Extra-intestinal calcium handling contributes to normal serum calcium levels when intestinal calcium absorption is suboptimal

The active form of vitamin D, 1,25(OH)₂D, is a crucial regulator of calcium homeostasis, especially through stimulation of intestinal calcium transport. Lack of intestinal vitamin D receptor (VDR) signaling does however not result in hypocalcemia, because the increased 1,25(OH)₂D levels stimulate calcium handling in extra-intestinal tissues. Systemic VDR deficiency, on the other hand, results in hypocalcemia because calcium handling is impaired not only in the intestine, but also in kidney and bone. It remains however unclear whether low intestinal VDR activity, as observed during aging, is sufficient for intestinal calcium transport and for mineral and bone homeostasis. To this end, we generated mice that expressed the Vdr exclusively in the gut, but at reduced levels. We found that ~ 15% of intestinal VDR expression greatly prevented the Vdr null phenotype in young-adult mice, including the severe hypocalcemia. Serum calcium levels were, however, in the low-normal range, which may be due to the suboptimal intestinal calcium absorption, renal calcium loss, insufficient increase in bone resorption and normal calcium incorporation in the bone matrix. In conclusion, our results indicate that low intestinal VDR levels improve intestinal calcium absorption compared to Vdr null mice, but also show that 1,25(OH)₂D-mediated fine-tuning of renal calcium reabsorption and bone mineralization and resorption is required to maintain fully normal serum calcium levels.     

Vitamin D signaling in osteocytes: Effects on bone and mineral homeostasis

The active form of vitamin D [1,25(OH)₂D] is an important regulator of calcium and bone homeostasis, as evidenced by the consequences of 1,25(OH)₂D inactivity in man and mice, which include hypocalcemia, hypophosphatemia, secondary hyperparathyroidism and bone abnormalities. The recent generation of tissue-specific (intestine, osteoblast/osteocyte, chondrocyte) vitamin D receptor (Vdr) null mice has provided mechanistic insight in the cell-specific actions of 1,25(OH)₂D and their contribution to the integrative physiology of VDR signaling that controls bone and mineral metabolism. These studies have demonstrated that even with normal dietary calcium intake, 1,25(OH)₂D is crucial to maintain normal calcium and bone homeostasis and accomplishes this primarily through stimulation of intestinal calcium transport. When, moreover, insufficient calcium is acquired from the diet (severe dietary calcium restriction, lack of intestinal VDR activity), 1,25(OH)₂D levels will increase and will directly act on osteoblasts and osteocytes to enhance bone resorption and to suppress bone matrix mineralization. Although this system is essential to maintain normal calcium levels in blood during a negative calcium balance, the consequences for bone are disastrous and generate an increased fracture risk. These findings evidently demonstrate that preservation of serum calcium levels has priority over skeletal integrity. Since vitamin D supplementation is an essential part of anti-osteoporotic therapy, mechanistic insight in vitamin D actions is required to define the optimal therapeutic regimen, taking into account the amount of dietary calcium supply, in order to maximize the targeted outcome and to avoid side-effects. We will review the current understanding concerning the functions of osteoblastic/osteocytic VDR signaling which not only include the regulation of bone metabolism, but also comprise the control of calcium and phosphate homeostasis via fibroblast growth factor (FGF) 23 secretion and the maintenance of the hematopoeitic stem cell (HSC) niche, with special focus on the experimental data obtained from systemic and osteoblast/osteocyte-specific Vdr null mice.This article is part of a Special Issue entitled "The Osteocyte".     

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

1,25 Dihydroxyvitamin D₃ (1,25(OH)₂D) increases intestinal Ca absorption when dietary Ca intake is low by inducing gene expression through the vitamin D receptor (VDR). 1,25(OH)₂D‐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)₂D and Ca levels. Despite elevated urinary Ca loss (eightfold higher in CDX2‐KO) and reduced colonic target genes TRPV6 (–90%) and CaBPD_9k (–80%) mRNA levels, CDX2‐KO mice had only modestly lower femoral bone density. Interestingly, duodenal TRPV6 and CaBPD_9k 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. © 2015 American Society for Bone and Mineral Research.     

