Intestinal resistance to 1,25 dihydroxyvitamin D in mice heterozygous for the vitamin D receptor knockout allele

Abstract

We tested the hypothesis that low vitamin D receptor (VDR) level causes intestinal vitamin D resistance and intestinal calcium (Ca) malabsorption. To do so, we examined vitamin D regulated duodenal Ca absorption and gene expression [transient receptor potential channel, vallinoid subfamily member 6 (TRPV6), 24-hydroxylase, calbindin D(9k) (CaBP) mRNA, and CaBP protein] in wild-type mice and mice with reduced tissue VDR levels [i.e. heterozygotes for the VDR gene knockout (HT)]. Induction of 24-hydroxylase mRNA levels by 1,25 dihydroxyvitamin D(3) [1,25(OH)(2) D(3)] injection was significantly reduced in the duodenum and kidney of HT mice in both time-course and dose-response experiments. TRPV6 and CaBP mRNA levels in duodenum were significantly induced after 1,25(OH)(2) D(3) injection, but there was no difference in response between wild-type and HT mice. Feeding a low-calcium diet for 1 wk increased plasma PTH, renal 1alpha-hydroxylase (CYP27B1) mRNA level, and plasma 1,25(OH)(2) D(3), and this response was greater in HT mice (by 88, 55, and 37% higher, respectively). In contrast, duodenal TRPV6 and CaBP mRNA were not higher in HT mice fed the low-calcium diet. However, the response of duodenal Ca absorption and CaBP protein to increasing 1,25(OH)(2) D(3) levels was blunted by 40% in HT mice. Our data show that low VDR levels lead to resistance of intestinal Ca absorption to 1,25(OH)(2) D(3), and this resistance may be due to a role for the VDR (and VDR level) in the translation of CaBP.

FIG. 1

Effect of genotype on tissue VDR expression. Duodenal VDR mRNA (A) or renal VDR protein levels (B) from WT, VDR KO, and HT mice. For the VDR, mRNA analysis bars represent the mean ± SEM (n = 6 per group) and bars with different letter superscripts are significantly different from one another (Fisher's protected LSD, P < 0.05). For the VDR protein analysis, data are representative and the VDR standard is a recombinant human protein.

FIG. 2

Effect of VDR level on the induction of duodenal (Dd) and renal (Kd) gene expression. Ninety-day-old WT and HT mice were injected with a single dose of 1,25(OH)₂ D₃ (200 ng/100 g BW) or vehicle (control). Animals were killed at indicated time and tissues were harvested for mRNA analysis by real-time PCR. Values for specific targets were normalized to the expression of the constitutively expressed gene GAPDH: A, Dd TRPV6; B, Dd calbindin D_9k; C, Dd CYP24; and D, Kd CYP24 mRNA. Gender was balanced across genotype and diet groups and used as a covariate to correct for the higher efficiency of calcium absorption previously seen in female mice (35). Values are expressed as fold change relative to the 0 h time point. Symbols represent the mean ± SEM (n = 6). *, Value different from 0 h for both groups; #, HT value significantly lower than WT value (Fisher's protected LSD, P < 0.05).

FIG. 3

Effect of VDR level on the hormonal adaptation to variations in dietary calcium intake. Ninety-day-old mice were adapted to diets containing different calcium contents (0.5% Ca, 0.02% Ca, or 2.0% Ca) for a 7-d period before the experiment. Plasma was prepared from blood and analyzed for PTH (A) and 1,25(OH)₂ D₃ (C) using commercial assays. RNA was isolated from kidneys and analyzed for renal (Kd) CYP27B1 mRNA levels by real time RT-PCR and normalized for the expression of GAPDH (B). Gender was balanced across genotype and diet groups and used as a covariate to correct for previously reported gender differences (35). Values are expressed relative to the WT mice fed the 0.5% Ca diet. Bars represent the mean ± SEM (n = 8-11 for PTH and 1,25(OH)₂ D₃, n = 6 for CYP27B1). Within a panel, bars with different letter superscripts are significantly different from one another (Fisher's protected LSD, P < 0.05).

FIG. 4

Effect of VDR level on the relationship between intestinal calcium absorption and plasma 1,25(OH)₂ D₃ levels. Ninety-day-old mice were fed diets containing different levels of calcium (0.5% Ca, 0.02% Ca, or 2.0% Ca) for a 7-d period before the experiment to modulate plasma levels of 1,25(OH)₂ D₃ in the mice (n = 8-11/diet group). Gender was balanced across genotype and diet groups and used as a covariate to correct for the higher efficiency of calcium absorption previously seen in female mice (35). Calcium absorption values and plasma 1,25(OH)₂ D₃ data for individual mice was plotted and regression analysis was conducted. Covariate corrected regression lines are presented by solid (WT) or dashed (HT) lines. WT calcium absorption = 0.31 * 1,25(OH)₂ D₂ - 11.2 (r² = 0.85); HT calcium absorption = 0.19 * 1,25(OH)₂ D₂ - 2.3 (r² = 0.77, WT vs. HT slope, P < 0.01).

FIG. 5

Effect of VDR level on the relationship between calbindin D_9k protein levels and plasma 1,25(OH)₂ D₃ levels. Ninety-day-old mice were fed diets containing different levels of calcium (0.5% Ca, 0.02% Ca, or 2.0% Ca) for a 7-d period before the experiment to modulate plasma levels of 1,25(OH)₂ D₃ in the mice (n = 5 males/diet group). Calbindin D_9k values and plasma 1,25(OH)₂ D₃ data were plotted by individual mouse or, as shown in the figure, by the mean value for each diet and genotype group, and regression analysis was conducted. Regression lines are presented by solid (WT) or dashed (HT) lines. WT calbindin D_9k = 0.046 * 1,25(OH)₂ D₃ + 5.1 (r² = 0.76 for individual data points, r² = 0.98 for means); HT calbindin D_9k = 0.026 * 1,25(OH)₂ D₃ + 5.8 (r² = 0.51 individual/r² = 0.99 means, WT vs. HT slope, P = 0.054).