Is vitamin D indispensable for Ca2+ homeostasis: lessons from knockout mouse models?

Joost G. J. Hoenderop and René J. M. Bindels

Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, The Netherlands

Correspondence and offprint requests to: René J. M. Bindels, 160 Physiology, Radboud University Nijmegen Medical Centre, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands. Email: R.Bindels{at}ncmls.ru.nl

Keywords: calcium diet; calcium reabsorption; 25-hydroxyvitamin D3-1{alpha}-hydroxylase; microarray; TRPV5



   Role of vitamin D in the maintenance of Ca2+ balance
 Top
 Role of vitamin D...
 Vitamin D-deficient knockout...
 Gene products involved in...
 Is vitamin D indispensable?
 References
 
Calcium (Ca2+) is undoubtedly one of the most tightly regulated ions in plasma of higher animals. Ca2+ is involved in the normal functioning of a wide variety of tissues and physiological processes which include bone formation, muscle contraction, blood clotting, nerve transmission and as a second messenger regulating the actions of many hormones. The homeostasis of Ca2+ is complex because the gastrointestinal tract, the bones and the kidneys all affect the Ca2+ balance. Furthermore, the vitamin D endocrine system is critical for the proper development and maintenance of this Ca2+ homeostatic system. Once vitamin D is absorbed from the diet or made in the skin by the action of sunlight, it is metabolized in the liver to 25-hydroxyvitamin D and then the kidney serves as the endocrine gland to produce the biologically active form of vitamin D. This active form of vitamin D, 1{alpha},25-dihydroxyvitamin D3 [1,25(OH)2D3], is synthesized in the proximal tubule by the renal cytochrome P450 enzyme 25-hydroxyvitamin D3-1{alpha}-hydroxylase (1{alpha}-OHase) [1]. The importance of this enzyme is underlined by severe disorders in Ca2+ homeostasis caused by mutations in the 1{alpha}-OHase gene, including vitamin D-dependent rickets type I (VDDR-I). 1,25(OH)2D3 remains the most potent active form of vitamin D known to date. The intestine and kidney are the main target organs for the action of this hormone. The biological effects of 1,25(OH)2D3 on these target organs are mediated by both genomic and rapid post-transcriptional mechanisms [1]. 1{alpha},25(OH)2D3 transcriptionally controls the expression of a particular set of target genes mediated through a nuclear vitamin D receptor (VDR) acting as a ligand-inducible factor. Upon binding 1,25(OH)2D3, the VDR undergoes a conformational change and forms a complex with a retinoid X receptor (RXR). This VDR–RXR complex binds to DNA elements in the promoter regions of target genes described as vitamin D response elements (VDREs). Binding to these VDREs controls the rate of gene transcription. The rapid response presumably utilizes another signal transduction pathway that is probably linked to putative plasma membrane receptors for 1,25(OH)2D3, but its physiological role is not well understood.



   Vitamin D-deficient knockout mice models
 Top
 Role of vitamin D...
 Vitamin D-deficient knockout...
 Gene products involved in...
 Is vitamin D indispensable?
 References
 
Targeted deletion of genes encoding 1{alpha}-OHase [2,3] and of the nuclear VDR [4,5] have provided useful mice models of inherited human diseases such as VDDR-I (also known as pseudovitamin D-deficiency rickets; PDDR) and VDDR-II. Mice in which the 1{alpha}-OHase gene was inactivated presented the same clinical phenotype as patients with PDDR, including undetectable levels of 1,25(OH)2D3, rickets and secondary hyperparathyroidism [2,3]. On a normal diet, 1{alpha}-OHase knockout mice have an average life span of 12± 2 weeks [3,6]. Previous studies indicated that daily injections of 1,25(OH)2D3 completely rescued these 1{alpha}-OHase knockout mice [7]. Bone histology and histomorphometry confirmed that the rickets and osteomalacia were cured by this 1,25(OH)2D3 supplementation. Blood biochemistry analysis revealed that the rescue treatment corrected the hypocalcaemia and secondary hyperparathyroidism. Interestingly, these 1{alpha}-OHase knockout mice were also rescued by a Ca enriched diet (2% w/w) [8]. Dietary Ca normalized the hypocalcaemia, secondary hyperparathyroidism and the biomechanical properties of the bone tissue. Comparable results were obtained in VDR knockout mice from which the bone phenotype could be completely rescued by feeding the animals an enriched Ca2+, phosphorus and lactose diet, suggesting that vitamin D deficiencies can be rescued by dietary Ca in a vitamin D-independent manner [4]. Other studies have however indicated that exogenous Ca may not entirely compensate for 1,25(OH)2D3 deficiency in mice and piglets [9,10]. In humans, beneficial effects of Ca infusions were reported in a child with hereditary resistance to 1,25(OH)2D3 and alopecia [11]. Ca infusions may be an efficient alternative for the management of patients with this condition who are unresponsive to large doses of vitamin D derivatives. However, it is not completely clear whether dietary Ca is effective in humans, and studies are needed in which vitamin D-deficient subjects are treated with Ca enriched diets.