Effect of Vitamin D Receptor Genotypes on Calcium Absorption,Duodenal Vitamin D Receptor Concentration,and Serum 1,25 Dihydroxyvitamin D Levels in Normal Women

It is well established that bone mineral density is under strong genetic control. Recently it was reported that the Bsm I restriction fragment length polymorphism of the vitamin D receptor (VDR) gene could account for up to 75% of the genetic variance in bone mineral density. However, the physiological basis for such an effect has not been established. The VDR gene codes for the vitamin D receptor protein which regulates intestinal calcium absorption. In order to assess the biochemical basis we studied the effect of common allelic variation of the VDR gene on intestinal VDR protein concentration, calcium absorption, and serum 1,25 dihydroxyvitamin D (1,25(OH)₂D). Ninety-two Caucasian women were genotyped for Bsm I and Taq I polymorphism at the VDR gene locus. From these we compared 49 young women aged 25–35 years and 43 elderly women aged 65–83 years, who had all three measurements performed. There were no significant differences in intestinal VDR protein concentration, serum 1,25(OH)₂D, or radioactive calcium absorption among VDR genotype groups. Therefore, the small intestine does not seem to be a target for VDR gene polymorphism. Received: 12 August 1996 / Accepted: 3 January 1997     

Immobilization decreases duodenal calcium absorption through a 1,25-dihydroxyvitamin D-dependent pathway

Immobilization induces significant and progressive bone loss, with an increase in urinary excretion and a decrease in intestinal absorption of calcium. These actions lead to negative calcium balance and the development of disuse osteoporosis. The aims of this study were to evaluate the molecular mechanisms of decreased intestinal calcium absorption and to determine the effect of dietary 1,25-dihydroxyvitamin D [1,25(OH)₂D] and a high-calcium diet on bone loss due to immobilization. The immobilized rat model was developed in the Bollman cage III to induce systemic disuse osteoporosis in the animals. There was a significant decrease in lumbar bone mineral density (BMD) and intestinal calcium absorption in the immobilized group compared with the controls. Serum 25-hydroxyvitamin D concentration did not change, but 1,25(OH)₂D concentration decreased significantly. The mRNA levels of renal 25-hydoxyvitamin D 24-hydroxylase (24OHase) increased, whereas those of renal 25-hydroxyvitamin D 1-alpha hydroxylase (1α-hydroxylase), duodenal transient receptor potential cation channel, subfamily V, member 6 (TRPV6), TRPV5, and calbindin-D9k were all decreased. A high-calcium diet did not prevent the reduction in lumbar BMD or affect the mRNA expression of proteins related to calcium transport. Dietary administration of 1,25(OH)₂D increased the intestinal calcium absorption that had been downregulated by immobilization. TRPV6, TRPV5, and calbindin-D9k mRNA levels were also upregulated, resulting in prevention of the reduction in lumbar BMD. Therefore, it is concluded that dietary 1,25(OH)₂D prevented decreases in intestinal calcium absorption and simultaneously prevented bone loss in immobilized rats. However, it remains unclear that calcium absorption and expression of calcium transport proteins are essential for the regulation of lumbar BMD.     

Compound Heterozygous Mutations in the Vitamin D Receptor in a Patient With Hereditary 1,25‐Dihydroxyvitamin D‐Resistant Rickets With Alopecia

