Compensatory Changes in Calcium Metabolism Accompany the Loss of Vitamin D Receptor (VDR) From the Distal Intestine and Kidney of Mice

Abstract

1,25 Dihydroxyvitamin D3 (1,25(OH)2 D) increases intestinal Ca absorption when dietary Ca intake is low by inducing gene expression through the vitamin D receptor (VDR). 1,25(OH)2 D-regulated Ca absorption has been studied extensively in the small intestine, but VDR is also present in the large intestine. Our goal was to determine the impact of large intestinal VDR deletion on Ca and bone metabolism. We used transgenic mice expressing Cre-recombinase driven by the 9.5-kb human caudal type homeobox 2 (CDX2) promoter to delete floxed VDR alleles from the caudal region of the mouse (CDX2-KO). Weanling CDX2-KO mice and control littermates were fed low (0.25%) or normal (0.5%) Ca diets for 7 weeks. Serum and urinary Ca, vitamin D metabolites, bone parameters, and gene expression were analyzed. Loss of the VDR in CDX2-KO was confirmed in colon and kidney. Unexpectedly, CDX2-KO had lower serum PTH (-65% of controls, p < 0.001) but normal serum 1,25(OH)2 D and Ca levels. Despite elevated urinary Ca loss (eightfold higher in CDX2-KO) and reduced colonic target genes TRPV6 (-90%) and CaBPD9k (-80%) mRNA levels, CDX2-KO mice had only modestly lower femoral bone density. Interestingly, duodenal TRPV6 and CaBPD9k mRNA expression was fourfold and threefold higher, respectively, and there was a trend toward increased duodenal Ca absorption (+19%, p = 0.076) in the CDX2-KO mice. The major finding of this study is that large intestine VDR significantly contributes to whole-body Ca metabolism but that duodenal compensation may prevent the consequences of VDR deletion from large intestine and kidney in growing mice.

Keywords: CA METABOLISM; CDX2P9.5; LARGE INTESTINE; VITAMIN D; VITAMIN D RECEPTOR.

Fig. 1. Confirmation of VDR deletion in CDX2-KO mice

(A) Measurement of mRNA from full length and knockout VDR alleles by RT-PCR; CDX2-KO mice (KO), VDR^f/f mice (Ctrl); KO allele transcript = 180 bp; floxed allele transcript = 350 bp; (+) = cDNA from C57BL/6J mice; (−) = cDNA from global VDRKO mice; PCR= PCR sample lacking cDNA, 1Kb = 1Kb DNA ladder. (B–E) mRNA level of 1,25(OH)₂D target genes in colon: (B) TRPV6 and (C) CaBPD_9k, or kidney (D) CaBPD_9k and (E) TRPV5. Bars represent the LSmean±SEM (n=20–24 per genotype) expressed as (%) relative to control. Different from control at **p<0.01.

Fig. 2. CDX2-KO Mice Have Normal Serum 1,25(OH)₂D But Low Serum PTH Levels

Serum levels of (A) PTH and (B) 1,25(OH)₂D in control, VDR^f/f (Ctrl) and CDX2-KO mice fed 0.25% or 0.5% Ca diets. Bars represent the LSmean±SEM (n=10–12 mice/diet/genotype). **Different from control at p<0.01. Groups with different letter superscripts are statistically different (Tukey-Kramer test, p<0.05).

Fig. 3. Renal phenotype of CDX2-KO mice

(A) CYP24A1, (B) CYP27B1 and (C) CaSR renal mRNA levels in control, VDR^f/f (Ctrl) and CDX2-KO mice fed 0.25% or 0.5% Ca diets. Data are expressed as (%) relative to controls on the 0.5% Ca diet. (D) Urinary Ca/creatinine ratio. Bars reflect the LSmean±SEM (n=10–12 mice/diet/genotype). Different from control at **p<0.01, *p<0.05. Groups with different letter superscripts are statistically different (Tukey–Kramer test, p<0.05).

Fig. 4. Duodenal phenotype of the CDX2-KO mice

Duodenal (A) TRPV6 and (B) CaBPD_9k mRNA in control, VDR^f/f (Ctrl) and CDX2-KO mice fed 0.25% or 0.5% Ca diets. Bars reflect the LSmean±SEM (n=10–12 mice/diet/genotype), expressed as (%) relative to controls on the 0.5% Ca diet. Groups with different letter superscripts are statistically different (Tukey–Kramer test, p<0.05). (C) Ca absorption assessed by oral gavage in mice fed the 0.5% Ca diet (n=12–14 mice per genotype). (D) VDR mRNA. Bars reflect the LSmean±SEM (n=20–24 mice per genotype). Data is expressed as (%) relative to controls. Different from control at ^#p<0.1. (E) Western blot analysis of VDR on pooled duodenal sample from each genotype (Ctrl= control, KO= CDX2-KO, STD= VDR Standard).

Fig. 5. Characterization of intestinal mRNA expression of 1,25(OH)₂D-target genes regulated tight junction function

(A) CLDN2, (B) CLDN12 and (C) ZO-1 mRNA expression in duodenum, and proximal and distal colon. Each data point represents the normalized LSmean±SEM (n=20–24 mice per genotype) for the specific target gene using duodenum mRNA levels of the control mice as reference. Different from control at *p<0.05.