Am J Physiol Gastrointest Liver Physiol 300: G191–G201, 2011.
First published November 18, 2010; doi:10.1152/ajpgi.00496.2010.
Review
Advances in the understanding of mineral and bone metabolism in
inflammatory bowel diseases
Fayez K. Ghishan1 and Pawel R. Kiela1,2
1
Department of Pediatrics, Steele Children’s Research Center, and 2Department of Immunobiology, University of Arizona
Health Sciences Center, Tucson, Arizona
Submitted 4 November 2010; accepted in final form 11 November 2010
bone mineral density; Crohn’s disease; osteopenia; osteoporosis; ulcerative colitis
(IBDs) represent a group of
chronic inflammatory disorders of the intestinal tract that to
this date remains idiopathic. More recent basic and clinical
research indicates that the chronic inflammatory reaction of the
intestinal mucosa is directed against the gut microbiota in
individuals with genetic and/or environmental susceptibilities.
Disease onset occurs typically during young adulthood (25–35
yr), although ⬃20 –25% of cases are diagnosed during childhood (93). On a clinical basis, two major subtypes of IBD are
defined as Crohn’s disease (CD), which potentially affects any
part of the gastrointestinal tract from the mouth to the anus, and
ulcerative colitis (UC), an inflammatory condition limited to
the colonic mucosa. However, IBD does not affect just a single
organ and should be considered a systemic disease with several
extraintestinal manifestations, which occur in a large proportion of IBD patients (60). Osteopenia and osteoporosis are two
of the more common extraintestinal symptoms with a general
consensus that IBD patients are at a significantly higher risk of
developing metabolic bone disease and low bone mineral
INFLAMMATORY BOWEL DISEASES
Address for reprint requests and other correspondence: P. R. Kiela, Dept. of
Pediatrics, Steele Children’s Research Center, Univ. of Arizona Health Sciences Center; 1501 N. Campbell Ave., Tucson, AZ 85724 (e-mail:
[email protected]).
http://www.ajpgi.org
density (BMD) than the healthy subjects. The relative risk of
fracture in IBD patients has been estimated to be 40% higher
than in general population (6), with the prevalence of osteopenia and osteoporosis varying significantly depending on the
study populations, geographic location, and study design, with
the reported range of osteopenia at 22–77% and osteoporosis at
17– 41% (6). Among the many risk factors predisposing for the
loss of BMD in general population, several are more pertinent
in IBD patient population. Some of them are listed in Table 1.
Although the data regarding patients with early (pediatric)
onset IBD are limited, a recent study clearly demonstrated low
BMD associated with IBD in children (especially in CD) (98).
Pediatric IBD patients form a separate group not only because
of distinct disease characteristics and perhaps distinct genetic
predispositions (93), but also because of differences in their
bone metabolism. During the prepubescent and early pubescent
period, bone modeling is the predominant form of skeletal
growth, and the activities of osteoblasts and osteoclasts are not
coupled. Therefore, the principles of bone loss observed in
adult patients with chronic inflammation or during postmenopausal osteopenia/osteoporosis are not directly applicable (97).
Although the risk of fracture in pediatric patients with IBD is
not well established and not without controversy (44), a more
important question is whether chronic, even subclinical inflam-
0193-1857/11 Copyright © 2011 the American Physiological Society
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Ghishan FK, Kiela PR. Advances in the understanding of mineral and bone
metabolism in inflammatory bowel diseases. Am J Physiol Gastrointest Liver
Physiol 300: G191–G201, 2011. First published November 18, 2010;
doi:10.1152/ajpgi.00496.2010.—Chronic inflammatory disorders such as inflammatory bowel diseases (IBDs) affect bone metabolism and are frequently associated
with the presence of osteopenia, osteoporosis, and increased risk of fractures.
Although several mechanisms may contribute to skeletal abnormalities in IBD
patients, inflammation and inflammatory mediators such as TNF, IL-1␤, and IL-6
may be the most critical. It is not clear whether the changes in bone metabolism
leading to decreased mineral density are the result of decreased bone formation,
increased bone resorption, or both, with varying results reported in experimental
models of IBD and in pediatric and adult IBD patients. New data, including our
own, challenge the conventional views, and contributes to the unraveling of an
increasingly complex network of interactions leading to the inflammationassociated bone loss. Since nutritional interventions (dietary calcium and vitamin D
supplementation) are of limited efficacy in IBD patients, understanding the pathophysiology of osteopenia and osteoporosis in Crohn’s disease and ulcerative colitis
is critical for the correct choice of available treatments or the development of new
targeted therapies. In this review, we discuss current concepts explaining the effects
of inflammation, inflammatory mediators and their signaling effectors on calcium
and phosphate homeostasis, osteoblast and osteoclast function, and the potential
limitations of vitamin D used as an immunomodulator and anabolic hormone in
IBD.