   Gene products involved in high dietary Ca rescue of 1{alpha}-OHase knockout mice
 Top
 Role of vitamin D...
 Vitamin D-deficient knockout...
 Gene products involved in...
 Is vitamin D indispensable?
 References
 
The kidney has a predominant role in maintaining the Ca2+ balance because it determines the final excretion of Ca2+ in the urine. Active Ca2+ reabsorption in the distal convoluted and connecting tubule comprises a sequence of processes involving apical Ca2+ entry via transient receptor potential channel V5 (TRPV5), translocation of Ca2+ through the cytosol by calbindins and extrusion over the basolateral membrane by the Na+/Ca2+ exchanger (NCX1) and plasma membrane Ca2+ ATPase (PMCA1b) [12] (Figure 1). Recently, it was demonstrated that the expression of these renal Ca2+ transport proteins, with the exception of PMCA1b, is significantly downregulated in kidneys of 1{alpha}-OHase knockout mice, which is in line with a diminished Ca2+ reabsorption capacity contributing to the development of the observed hypocalcaemia [6]. Intriguingly, high dietary Ca intake restored the decreased expression of the Ca2+ transport proteins independently from 1,25(OH)2D3 [6]. In order to identify gene products in the kidney that are regulated by high dietary Ca and/or 1,25(OH)2D3, cDNA microarray analysis (15 000 cDNAs) was performed on kidney samples from 1,25(OH)2D3- and high dietary Ca-treated 1{alpha}-OHase knockout mice. In this study, 1,25(OH)2D3 induced a significant regulation of ~1000 genes, whereas dietary Ca supplementation of the 1{alpha}-OHase knockout mice revealed ~2000 controlled genes as indicated in the Venn diagram (Figure 2) [13]. Interestingly, ~600 transcripts were regulated in both situations, suggesting the involvement in the dietary Ca-mediated rescue mechanism of these vitamin D-deficient mice (Figure 2; for overview data sheets, please see: http://www.genomics.med.uu.nl/pub/bb/kidney/). Conspicuous regulated genes encoded ion channels, channel-interacting proteins, kinases and other signalling molecules, and importantly Ca2+-transporting proteins including the NCX1, calbindin-D28K and the Ca2+ sensor calmodulin. Dietary Ca supplementation in the 1{alpha}-OHase knockout mice had a maximum effect on NCX1 expression, suggesting that this basolateral protein is an important extrusion mechanism in the process of transcellular Ca2+ reabsorption. Interestingly, several transcripts, previously not known to be involved in Ca2+ homeostasis, were significantly regulated. An intriguing question is how dietary Ca can regulate gene transcription. First, an increased dietary Ca load might increase the intracellular Ca2+ concentration in Ca2+-transporting kidney cells. Previous studies already indicated Ca2+-responsive elements in the promoter of calbindin-D28K and calmodulin [14]. Second, other reports point to a role for the Ca2+-sensing receptor in the kidney that senses the ambient Ca2+ concentration and transduces signals into the cell at the level of gene transcription (Figure 1) [15,16]. Functional analysis should reveal the regulatory pathways of the Ca2+-sensitive proteins with respect to dietary Ca-mediated rescue of the disturbed Ca2+ balance in vitamin D-deficient animals. The emerging tools of genomics and proteomics are enabling the in-depth study of relationships between diet, genetics and function.



View larger version (103K):
[in this window]
[in a new window]
 
Fig. 1. Cellular model of renal epithelial Ca2+ transport. Active and transcellular Ca2+ transport is carried out as a three-step process. Following entry of Ca2+ through the epithelial Ca2+ channels, TRPV5 and TRPV6, Ca2+ bound to calbindin diffuses to the basolateral membrane. At the basolateral membrane, Ca2+ is extruded via an ATP-dependent Ca2+-ATPase (PMCA1b) and an Na+–Ca2+ exchanger (NCX1). In this way, there is net Ca2+ absorption from the luminal space to the extracellular compartment. Dietary Ca and the active form of vitamin D, 1,25(OH)2D3, stimulate the individual steps of transcellular Ca2+ transport by increasing the expression levels of the luminal Ca2+ channels, calbindins and the extrusion systems. The extracellular Ca2+ concentration is sensed by the calcium-sensing receptor (CaR) that might be involved in the intracellular signalling to regulate Ca2+-responsive genes.

