 1.20 ± 0.05 to 2.33 ± 0.10 mM. Like dietary Ca²⁺, 1,25(OH)₂D₃ repletion resulted in increased expression of the Ca²⁺ transport proteins and normalization of serum Ca²⁺ concentrations. Immunohistochemistry showed that ECaC1, calbindin-D28K and calbindin-D9K are co-expressed in the majority of the distal tubules of 1α-OHase^-/- mice. In addition, 1α-OHase^-/- mice, exposed to the high Ca²⁺-enriched diet, contained immunopositive distal tubules expressing calbindin-D_28K, whereas these tubules did not contain calbindin-D_9K. Importantly, confocal microscopy demonstrated that the localization of the Ca²⁺ transport proteins is clearly polarized in the renal cell in which ECaC1 is localized along the apical membrane, calbindin-D_28K in the cytoplasm and calbindin-D_9K along the basolateral membrane resulting in a comprehensive mechanism facilitating renal transcellular Ca²⁺ transport.

All procedures accord with current National and local guidelines.