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Table 1. Risk factors for osteopenia/osteoporosis in
inflammatory bowel disease
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Malnutrition
Malabsorption of vitamin D, calcium, and vitamin K
Low body mass index
Low bone mineral intensity peak in IBD patients with pediatric onset
Chronic inflammatory state
Type of IBD (CD vs. UC; small intestinal involvement)
Increasing age
Female gender
Immobilization
Use of corticosteroids
Previous fragility fracture
Hypogonadism
Smoking
Family history of osteoporosis
mation predisposes the child to increased risk of future fractures later in life, as a result of low bone accrual and lower
peak BMD reached during early adulthood. Although it has
been long known that poor bone health in childhood often leads
into osteopenia/osteoporosis and increased risk of fracture in
adulthood (50), longitudinal studies with pediatric IBD patients
are yet to be performed.
Prevalence, risk factors, clinical evaluation, and prevention
and treatment of bone loss associated with IBD were reviewed
very well by Lichtenstein et al. (62) and Mascarenhas and
Thayu (68). It has to be acknowledged, however, that the
majority of knowledge on pathogenesis and pharmacological
interventions into bone metabolism in IBD is not tailored
specifically to unique aspects of this group of diseases. The
mechanisms and contribution of altered osteoblast and/or os-
Fig. 1. Factors and mechanisms associated with bone mass loss and increased risk of fractures in patients with inflammatory bowel diseases. IBD, inflammatory
bowel disease; RANKL, receptor activator of NF-␬B ligand; OPG, osteoprotegerin.
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IBD, inflammatory bowel disease; CD, Crohn’s disease; UC, ulcerative
colitis.
teoclast function in IBD have not been clearly defined and
many of the proposed mechanisms and the proposed clinical
approaches are not the result of direct experimentation, but
rather of an extrapolation of the growing field of osteoimmunology and rheumatoid arthritis (73) to the known cellular and
soluble inflammatory mediators involved in the pathogenesis
of IBD. The goal of this review is to focus on recent preclinical
studies with animal models of IBD as well as findings derived
directly from IBD patients. Studies addressing specific issues
related to mineral metabolism and bone health in IBD will
likely point to mechanisms common with other disorders as
well as unique to IBD (perhaps unique to their different forms
and time of onset) that may be explored in tailoring therapies
aimed at bone loss prevention.
The pathogenesis of osteopenia and osteoporosis in IBD has
been suggested to result from altered rates of bone formation
and resorption secondary to multifactorial but still poorly
characterized set of mechanisms. These are typically sorted
into two major groups: poor nutritional status and malabsorption (particularly affecting vitamin D, vitamin K, and Ca2⫹
homeostasis), and inflammation with IBD-associated inflammatory infiltrate and soluble mediators (IL-1, IL-6, TNF)
affecting bone more directly. More recent studies in animal
models of IBD indicate that malabsorption and inflammation
are not two independent contributors to IBD-associated BMD
loss and that inflammation and inflammatory cytokines actively
contribute to the abnormalities in the intestinal and renal
mineral and vitamin absorption. In this review, we will outline
current knowledge about the effects of intestinal inflammation
on epithelial mineral handling, vitamin D and K absorption and
metabolism, and the role of inflammation in bone modeling
and remodeling. The described relationships are schematically
illustrated in Fig. 1.
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SUPPLY OF CA2ⴙ AND PO2ⴚ
4 : THE BUILDING BLOCKS OF
THE INORGANIC COMPONENT OF THE BONE MINERAL
MATRIX
Intestinal and Renal Ca2⫹ (Re)absorption
Intestinal malabsorption has been intuitively linked to the
pathogenesis of bone loss in IBD and celiac disease patients.
Several factors likely contribute to this aspect of diminished
Ca2⫹ supply. Avoidance of milk and dairy food consumption
and relatively common lactose intolerance may prevent the
achievement of an adequate peak bone mass and may predispose to osteoporosis (31). In the intestinal tract, calcium is
primarily absorbed in the duodenum and proximal jejunum.
Depending on the segment and the luminal Ca2⫹ concentration, it is absorbed through a weakly regulated paracellular
route, or by highly regulated and vitamin D3-sensitive transcellular pathway. When dietary Ca2⫹ intake and luminal concentration is low (⬍20 mM), active transcellular transport in
the duodenum predominates and accounts for ⬃80% of the
total Ca2⫹ absorption. At higher Ca2⫹ supply (ⱖ50 mM), the
contribution of active transport diminishes to below 10%,
largely because of the short duodenal transit time and because
of downregulation of the key molecular components of the
transcellular Ca2⫹ transport proteins.