Hereditary vitamin D‐resistant rickets (HVDRR) is a rare recessive genetic disorder caused by mutations in the vitamin D receptor (VDR). In this study, we examined the VDR in a young girl with clinical features of HVDRR including rickets, hypophosphatemia, and elevated serum 1,25(OH)₂D. The girl also had total alopecia. Two mutations were found in the VDR gene: a nonsense mutation (R30X) in the DNA‐binding domain and a unique 3‐bp in‐frame deletion in exon 6 that deleted the codon for lysine at amino acid 246 (ΔK246). The child and her mother were both heterozygous for the 3‐bp deletion, whereas the child and her father were both heterozygous for the R30X mutation. Fibroblasts from the patient were unresponsive to 1,25(OH)₂D₃ as shown by their failure to induce CYP24A1 gene expression, a marker of 1,25(OH)₂D₃ responsiveness. [³H]1,25(OH)₂D₃ binding and immunoblot analysis showed that the patient's cells expressed the VDRΔK246 mutant protein; however, the amount of VDRΔK246 mutant protein was significantly reduced compared with wildtype controls. In transactivation assays, the recreated VDRΔK246 mutant was unresponsive to 1,25(OH)₂D₃. The ΔK246 mutation abolished heterodimerization of the mutant VDR with RXRα and binding to the coactivators DRIP205 and SRC‐1. However, the ΔK246 mutation did not affect the interaction of the mutant VDR with the corepressor Hairless (HR). In summary, we describe a patient with compound heterozygous mutations in the VDR that results in HVDRR with alopecia. The R30X mutation truncates the VDR, whereas the ΔK246 mutation prevents heterodimerization with RXR and disrupts coactivator interactions.     

Interrelation of cortisone and 1,25 dihydroxycholecalciferol on intestinal calcium and phosphate absorption

Summary The interrelation of glucocorticoids and 1,25 dihydroxycholecalciferol (1,25(OH)₂D₃) on intestinal calcium and phosphate absorption was investigated. The active and passive transport of calcium and phosphate was evaluated by thein situ intestinal loop technique. Administration of cortisone resulted in a decrease of the luminal fluid and an increase of the luminal calcium and phosphate concentration. Under active transport conditions, administration of cortisone resulted in a decrease of net calcium absorption through two mechanisms: (1) depressed vitamin D-dependent calcium absorption, (2) increased vitamin D-independent calcium backflux. The enhancement of bidirectional phosphate flux by cortisone was independent of 1,25(OH)₂D₃. An enhancement of water movement by cortisone resulted in an increase of luminal calcium and phosphate concentration which favors the passive diffusion of these ions. Enhanced calcium diffusion by cortisone compensates for the inhibitory effect of cortisone on vitamin D-dependent calcium transport. However, enhanced phosphate diffusion by cortisone is additive to the effect of 1,25(OH)₂D₃.     

Mechanism and function of high vitamin D receptor levels in genetic hypercalciuric stone-forming rats.

The functional status and mechanism of increased VDR in GHS rats were investigated. Basal VDR and calbindins were increased in GHS rats. 1,25(OH)(2)D(3) increased VDR and calbindins in controls but not GHS rats. VDR half-life was prolonged in GHS rats. This study supports the mechanism and functional status of elevated VDR in GHS rats. INTRODUCTION: Genetic hypercalciuric stone-forming (GHS) rats form calcium kidney stones from hypercalciuria arising from increased intestinal calcium absorption and bone resorption and decreased renal calcium reabsorption. Normal serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels and increased vitamin D receptor (VDR) protein suggest that high rates of expression of vitamin D-responsive genes may mediate the hypercalciuria. The mechanism of elevated VDR and state of receptor function are not known. MATERIALS AND METHODS: GHS and non-stone-forming control (NC) male rats (mean, 249 g), fed a normal calcium diet, were injected intraperitoneally with 1,25(OH)2D3 (30 ng/100 g BW) or vehicle 24 h before cycloheximide (6 mg/100 g, IP) and were killed 0-8 h afterward. Duodenal VDR was measured by ELISA and Western blot, and duodenal and kidney calbindins (9 and 28 kDa) were measured by Western blots. RESULTS AND CONCLUSIONS: Duodenal VDR protein by Western blot was increased 2-fold in GHS versus NC rats (633 +/- 62 versus 388 +/- 48 fmol/mg protein, n = 4, p < 0.02), and 1,25(OH)2D3 increased VDR and calbindins (9 and 28 kDa) further in NC but not GHS rats. Duodenal VDR half-life was prolonged in GHS rats (2.59 +/- 0.2 versus 1.81 +/- 0.2 h, p < 0.001). 1,25(OH)2D3 prolonged duodenal VDR half-life in NC rats to that of untreated GHS rats (2.59 +/- 0.2 versus 2.83 +/- 0.3 h, not significant). This study supports the hypothesis that prolongation of VDR half-life increases VDR tissue levels and mediates increased VDR-regulated genes that result in hypercalciuria through actions on vitamin D-regulated calcium transport in intestine, bone, and kidney.     