 


View larger version (79K):
[in this window]
[in a new window]
 
Fig. 2. Venn diagram depicting the number of regulated genes in 1{alpha}-OHase mice during dietary treatment. The number of genes regulated when comparing the three conditions 1{alpha}-OHase knockout mice vs 1{alpha}-OHase control mice (left), 1{alpha}-OHase knockout mice vs 1{alpha}-OHase knockout mice supplemented with dietary Ca (middle), and 1{alpha}-OHase control mice vs Ca2+-supplemented 1{alpha}-OHase control mice (right). The number of total regulated genes on the DNA chip is depicted. Genes are shown in the overlapping regions that are regulated in two conditions.

 


   Is vitamin D indispensable?
 Top
 Role of vitamin D...
 Vitamin D-deficient knockout...
 Gene products involved in...
 Is vitamin D indispensable?
 References
 
Several studies supported the notion that vitamin D and Ca supplementation may prevent osteoporotic fractures in people known to be vitamin D deficient [17]. Osteoporosis, a systemic skeletal disease characterized by a low bone mass, is a major public health problem [18]. Nutritional deficiencies have a significant influence on the cause of osteoporosis. Previous studies indicated that a reduced supply of Ca2+ is associated with decreased bone mass and osteoporosis, whereas a chronic and severe vitamin D deficiency leads to osteomalacia, a metabolic bone disease characterized by a decreased mineralization of bone [18]. Results of various clinical trials suggested that Ca2+ supplementation may prevent vertebral fractures in the elderly. As outlined above, Ca supplementation in vitamin D-deficient mice models normalized the hypocalcaemia and restored the biomechanical properties of bone [3,4,8]. This treatment, however, does not appear as effective as 1,25(OH)2D3 replacement therapy, since bone growth remained impaired [8]. Hendy and co-workers demonstrated in 1{alpha}-OHase and/or VDR knockout mice that optimal dietary Ca absorption requires 1,25(OH)2D3/VDR, whereas skeletal mineralization was dependent on adequate ambient Ca2+ and did not require the 1,25(OH)2D3/VDR system [19]. Together, these studies indicate that Ca2+ cannot entirely substitute vitamin D in mineral and skeletal homeostasis, but the two agents have discrete and complementary functions.

In various clinical conditions associated with a disturbed Ca homeostasis, vitamin D analogues are administered. For instance, the treatment of choice for PDDR and for patients with chronic renal failure is long-term replacement therapy with 1,25(OH)2D3. Notably, the currently applied strategy of vitamin D and Ca2+ supplementation to patients with chronic renal failure has been associated with adverse effects, such as vascular calcification and calciphylaxis. It would be interesting to compare the effectiveness of Ca supplementation with the treatment with vitamin D analogues in these patient groups. Based on present evidence, chelated Ca2+ may be safely and effectively ingested by most people at doses generally recommended for treatment or prevention of Ca2+-related disorders [20]. Further studies on potential dietary Ca-sensitive targets will provide insight into the molecular rescue mechanisms of dietary Ca supplementation.



   Acknowledgments
 
This work was supported by grants from the Dutch Organization of Scientific Research (Zon-Mw 016.006.001) and the Dutch Kidney Foundation (C03.6017).

Conflict of interest statement. None declared.



   References
 Top
 Role of vitamin D...
 Vitamin D-deficient knockout...
 Gene products involved in...
 Is vitamin D indispensable?
 References
 