Apical Ca2⫹ entry. Calcium enters the epithelial cells via
selective non-voltage-gated Ca2⫹ channels at the luminal
membrane under the influence of a steep, inwardly directed
electrochemical gradient. This facilitated diffusion is mediated
by two major members of the TRPV subfamily (transient
receptor potential vanilloid channels), TRPV5 (predominant in
the kidney) and TRPV6 (predominant in the duodenum). Both
channels are extensively regulated by transcriptional mechanisms [calciotropic hormones including 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), parathyroid hormone, and estrogens],
by pH- and Ca2⫹-dependent regulatory mechanisms, by trafficking of TRPV5 and TRPV6 to the luminal membrane, and
by physical interactions with associated proteins. Several comprehensive reviews on this subject have been recently published (30, 51). Results from knockout mice highlight the
importance of these two transport proteins in Ca2⫹ homeostasis. TRPV5⫺/⫺ mice show robust renal Ca2⫹ wasting, with
active Ca2⫹ reabsorption in distal convoluted tubules and
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The inorganic component makes up 60% of the dry weight
of bone and is composed primarily of calcium hydroxyapatite,
Ca10(PO4)6(OH)2. Calcium hydroxyapatite crystals are arranged parallel to collagen fibers, which maximizes the bone’s
resistance to both tensile (stretch) and compressive forces.
Bone is highly dynamic and it undergoes constant remodeling
throughout life. The remodeling involves coupled resorption of
existing bone and the formation of new bone. The supply of
calcium and inorganic phosphate available for bone formation
is a result of highly regulated and complex homeostatic mechanism involving paracellular and transcellular intestinal and
renal (re)absorption. In this review, we provide only a brief
overview of those physiological mechanisms and focus on their
regulation in inflammatory states as pertinent to bone loss in
IBD patients. For more detailed description of intestinal and
renal calcium and phosphate transport mechanism, the reader is
referred to recent literature reviews and references therein (20,
30, 51, 66, 81).
connecting tubules effectively abolished, and with a reduced
bone thickness despite the compensatory intestinal Ca2⫹ hyperabsorption accompanied by significantly enhanced duodenal TRPV6 expression (82). TRPV6⫺/⫺ mice display a consistent decrease in Ca2⫹ absorption over time, increased urinary excretion, and decreased BMD (15).
Transcellular Ca2⫹ movement. Free ionized cytoplasmic
Ca2⫹ concentration has to be tightly controlled because of its
cytotoxic effects and because of its inhibitory effects on
TRPV5 and TRPV6-mediated Ca2⫹ currents. Some of these
effects are mediated by associated proteins like calmodulin and
protein kinase C substrate 80K-H. There is a large body of
literature on the role of calbindins D9K and D28K acting both
as Ca2⫹ buffers and ferries facilitating transcytoplasmic movement of Ca2⫹ to the basolateral site. Despite extensive research, the role of the two calbindins in both functions remains
controversial. Calbindin D9K knockout mice have normal
plasma Ca2⫹, the same increase in the efficiency of Ca2⫹
absorption when fed low Ca2⫹ diet, and display no difference
in 1,25(OH)2D3-induced transcellular Ca2⫹ transport (4, 58).
The phenotype of calbindin D28K knockout mice is also
negligible. These mice are not different from the wild-type
littermates (growth, life span, fertility), have normal plasma
Ca2⫹ and Pi levels, and display only mild impairment in motor
coordination/motor learning (abnormal Ca2⫹ oscillations in
Purkinje cells of the cerebellum) (2). Moreover, D28K knockout mice had a measurable increase in bone volume and
stiffness, indicating that calbindin-D28K plays an important
role in bone remodeling (65).
An alternative vesicular transport model has also been proposed that may partially explain the lack of a significant
phenotype in calbindin-deficient mice. According to this
model, apical formation of Ca2⫹-loaded vesicles is initiated
upon TRPV-mediated Ca2⫹ uptake. These vesicles can then be
transported vectorially by microtubules, or they can fuse with
endoplasmic reticulum (ER) followed by passive diffusion
with the ER. Ca2⫹ is then released from the ER at the
basolateral membrane through calcium release channels and
extruded by NCX1 and PMCA1b (see Basolateral Ca2⫹ exit).
Alternatively, these vesicles may fuse with the lysosomes,
which move laterally to eventually coalesce with the lateral
membrane and release the content through exocytosis. This
route is also stimulated by 1,25(OH)2D3. Net epithelial Ca2⫹ is
inhibited by chloroquine, suggesting that this may be the main
route of transcellular Ca2⫹ trafficking (51).
Basolateral Ca2⫹ exit. Two transport proteins have been
implicated in the cellular exit of Ca2⫹. One of them is a
bidirectional Na⫹/Ca2⫹ exchanger, NCX1, which under physiological electrochemical gradients is responsible for basolateral Ca2⫹ extrusion. It is widely expressed in the absorptive
epithelia, although it is considered to be of primary importance
in the renal distal convoluted tubules (16) and of minor
significance in the intestinal epithelium (45). The physiological
importance of this exchanger is highlighted by the embryonic
lethality of NCX1-null knockout mice (55). In heterozygous
mice, expression of NCX1 protein in the tubular epithelial cells
and Ca2⫹ influx via NCX1 in renal tubules are markedly
attenuated. The second transporter involved in basolateral
Ca2⫹ extrusion is a P-type ATPase, a high-affinity Ca2⫹ efflux
pump PMCA1b, abundantly expressed in the intestine (45).
Calcium is expelled through a channel-like opening formed by
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the transmembrane elements, and phosphorylation is believed
to bring about the necessary conformational change such that
Ca2⫹ bound to the enzyme is propelled through the opening.