Growth hormone and intestinal calcium transport in the rat

The everted sac technique was used to determine if growth hormone (GH) can increase intestinal calcium transport Independently of its action on vitamin D metabolism. Rats maintained on a vitamin D deficient diet, low in calcium and phosphate, were divided into 6 groups : 2 groups received no vitamin D metabolites (one treated with bovine GH (bGH)) and 4 groups received either 25 hydroxyvitamin D₃ (250HD₃) or 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃) in the presence or absence of bGH treatment.Calcium transport was reduced to a basal level after vitamin D depletion and was significantly increased by either a single dose (300 p-mole) of 250HD₃ or 1,25(OH)₂D₃. Pretreatment with bGH resulted in an increase in calcium transport under each of the vitamin D conditions studied. Factorial analysis of variance indicated a significant effect of GH treatment in addition to the effect of exogenous 1,25(OH)₂D₃ and also suggested that there is a significant interaction between GH and 1,25(OH)₂D₃ on calcium transport.A single dose of ³H-25OHD₃ was administered intraperitoneally to the animals and the labeled metabolites in serum and intestinal mucosa were analyzed. The data indicated that bGH did not affect the concentration of endogenous ³H-1,25(OH)₂D₃ in serum or intestinal mucosa under the conditions of this study.Since vitamin D depletion minimized the availability of endogenous 250HD₃ and the synthesis of 1,25(OH)₂D₃ was shown not to be affected by bGH, GH stimulation of intestinal calcium transport does not appear to be solely mediated by an alteration of vitamin D metabolism but also by some other mechanism.     

Vitamin D receptor polymorphism and prostate cancer prognosis

BackgroundProstatic epithelial cells synthesize the active form of vitamin D (1,25-dihydroxyvitamin D₃), which participates in regulating prostate growth. Calcitriol, a synthetic form of vitamin D₃, exhibits antiproliferative and prodifferentiation activities in prostate cancer. The function of 1,25-dihydroxyvitamin D₃ is mediated by its binding to vitamin D receptor (VDR). VDR forms a heterodimer, typically with retinoid X receptor, to regulate vitamin D target genes. We evaluated the relationship between VDR polymorphism and clinical characteristics associated with prostate cancer risk and prognosis among Egyptian men.Materials and methodsThis case-control study included 2 groups of patients: group A, a control group of 50 subjects with benign prostate hyperplasia, and group B, 50 subjects newly diagnosed with prostate cancer. All participants performed complete blood count, liver and kidney function tests, prostate specific antigen measurement, histopathological analysis and immunohistochemistry for Dickkopf Homolog 3. Restriction fragment length polymorphism-polymerase chain reaction as performed to detect VDR polymorphism.ResultsPatients with prostate cancer and controls showed a significantly different CA genotype frequency (p = 0.007). Furthermore, prostate-specific antigen levels were significantly different in different genotypes in patients with prostate cancer (p < 0.001). Finally, T stage and the VDR ApaI C/A polymorphism were significantly associated (p < 0.041).ConclusionThe VDR ApaI C/A polymorphism may be a diagnostic and prognostic marker for prostate cancer in Egyptian men.     

