NDT Advance Access originally published online on March 15, 2005
Nephrology Dialysis Transplantation 2005 20(5):864-867; doi:10.1093/ndt/gfh587
© The Author [2005]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org
Editorial Comment
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
-hydroxylase; microarray; TRPV5
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Role of vitamin D in the maintenance of Ca2+ balance
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Calcium (Ca
2+) is undoubtedly one of the most tightly regulated
ions in plasma of higher animals. Ca
2+ 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 Ca
2+ is complex
because the gastrointestinal tract, the bones and the kidneys
all affect the Ca
2+ balance. Furthermore, the vitamin D endocrine
system is critical for the proper development and maintenance
of this Ca
2+ 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
,25-dihydroxyvitamin
D
3 [1,25(OH)
2D
3], is synthesized in the proximal tubule by the
renal cytochrome P450 enzyme 25-hydroxyvitamin D
3-1
-hydroxylase
(1
-OHase) [
1]. The importance of this enzyme is underlined by
severe disorders in Ca
2+ homeostasis caused by mutations in
the 1
-OHase gene, including vitamin D-dependent rickets type
I (VDDR-I). 1,25(OH)
2D
3 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)
2D
3 on these target organs are mediated by
both genomic and rapid post-transcriptional mechanisms [
1].
1
,25(OH)
2D
3 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)
2D
3,
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)
2D
3, but its physiological role is not well understood.
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Vitamin D-deficient knockout mice models
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Targeted deletion of genes encoding 1
-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
-OHase gene was
inactivated presented the same clinical phenotype as patients
with PDDR, including undetectable levels of 1,25(OH)
2D
3, rickets
and secondary hyperparathyroidism [
2,
3]. On a normal diet, 1
-OHase
knockout mice have an average life span of 12± 2 weeks
[
3,
6]. Previous studies indicated that daily injections of 1,25(OH)
2D
3 completely rescued these 1
-OHase knockout mice [
7]. Bone histology
and histomorphometry confirmed that the rickets and osteomalacia
were cured by this 1,25(OH)
2D
3 supplementation. Blood biochemistry
analysis revealed that the rescue treatment corrected the hypocalcaemia
and secondary hyperparathyroidism. Interestingly, these 1
-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 Ca
2+, 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)
2D
3 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)
2D
3 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.
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Gene products involved in high dietary Ca rescue of 1-OHase knockout mice
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The kidney has a predominant role in maintaining the Ca
2+ balance
because it determines the final excretion of Ca
2+ in the urine.
Active Ca
2+ reabsorption in the distal convoluted and connecting
tubule comprises a sequence of processes involving apical Ca
2+ entry via transient receptor potential channel V5 (TRPV5), translocation
of Ca
2+ through the cytosol by calbindins and extrusion over
the basolateral membrane by the Na
+/Ca
2+ exchanger (NCX1) and
plasma membrane Ca
2+ ATPase (PMCA1b) [
12] (
Figure 1). Recently,
it was demonstrated that the expression of these renal Ca
2+ transport proteins, with the exception of PMCA1b, is significantly
downregulated in kidneys of 1
-OHase knockout mice, which is
in line with a diminished Ca
2+ reabsorption capacity contributing
to the development of the observed hypocalcaemia [
6]. Intriguingly,
high dietary Ca intake restored the decreased expression of
the Ca
2+ transport proteins independently from 1,25(OH)
2D
3 [
6].
In order to identify gene products in the kidney that are regulated
by high dietary Ca and/or 1,25(OH)
2D
3, cDNA microarray analysis
(15 000 cDNAs) was performed on kidney samples from 1,25(OH)
2D
3-
and high dietary Ca-treated 1
-OHase knockout mice. In this study,
1,25(OH)
2D
3 induced a significant regulation of
1000 genes,
whereas dietary Ca supplementation of the 1
-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
Ca
2+-transporting proteins including the NCX1, calbindin-D
28K and the Ca
2+ sensor calmodulin. Dietary Ca supplementation in
the 1
-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 Ca
2+ reabsorption.
Interestingly, several transcripts, previously not known to
be involved in Ca
2+ 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
Ca
2+ concentration in Ca
2+-transporting kidney cells. Previous
studies already indicated Ca
2+-responsive elements in the promoter
of calbindin-D
28K and calmodulin [
14]. Second, other reports
point to a role for the Ca
2+-sensing receptor in the kidney
that senses the ambient Ca
2+ 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 Ca
2+-sensitive proteins with respect to dietary Ca-mediated
rescue of the disturbed Ca
2+ 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.
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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.
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Is vitamin D indispensable?
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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 Ca
2+ 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 Ca
2+ 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)
2D
3 replacement therapy, since bone growth remained impaired [
8].
Hendy and co-workers demonstrated in 1
-OHase and/or VDR knockout
mice that optimal dietary Ca absorption requires 1,25(OH)
2D
3/VDR,
whereas skeletal mineralization was dependent on adequate ambient
Ca
2+ and did not require the 1,25(OH)
2D
3/VDR system [
19]. Together,
these studies indicate that Ca
2+ 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.
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Acknowledgments
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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.
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