Similarly to NCX1, PMCA1b-null knockout is embryonically
lethal (75).
Ca2⫹ Transport in IBD
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Intestinal and Renal Pi (Re)absorption
Phosphate is one the most abundant dietary constituents and
one of the most abundant minerals in the organism. Complex
homeostatic mechanism evolved in mammals to maintain the
extracellular and intracellular Pi levels within a narrow range
necessary for many biological processes, including skeletal
development and BMD. Prolonged phosphate deficiency and
hypophosphatemia resulting from malnutrition or malabsorption may, among other complications, lead to bone demineralization, leading to skeletal defects such as rickets in children
and osteomalacia in adults. Phosphate homeostasis is maintained through a concerted action of sodium-dependent phosphate transporters forming the SLC34 solute carrier family.
Most of the active renal Pi reabsorption occurs in the proximal
convoluted tubules via NaPi-IIa (SLC34A1) and NaPi-IIc
(SLC34A3), whereas NaPi-IIb (SLC34A2) is the predominant
intestinal isoform. More recent data also implicate members of
the SLC20 family, or type III transporters, specifically PiT1
and PiT2, initially identified as retroviral receptors, in the
intestinal and renal Pi transport (26). Contrary to initial findings, they are now believed to be expressed at the intestinal
(PiT1) (40) and renal brush border membrane (BBM) (PiT2)
(106).
Very little is known about the effects of intestinal inflammation on intestinal or renal Pi (re)absorption. Similar to Ca2⫹,
serum level of Pi is not a good indicator of modest changes in
active mineral transport, unless more severe malabsorption and
cachexia are present. Constant serum level of Ca2⫹ and Pi are
the primary determinants of the homeostatic mechanisms controlling both minerals, with bone being the disposable reservoir
for the systemic demand for these two minerals. It is therefore
not surprising that little to no changes in serum Ca2⫹ or Pi are
observed in IBD patients. Urinary excretion is rarely reported
and mostly limited to patients at risk for or diagnosed with uroor nephrolithiasis. In a recent study, our group has demonstrated that trinitrobenzene sulfonic acid (TNBS)-induced colitis resulted in significant inhibition of NaPi-IIb-mediated
intestinal Pi absorption in isolated BBM vesicles (23). This was
accompanied by an ⬃50% decrease in NaPi-IIb mRNA and
protein expression. This inhibition could be recapitulated in
TNF-treated Caco-2 cells, with the transcriptional mechanism
potentially involving epidermal growth factor receptor, and
ERK1/2-dependent decrease in NF1 transcription factor interaction with the basal region of the human NaPi-IIb gene
promoter (23). The extent to which this inhibition contributes
to the limited supply of Pi for the skeletal system in IBD
patients remains unknown. One report questioned the significance of Na⫹-dependent Pi absorption in the gut (108), an
observation seemingly in agreement with a lack of significant
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Surprisingly little is known on the effects of acute and
chronic inflammation on intestinal and renal calcium handling.
In recent comprehensive reviews of the mechanism and regulation of epithelial Ca2⫹ absorption in health and disease, the
role of inflammation and inflammatory mediators is not even
mentioned (71, 96). On the other hand, malnutrition and
negative systemic Ca2⫹ balance in conjunction with vitamin D
insufficiency are widely considered as important factors in the
pathogenesis of bone loss in IBD patients. However, in a
randomized, placebo-controlled trial in glucocorticoid-treated
patients with IBD, the intake of 250 IU of vitamin D and 1,000
mg/day calcium had no significant benefit in bone density at 1
year of follow-up (14). Similarly, a study with pediatric IBD
patients supplemented with calcium with or without vitamin D
failed to demonstrate any effect on BMD accrual (12). It
appears, therefore, that the supplementation with calcium and
vitamin D, although accepted as a cost-effective medication,
may be insufficient in the prevention and treatment of IBDassociated osteoporosis. It is not inconceivable that changes in
expression and activity of key Ca2⫹ transport proteins in the
gut and kidney, induced by associated inflammatory mediators,
are the main reason for failure of dietary Ca2⫹ and vitamin D
supplementation in IBD. Indeed, a very recent report (47) from
the group of Joost Hoenderop in the Netherlands demonstrated
for the first time that in mouse model of Crohn’s-like ileitis
(TNF-␣-overexpressing TNF⌬ARE mice), both duodenal and
renal Ca2⫹ absorptive epithelia displayed significant downregulation of TRPV6, calbindin D9K, PMCA1b, as well as
calbindin D28K and NCX1, respectively (47). In this report,
changes in expression of these Ca2⫹ transport genes were
accompanied with increased bone resorption, as documented
by tomography scanning (reduced trabecular and cortical bone
thickness and volume) and increased total deoxypyridinoline in
serum of the TNF⌬ARE mice.