Comparison of vitamin D metabolism in early healthy and late osteoporotic postmenopausal women

Summary We studied 20 healthy premenopausal women aged 36.5±4.0 years (mean±1 SD), 123 healthy postmenopausal women aged 50.0±2.4 years, and 103 postmenopausal women aged 65.1±5.6 years with symptomatic osteoporosis (forearm and spinal fracture). Serum levels of vitamin D metabolites [25(OH)D, 24,25(OH)₂D₃, and 1,25(OH)₂D] were compared with (1) bone mass in the forearm (single photon absorptiometry) and in the spine (dual photon absorptiometry); (2) biochemical indices of bone formation (serum alkaline phosphatase, plasma bone Gla protien), and bone resorption (fasting urinary hydroxyproline); and (3) other biochemical estimates of calcium metabolism (serum calcium, serum phosphate, 24-hour urinary calcium, intestinal absorption of calcium). The present study revealed no difference in any of the vitamin D metabolites between the premenopausal women, the healthy postmenopausal women and the osteoporotic women as a group. The concentrations of 1,25(OH)₂D and 25(OH)D were significantly lower in patients with spinal fracture than in those with forearm fracture. In the early postmenopausal women, serum 1,25(OH)₂D was related to forearm bone mass (r=−0.20;P<0.05), intestinal calcium absorption (r=0.18;P<0.05), and 24-hour urinary calcium (r=0.21;P<0.05); serum 25(OH)D was related to spinal bone mass (r=0.23;P<0.01). In the osteoporotic women, serum vitamin D metabolites were not related to bone mass, but 1,25(OH)₂D was related to bone Gla protein (r=0.33;P<0.001), serum phosphate (r=−0.27;P<0.01), and 24-hour urinary calcium (r=0.43;P<0.001). The present study demonstrates that in a population that is apparently not deficient in vitamin D, a disturbance of the vitamin D metabolism is not likely to play a pathogenetic role in early postmenopausal bone loss. Patients with spinal fractures have low levels of vitamin D metabolites, which may aggravate their osteoporosis.     

Vitamin D and calcium receptors: links to hypercalciuria

PURPOSE OF REVIEW: In idiopathic hypercalciuria, patients have increased intestinal Ca absorption and decreased renal Ca reabsorption, with either elevated or normal serum levels of 1,25-dihydroxyvitamin D. As 1,25-dihydroxyvitamin D exerts its biologic effects through interactions with the vitamin D receptor, we examine the actions of this receptor and 1,25-dihydroxyvitamin D in animals with genetic hypercalciuria. RECENT FINDINGS: In genetic hypercalciuric stone-forming rats intestinal calcium transport is increased and renal calcium reabsorption is reduced, yet serum 1,25-dihydroxyvitamin D levels are normal. Elevated intestinal and kidney vitamin D receptors suggest that increased tissue 1,25-dihydroxyvitamin D-vitamin D receptor complexes enhance 1,25-dihydroxyvitamin D actions on intestine and kidney, and vitamin D-dependent over-expression of renal calcium-sensing receptor alone can decrease tubule calcium reabsorption. In TRPV5-knockout mice, ablation of the renal calcium-influx channel decreases tubular calcium reabsorption, and secondary elevations in 1,25-dihydroxyvitamin D increase intestinal calcium transport. SUMMARY: 1,25-Dihydroxyvitamin D or vitamin D receptor may change intestinal and renal epithelial calcium transport simultaneously or calcium-transport changes across renal epithelia may be primary with a vitamin D-mediated secondary increase in intestinal transport. The extent of homology between the animal models and human idiopathic hypercalciuria remains to be determined.     