  1. Jones G, Strugnell SA, DeLuca HF. Current understanding of the molecular actions of vitamin D. Physiol Rev 1998; 78: 1193–1231[Abstract/Free Full Text]
  2. Panda DK, Miao D, Tremblay ML et al. Targeted ablation of the 25-hydroxyvitamin D 1alpha-hydroxylase enzyme: evidence for skeletal, reproductive, and immune dysfunction. Proc Natl Acad Sci USA 2001; 98: 7498–7503[Abstract/Free Full Text]
  3. Dardenne O, Prud'homme J, Arabian A, Glorieux FH, St-Arnaud R. Targeted inactivation of the 25-hydroxyvitamin D3-1(alpha)-hydroxylase gene (CYP27B1) creates an animal model of pseudovitamin D-deficiency rickets. Endocrinology 2001; 142: 3135–3141[Abstract/Free Full Text]
  4. Van Cromphaut SJ, Dewerchin M, Hoenderop JG et al. Duodenal calcium absorption in vitamin D receptor-knockout mice: functional and molecular aspects. Proc Natl Acad Sci USA 2001; 98: 13324–13329[Abstract/Free Full Text]
  5. Takeyama K, Kitanaka S, Sato T, Kobori M, Yanagisawa J, Kato S. 25-Hydroxyvitamin D3 1alpha-hydroxylase and vitamin D synthesis. Science 1997; 277: 1827–1830[Abstract/Free Full Text]
  6. Hoenderop JG, Dardenne O, Van Abel M et al. Modulation of renal Ca2+ transport protein genes by dietary Ca2+ and 1,25-dihydroxyvitamin D3 in 25-hydroxyvitamin D3-1alpha-hydroxylase knockout mice. FASEB J 2002; 16: 1398–1406[Abstract/Free Full Text]
  7. Dardenne O, Prudhomme J, Hacking SA, Glorieux FH, St-Arnaud R. Rescue of the pseudo-vitamin D deficiency rickets phenotype of CYP27B1-deficient mice by treatment with 1,25-dihydroxyvitamin D3: biochemical, histomorphometric, and biomechanical analyses. J Bone Miner Res 2003; 18: 637–643[ISI][Medline]
  8. Dardenne O, Prud'homme J, Hacking SA, Glorieux FH, St-Arnaud R. Correction of the abnormal mineral ion homeostasis with a high-calcium, high-phosphorus, high-lactose diet rescues the PDDR phenotype of mice deficient for the 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1). Bone 2003; 32: 332–340[CrossRef][ISI][Medline]
  9. Goltzman D, Miao D, Panda DK, Hendy GN. Effects of calcium and of the vitamin D system on skeletal and calcium homeostasis: lessons from genetic models. J Steroid Biochem Mol Biol 2004; 89–90: 485–489
  10. Schlumbohm C, Harmeyer J. Dietary additions of lactose, casein and soy protein exerted only moderate effects on calcium homeostasis in calcitriol deficient piglets. J Steroid Biochem Mol Biol 2004; 89–90: 605–609[CrossRef]
  11. Balsan S, Garabedian M, Larchet M et al. Long-term nocturnal calcium infusions can cure rickets and promote normal mineralization in hereditary resistance to 1,25-dihydroxyvitamin D. J Clin Invest 1986; 77: 1661–1667[ISI][Medline]
  12. Hoenderop JG, Nilius B, Bindels RJ. Calcium absorption across epithelia. Physiol Rev 2005; 85: 373–422[Abstract/Free Full Text]
  13. Hoenderop JG, Chon H, Gkika D et al. Regulation of gene expression by dietary Ca2+ in kidneys of 25-hydroxyvitamin D3-1 alpha-hydroxylase knockout mice. Kidney Int 2004; 65: 531–539[CrossRef][ISI][Medline]
  14. Arnold DB, Heintz N. A calcium responsive element that regulates expression of two calcium binding proteins in Purkinje cells. Proc Natl Acad Sci USA 1997; 94: 8842–8847[Abstract/Free Full Text]
  15. Brown EM, MacLeod RJ. Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev 2001; 81: 239–297[Abstract/Free Full Text]
  16. Drueke TB. Modulation and action of the calcium-sensing receptor. Nephrol Dial Transplant 2004; 19 [Suppl 5]: V20–V26[Medline]
  17. Larsen ER, Mosekilde L, Foldspang A. Vitamin D and calcium supplementation prevents osteoporotic fractures in elderly community dwelling residents: a pragmatic population-based 3-year intervention study. J Bone Miner Res 2004; 19: 370–378[ISI][Medline]
  18. Gennari C. Calcium and vitamin D nutrition and bone disease of the elderly. Public Health Nutr 2001; 4: 547–559[Medline]
  19. Panda DK, Miao D, Bolivar I et al. Inactivation of the 25-hydroxyvitamin D 1alpha-hydroxylase and vitamin D receptor demonstrates independent and interdependent effects of calcium and vitamin D on skeletal and mineral homeostasis. J Biol Chem 2004; 279: 16754–16766[Abstract/Free Full Text]
  20. Whiting SJ, Wood R, Kim K. Calcium supplementation. J Am Acad Nurse Pract 1997; 9: 187–192[Medline]




This Article
Extract
FREE Full Text (PDF)
All Versions of this Article:
20/5/864    most recent
gfh587v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Hoenderop, J. G. J.
Articles by Bindels, R. J. M.
PubMed
PubMed Citation
Articles by Hoenderop, J. G. J.
Articles by Bindels, R. J. M.