Contrary to the initially widespread notion, loss of bone
density in IBD patients poorly correlates with cumulative
steroid exposure and is frequently observed in newly diagnosed
steroid-naive patients. However, corticosteroids undoubtedly
contribute to bone loss in chronically ill IBD patients by
directly influencing the balance of bone formation and resorption. Additional likely mechanism pertinent to intestinal Ca2⫹
absorption was described by Huybers et al. (48), who demonstrated that in mice orally administered with methylprednisolone, duodenal absorption of Ca2⫹ was significantly decreased. This decrease coincided with inhibited expression of
TRPV6 and calbindin D9K mRNA and protein, with unaltered
serum Ca2⫹ or 1,25(OH)2D3 levels (48).
These murine observations are yet to be translated to the
clinical settings. Kumari et al. (56) recently demonstrated in a
small pilot study with four CD patients in remission that
fractional intestinal Ca2⫹ absorption at baseline is similar to
that of control subjects. Moreover, both groups of patients
responded similarly to oral 1,25(OH)2D3 (0.25 or 0.5 ␮g twice
daily) given for 7 days (56). Keeping the small sample size in
mind, these findings suggest that in patients with mild CD or in
patients in remission intestinal Ca2⫹ is not impaired and that
there may not be a need for high-dose calcitriol supplementation. Similar well-designed studies with higher numbers of
patients with both active and quiescent IBD are needed to
verify these preliminary findings. It is also plausible that
supraphysiological vitamin D3 supplementation may be detrimental for the overall bone health in active inflammation (see
discussion in Vitamin D Status in IBD below).
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Role of Klotho in Epithelial Ca2⫹ and Pi Transport
In 1997, Kuro-o et al. (57) described transgenic mice obtained by accidental mutational insertion, which exhibited
symptoms typical of human aging: short lifespan, infertility,
arteriosclerosis, skin atrophy, severe osteoporosis, and emphysema. A then-unknown gene was disrupted or mutated in its
5=-flanking promoter region by the random insertion of an
exogenously introduced nonfunctional gene, the rabbit Na/H
exchanger under the control of the human elongation factor
promoter. The coding region of this mutated gene termed
Klotho was still preserved, but its expression was markedly
reduced, generating a mouse strain with a strong hypomorphic
allele. Klotho gene encodes a 130-kDa single pass transmembrane protein with ␤-glucuronidase activity and is primarily
expressed in the epithelial cells of renal distal tubules, in the
choroid plexus, and in the parathyroid gland.
Involvement of the Klotho gene in bone homeostasis was
suggested by recent genetic studies showing that certain allelic
variants of Klotho constitute one of the genetic factors influencing BMD in male adults (110) and in postmenopausal
women (83). All this evidence implies that Klotho is a multifunctional protein that regulates phosphate/calcium metabolism as well as aging. Klotho has also been directly implicated
in the regulation of transepithelial Ca2⫹ transport through
regulation of both apical entry as well as basolateral Ca2⫹ exit.
The existing homeostatic mechanisms provide for a fast and
active Ca2⫹ reabsorption when the extracellular Ca2⫹ concentration in the circulation decreases. A key component to create
such elevated Ca2⫹ influx is to increase TRPV5 and TRPV6
channels’ abundance at the epithelial cell surface. Shuttling of
TRPV5 to and from a subapical pool is a key component of this
regulation, and high Ca2⫹ influx may be obtained by prolonging the channel’s durability at the cell surface before inactivation or internalization. Klotho, through its glucuronidase activity, modulates N-glycosylation of TRPV5 at Asn358 by
removal of the capping sialic acid, which enables physical
interaction of galectin 1 with TRPV5 on the cell surface and
promotes its apical retention (89). Similar activating effects of
Klotho on TRPV6-mediated Ca2⫹ flux have also been shown
(63), although intestinal Ca2⫹ absorption has not been shown
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to date to be controlled by Klotho. Moreover, under decreased
extracellular Ca2⫹ concentration, intracellular Klotho physically interacts with endosomal pool of Na⫹/K⫹ ATPase and
enables active recruitment of the sodium pump to the basolateral
membrane. This, in turn, promotes the maintenance of the electrochemical Na⫹ gradient indispensable for the Na⫹/Ca2⫹ exchanger NCX1 to operate as a Ca2⫹ extrusion mechanism to
complete the transepithelial calcium transport.
In our laboratory’s recent study (101), we described that
renal Klotho is significantly downregulated in experimental
colitis. This was consistently observed in three distinct models
of murine IBD: TNBS colitis, SPF-associated germ-free IL10⫺/⫺ mice, and CD4⫹CD45RBHi T-cell transfer colitis. In
vitro, Klotho mRNA and protein expression was greatly and
synergistically reduced in immortalized distal convoluted tubule cells by TNF and IFN-␥. The latter cytokine contributed
to the reduction of Klotho by induction of NOS2 expression
and the production of NOx, which by itself significantly affected Klotho expression. The effects of both cytokines were
transcriptionally mediated as determined by unaltered transcript stability and decreased gene promoter activity (101).
Consistent with the role of Klotho in preventing tubular Ca2⫹
loss (5), colitis mice exhibit increased urinary fractional Ca2⫹
excretion accompanied with dramatically decreased TRPV5
protein, but not mRNA expression (P. R. Kiela and F. K.