Regulation of intestinal vitamin D receptor expression in experimental uraemia: effects of parathyroidectomy and administration of PTH

In this study, the effects of PTH on binding of [³H]-1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and on vitamin D receptor (VDR) mRNA concentration were assessed in intestinal mucosa of subtotally nephrectomized rats (Nx) and in intestinal mucosa sham-operated rats with normal kidney function (Intact). Intestinal 1,25(OH)2D3 binding capacity of Intact remained unchanged (I) after parathyroidectomy (PTx), (ii) after administration of PTH for up to 6 days, and (iii) after PTx and subsequent administration of PTH (n=4 experiments). In contrast, PTx of subtotally nephrectomized animals (Nx-PTx) decreased 1,25(OH)2D3 binding capacity from 757±95 fmol/mg protein in Nx to 417±42 in Nx-PTx (P<0.01, n=5). PTH administration had no effect on intestinal 1,25(OH)2D3 binding capacity in Nx. However, PTH administration to Nx-PTx resulted in re-elevation of 1,25(OH)2D3 binding capacity to a level (790±113 fmol/mg protein) which was comparable to Nx. Kd-values remained unaltered under all experimental conditions. The intestinal VDR mRNA concentration (normalized to {beta}-actin mRNA) was decreased, on average, by 23% in Nx-PTx (P<0.05 versus Nx). In further experiments, 1,25(OH)2D3 was administered to Nx-PTx. This resulted in upregulation of 1,25(OH)2D3 binding capacity as compared to vehicle-treated Nx-PTx (562±90 fmol/mg protein versus 249±32, P<0.01). The latter results could indicate that PTH-mediated stimulation of residual renal 1,25(OH)2D3 production was involved in PTH-mediated up-regulation of intestinal 1,25(OH)2D3 binding capacity in Nx-PTx. To rule out this possibility, PTH was administered to totally nephrectomized and parathyroidectomized rats (TNx-PTx). Since PTH caused an approximately 80% increase (P<0.05) in intestinal 1,25(OH)2D3 binding capacity under those experimental conditions a mediator role of 1,25(OH)2D3 could be excluded. Functional significance of decreased intestinal 1,25(OH)2D36 binding capacity in Nx-PTx as compared to Nx was demonstrated by significantly lower 1,25(OH)2D3-mediated stimulation of intestinal 25(OH)2D3-24-hydroxylase activity in Nx-PTx (209±68 pmol/mg protein) than in Nx (385±81, P<0.01). The modulation of intestinal 1,25(OH)2D3 binding capacity was not correlated with changes in calcium, phosphate or 1,25(OH)2D3 serum concentrations under our experimental conditions. Taken together, intact parathyroid gland function was required to maintain adequate intestinal VDR expression in experimental uraemia (but not in normal animals). The mechanism of the modulation of intestinal VDR by PTH remains to be elucidated although an indirect effect of PTH on VDR expression in intestinal mucosa seems most likely.     

Vitamin D Receptor Genotypes and Intestinal Calcium Absorption in Postmenopausal Women

Several studies have shown that bone mass and bone turnover are genetically determined. This genetic component is thought to be mediated in part by polymorphisms at the vitamin D receptor (VDR) locus, even though the underlying molecular mechanisms are still unknown. To evaluate a possible site of differential action of the VDR gene alleles we examined their correlation with intestinal calcium absorption in 120 Caucasian postmenopausal women (aged 61 ± 0.6 years). VDR gene polymorphisms for Apa I, Bsm I, and Taq I restriction endonucleases were assessed by Southern blotting analysis. The most common genotypes observed in our population were AaBbTt (37%), AABBtt (20%), aabbTT (15%), AabbTT (15%), and AABbTt (9%). Although there was some evidence of 13% higher lumbar BMD values in aabbTT genotype with respect to AABBtt genotype, this difference of approximately 0.1 g/cm² did not reach statistical significance, possibly because of the limited number of observations. On the contrary, no relationship was found between genotypes and femoral neck BMD values. Intestinal calcium absorption was significantly lower in BB and tt genotypes than, in bb and TT genotypes, respectively, and in AABBtt genotype than in either aabbTT or AaBbTt genotypes (P= 0.0015 ANOVA). No significant differences in intact PTH, alkaline phosphatase, 25OHD₃, and 1,25(OH)₂D₃ were found among subjects with different VDR genotypes. These results are consistent with a possible role of VDR alleles on intestinal calcium absorption.