Duodenal calcium absorption in vitamin D receptor-knockout mice: functional and molecular aspects

Abstract

Rickets and hyperparathyroidism caused by a defective vitamin D receptor (VDR) can be prevented in humans and animals by high calcium intake, suggesting that intestinal calcium absorption is critical for 1,25(OH)(2) vitamin D [1,25(OH)(2)D(3)] action on calcium homeostasis. We assessed the rate of serum (45)Ca accumulation within 10 min of oral gavage in two strains of VDR-knockout (KO) mice (Leuven and Tokyo KO) and observed a 3-fold lower area under the curve in both KO strains. Moreover, we evaluated the expression of intestinal candidate genes involved in transcellular calcium transport. The calcium transport protein1 (CaT1) was more abundantly expressed at mRNA level than the epithelial calcium channel (ECaC) in duodenum, but both were considerably reduced (CaT1>90%, ECaC>60%) in the two VDR-KO strains on a normal calcium diet. Calbindin-D(9K) expression was decreased only in the Tokyo KO, whereas plasma membrane calcium ATPase (PMCA(1b)) expression was normal in both VDR-KOs. In Leuven wild-type mice, a high calcium diet inhibited (>90%) and 1,25(OH)(2)D(3) injection or low calcium diet induced (6-fold) duodenal CaT1 expression and, to a lesser degree, ECaC and calbindin-D(9K) expression. In Leuven KO mice, however, high or low calcium intake decreased calbindin-D(9K) and PMCA(1b) expression, whereas CaT1 and ECaC expression remained consistently low on any diet. These results suggest that the expression of the novel duodenal epithelial calcium channels (in particular CaT1) is strongly vitamin D-dependent, and that calcium influx, probably interacting with calbindin-D(9K), should be considered as a rate-limiting step in the process of vitamin D-dependent active calcium absorption.

Figure 1

Targeting of the VDR gene. (A) Modification of the VDR gene. 1, Targeting vector pNT.VDR and VDR^WT allele; 2, Homologously recombined VDR allele (VDR^neo); 3, Modified VDR allele after Cre-excision of the floxed neo cassette; 4, VDR^min allele after Cre-excision of the floxed exon 2. exons ; intron sequences ; D external (1.5-kb XhoI fragment) and C internal (1-kb EcoRV-SacI fragment) hybridization probe. The observed size of the diagnostic restriction fragments, used to distinguish the WT and mutant alleles, corresponds to their expected size. (B) Southern analysis of EcoRV-digested genomic DNA from VDR^WT and VDR^neo embryonic stem cell clones by using the external 3′ probe D, which generates a 8.5-kb VDR^WT and a 10.5-kb VDR^neo fragment. (C) Southern analysis of EcoRV-digested genomic DNA from VDR^WT and VDR^+/lox mice by using the external 3′ probe D, which generates a 8.5-kb VDR^WT and a 16-kb VDR^lox fragment. (D) Southern analysis of SacI-digested genomic DNA from VDR^WT, VDR^+/− and VDR^KO mice by using the internal probe C. (E) RT-PCR analysis of RNA isolated from duodenum, kidney, and femur of VDR^WT and VDR^KO mice for VDR and HPRT RNA level expression.

Figure 2

Intestinal calcium absorption assay. Changes in serum calcium (ΔμMol) within 10 min after administration of ⁴⁵Ca: (A) orally in vehicle (■) and 1,25(OH)₂D₃ (▴) treated Swiss mice; (B) orally in Swiss mice after 1 week of normal (□), low calcium (▵), or rescue diet (◊); (C) orally (WT ● and KO ▾) and intravenously (WT ○ and KO ▿) in the Leuven strain; (D) orally (WT ● and KO ▾) in the Tokyo strain. ΔμMol is obtained by the equation: (cpm 10 μl serum/cpm 10 μl stock solution) × 10² μM. *, P < 0.001 vs. vehicle at same time point; †, P < 0.05 vs. normal diet at same time point; ‡, P < 0.001 vs. WT at same time point.

Figure 3

Gene expression patterns in duodenum and kidney of Leuven mice on different diets (n = 12/group) and of Tokyo mice on normal diet (n = 6/group). (A) Duodenal gene expression of CaT1 , calbindin-D_9K , and PMCA , assessed by qRT-PCR analysis, is calculated as a ratio to the HPRT RNA level and is expressed relatively to levels of WT mice on normal diet. (B) Calbindin-D_9K protein content in duodenum, measured by RIA, is expressed as % (mg/mg) of total duodenal protein. (C) Renal gene expression of ECaC , calbindin-D_9K , calbindin-D_28K , PMCA , and NCX , assessed by qRT-PCR analysis, is calculated as a ratio to the HPRT RNA level and expressed relatively to levels of WT mice on normal diet. *, P < 0.001, †, P < 0.01, and ‡, P < 0.05 vs. WT on normal diet.