Ghishan; unpublished observations). This is consistent with
earlier observations by Fries et al. (36), who observed increased urinary calcium excretion in rats with TNBS colitis
associated with decreased bone density.
The relationship between Klotho and renal Pi reabsorption is
also quite complex. Klotho functions as a coreceptor for
fibroblast growth factor (FGF) 23, a phosphaturic hormone
produced primarily by osteoblasts under the control of
1,25(OH)2D3 (54). FGF23 inhibits renal phosphate reabsorption by activating FGF receptor (FGFR) 1c in a Klothodependent fashion (105), leading to decreased expression of
NaPi-2a and NaPi-2c proteins and phosphate wasting (38). A
recent paper by Hu et al. (46) also shows that Klotho directly,
and independently of FGF23, inhibits Na⫹-dependent Pi reabsorption by acutely inhibiting NaPi-2a-mediated Pi transport
activity followed by decreased cell surface protein expression.
Whether downregulation of renal Klotho expression in colitis
(101) represents a compensatory mechanism for the observed
changes in NaPi-2a and NaPi-2c mRNA levels and contributes
to maintenance of renal and systemic Pi homeostasis remains to
be determined.
Vitamin D Status in IBD
The biochemistry, nutritional aspects, metabolism, and
physiological and therapeutic potential of vitamin D and its
analogs have been the subject of countless reviews and monographs. Vitamin D3 (cholecalciferol) is supplied from the diet,
mostly from fortified dairy products and fish oils, or is synthesized in the skin as previtamin D3 from 7-dehydrocholesterol
by ultraviolet irradiation. Previtamin D3 spontaneously but
slowly isomerizes to vitamin D3 (cholecalciferol), which is
then transported in the blood by the vitamin D binding protein
(DBP) to the liver, where it is hydroxylated at C-25 by one or
more cytochrome P-450 vitamin D 25-hydroxylases (CYP2R1,
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changes in serum Pi levels in patients with inactivating mutations of NaPi-IIb gene (27). However, pharmacological inhibition of Na⫹-dependent Pi absorption and genetic ablation of
the murine NaPi-IIb gene argue to the contrary (35, 88).
Interestingly, consistently with our comments above, NaPiIIb⫺/⫺ mice had increased fecal phosphate excretion and hypophosphaturia, with no change in serum phosphate concentration. Renal Pi reabsorption has not been systematically
studied in clinical or preclinical settings. Our unpublished
observations with TNBS model of colitis suggest significant
downregulation of NaPi-IIa and NaPi-IIc renal transcript level
(50 and 90% decrease, respectively). However, neither in
TNBS colitis nor in the model of adoptive naive T-cell transfer
colitis were we able to demonstrate a change in fractional Pi
excretion and phosphaturia (F. K. Ghishan and P. R. Kiela,
unpublished observations). This may indicate that compensatory mechanisms, perhaps involving type III phosphate transporter (s), counteract the observed changes in the expression of
NaPi-IIa and NaPi-IIc.
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deficiency has also been postulated to contribute to the overall
inflammatory state because of its diverse role in modulating the
immune response (43). For these reasons, vitamin D3 (and
Ca2⫹) supplementation has become the mainstream intuitive
approach to prevent inflammation-associated bone loss and is
widely recommended by gastroenterologists and promoted by
the American Gastroenterological Association and by the British Society of Gastroenterology. However, the clinical efficacy
of this approach in relationship with BMD loss or accrual in
patients with active IBD remains controversial, with several
published reports that failed to identify any clinical benefits
(12, 14). Obviously, more systematic studies in pediatric and
adult IBD patients need to be conducted to identify factors
determining the clinical response to vitamin D and Ca2⫹
supplementation, the form and route of administered vitamin
D, with careful monitoring of the parameters of bone mass,
bone turnover, and mineral homeostasis. The need for such
systematic approach is further justified by identification of at
least a subset of IBD patients with inappropriate hypercalcitriolemia [elevated serum 1,25(OH)2D3] (1, 87). The earlier
report by Rudnicki et al. (87) demonstrated high 1,25(OH)2D3
levels in CD patients undergoing ileal resection despite reduced levels of 25(OH)D3 and speculated that extrarenal 1␣hydroxylation, similar to that found in sarcoidosis, may contribute to this paradoxical elevation of the bioactive vitamin D
form. Indeed, Abreu et al. (1) reproduced these findings in 42%
of CD patients who had abnormally high (⬎60 pg/ml)
1,25(OH)2D3 levels, which inversely correlated with BMD
irrespective of glucocorticoid use. Consistent with the hypothesis postulated by Rudnicki et al., the latter study showed
elevated expression of 1␣-hydroxylase in colon from patients
with active CD, with the protein overexpressed primarily by
macrophages and multinucleated giant cells, similar to that
observed in sarcoidosis-associated granulomas (1). On the
basis of these studies, it is not inconceivable that, paradoxically, high levels of 1,25(OH)2D3 are an important risk factor
for the development of osteoporosis in patients with CD. In
such scenario, elevated 1,25(OH)2D3 superimposed on limited
Ca2⫹ and Pi supply and direct effect of proinflammatory
mediators on bone turnover could skew the ultimate effects of
1,25(OH)2D3 toward a proresorptive mechanism. Since in
clinical settings analysis of serum 1,25(OH)2D3 is rarely performed (because of limited stability, assay cost, and reliability), these findings beg the question whether indiscriminate use
of vitamin D supplementation in patients with active CD is
beneficial or destructive and further emphasize the need for
more comprehensive clinical studies. It also raises concerns for
potential use of large doses of vitamin D as an immunomodulator in IBD as proposed by some preclinical studies without
careful monitoring of the bone metabolism.
Vitamin K Status in IBD Patients
Vitamin K family members phylloquinone (vitamin K1) and
the menaquinones (vitamin K2) play prominent roles in bone
metabolism and have been considered beneficial in preventing
bone loss associated with menopause, Parkinson’s disease,
chronic steroid treatment, biliary cirrhosis, and other disorders
(106a). The primary mechanism of vitamin K action is through
␥-carboxylation of vitamin K-dependent proteins collectively
called Gla proteins. Bone contains three known targets of
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hydroxyvitamin D3 [25(OH)D3]. Due to its stability and ease of
assay, 25(OH)D3 is almost exclusively used clinically as a
measure of vitamin D status. 25(OH)D3 is transported by DBP
to the kidney, where it is endocytosed in the proximal tubules
and is further hydroxylated at position C-1 by 1␣(OH)ase,
resulting in the hormonally active form of vitamin D,
1,25(OH)2D3, which is responsible for most, if not all of the
biological effects of vitamin D (24). 24-hydroxylase [CYP24,
24(OH)ase], also a renal mitochondrial P-450 enzyme, can
hydroxylate both 25(OH)D3 and 1,25(OH)2D3, with preference
for the latter. Catabolism of 1,25(OH)2D3 to 1,24,25(OH)3D3
decreases its biological activity, whereas hydroxylation of
25(OH)D3 to 24,25(OH)2D3 leads to a reduction in the pool
of 25(OH)D3 available for 1␣-hydroxylation. Therefore,
24(OH)ase functions to inactivate vitamin D.
Vitamin D plays a key role in skeletal and mineral homeostasis. It is frequently viewed as a pro-bone anabolic
hormone because of its positive effects on intestinal and renal
Ca2⫹ and Pi (re)absorption and through its positive effects on
osteoblast differentiation and bone matrix synthesis. However,
a more appropriate view of vitamin D in bone metabolism is as
a regulator of bone turnover. Indeed, 1,25(OH)2D3 can also
increase the release of receptor activator of NF-␬B ligand
(RANKL) and decrease osteoprotegerin (OPG) release from
osteoblastic cells and stimulate osteoclastogenesis, resulting in
bone resorption. One example of such effect was provided by
24(OH)ase knockout mice, which demonstrated that defective
catabolic clearance of 1,25(OH)2D3 leads to impaired intramembranous bone mineralization (95). Another example
comes from Klotho-deficient mice. Klotho acts as the FGF23
coreceptor and contributes to FGF23-mediated negative feedback thus leading to suppression of 1␣(OH)ase expression and
decreased 1,25(OH)2D3 synthesis. In the absence of Klotho,
mice develop severe defects in mineral homeostasis accompanied with very high levels of 1,25(OH)2D3 and osteoporosis
(57). This defect in bone mineralization, as well as other
phenotypic elements of aging, could be eliminated by crossing
Klotho knockouts with 1␣(OH)ase-deficient mice (74). These
findings highlight the pleiotropic role of 1,25(OH)2D3 in mineral homeostasis and question the simplistic views of the
vitamin often promoted by science and product marketing as
“the fountain of youth” (17).
The 2002 report of the American Gastroenterological Association Committee on Osteoporosis in Gastrointestinal Disease
stated that “osteomalacia and vitamin D deficiency are not
common in IBD (including Crohn’s disease) and are unlikely
to be important causes of most cases of diminished bone
mineral density (BMD) in IBD” (7). Since then, numerous
studies demonstrated evidence for an increased prevalence of
vitamin D deficiency among IBD patients. 25(OH)D3 deficiency or insufficiency has been reported more frequently in
patients with ulcerative colitis and Crohn’s disease compared
with either control subjects or a healthy population. This was
documented for both children with IBD (e.g., Ref. 34) as well
as in many studies with adult IBD patients (e.g., Ref. 61).
Among the many suggested reasons for the impaired vitamin
status in IBD patients are reduced efficiency of intestinal
absorption of vitamin D, primarily in patients with small
intestinal involvement or ileal resection; a disrupted enterohepatic circulation of vitamin D; renal insufficiency; reduced
dietary intake; and reduced exposure to sunshine. Vitamin D
Review
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IBD-ASSOCIATED BONE LOSS
INFLAMMATORY MEDIATORS AND BONE MODELING AND
REMODELING
Bone modeling (construction) is the process by which bone
is formed by osteoblasts without prior bone resorption and is
predominant during growth, resulting in changes in bone size
and shape. Bone remodeling (reconstruction) occurs throughout life and involves a coordinated action of bone resorbing
osteoclasts followed by bone formation at the same location by
osteoblasts. Both of these processes are critical to achieve
strength and lightness for mobility by strategically depositing
bone in locations where it is needed and by removing bone to
avoid bulk in places where it is unnecessary. Osteoblasts and
osteoclasts form the basic metabolic unit and are engaged in a
complex cross talk network that can be easily disrupted by both
the physiological process of aging, as well as by hormonal and
inflammatory mediators in numerous clinical disorders (79).
One important example of such interaction is the osteoprotegerin-RANK-RANKL axis. RANKL is a member of the TNF
family. It is produced by osteoblasts and by activated T cells
and it interacts with its receptor (RANK) on osteoclast precursor cells and mature osteoclasts. The ligation of RANK with
AJP-Gastrointest Liver Physiol • VOL
RANKL results in the commitment of monocyte/macrophage
precursor cells to the osteoclast lineage and the activation of
mature osteoclasts and preventing their apoptosis, thus leading
to increased bone resorption. RANKL signaling is accelerated
by inflammatory cytokines abundant in rheumatoid arthritis
and IBD such as IL-1, IL-6, and TNF-␣. These mediators
potently induce RANKL on osteoblasts and synovial fibroblasts and are believed to directly contribute to the bone
destruction process. OPG, also known as osteoclastogenesis
inhibitory factor, is an osteoblast-derived soluble cytokine
receptor from the TNF receptor superfamily. OPG acts as a
decoy receptor for RANKL and inhibits osteoclast differentiation, blocks activation of mature osteoclasts, and permits
osteoclast apoptosis. OPG is therefore a natural antagonist for
RANKL and a powerful inhibitor of bone resorption. This
pathway has been under intense scrutiny for the development
of therapeutic approaches to prevent osteopenia and osteoporosis in postmenopausal women, in primary hyperparathyroidism, Paget’s disease, rheumatoid arthritis, periodontal bone
loss, and several other disorders (85). RANKL/OPG system is
known to be activated in IBD patients (13, 72) and can be
normalized with infliximab (69). Exogenous recombinant OPG
(OPG-Fc) reverses skeletal abnormalities and reduces colitis in
IL-2 knockout mice (10). OPG-Fc, soluble RANK-Fc IgG
fusion proteins, or monoclonal anti-RANKL antibody (denosumab) have not yet been tested in IBD patients.
Bone Formation vs. Resorption in Animal Models of IBD
HLA-B27 transgenic rats that spontaneously develop inflammatory bowel disease, peripheral arthritis, ankylosing
spondylitis, and skin lesions were also reported to have decreased bone strength (3). Papet et al. (76) showed that they
have normal levels of osteocalcin (marker of bone formation),
normal fractional protein synthesis in the bone, but significantly increased urinary excretion of deoxypyridinoline
(marker of bone resorption). In another recent study, Rauner et
al. (80) demonstrated increased RANKL-OPG ratio and increased osteocalcin mRNA in the bone of HLA-B27 rats.
However, serum levels of type I collagen C-telopeptides
(CTx), NH2-terminal propeptide of type I procollagen (P1NP),
RANKL (all markers of bone resorption), as well as OPG, and
osteocalcin did not differ significantly in this study. These data
suggest that in HLA-B27 transgenic rats inflammation-associated bone loss is driven mainly by increased bone resorption,
perhaps because of skewed RANKL-OPG ratio and that serum
markers of bone metabolism may not always adequately reflect
the mechanism of bone loss.
In murine studies, varying contributions of bone formation
and resorption defects have been reported. Using IL-10⫺/⫺
mice as a model of Crohn’s-like colitis, Drezner-Pollak et al.
(32) showed that the effect of intestinal inflammation on the
development of osteopenia and osteoporosis was primarily
mediated by deficient bone formation. This was evidenced by
decreased cancellous double-labeled surface, mineralizing surface, serum osteocalcin level, and mineralized nodule number
in bone marrow stromal cell cultures. The same group demonstrated later in dextran sodium sulfate (DSS) model of colitis
that both the impaired bone formation and increased bone
resorption contribute to the significant lowering of bone mass
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vitamin K-mediated ␥-carboxylation: osteocalcin (77), matrix
Gla protein (78), and the more recently described periostin
expressed by the bone mesenchymal stem cells (28). These
three Gla proteins upon ␥-carboxylation have dramatically
enhanced capacity to bind calcium, effecting mineralizing
nodule formation and play important roles in normal bone
development and repair (29). Vitamin K has also been shown
to have direct and ␥-carboxylation-independent effects on gene
expression in osteoblastic cells through activation of the steroid
and xenobiotic receptor SXR (49, 99). Moreover, vitamin K2
inhibits RANKL mRNA expression and inhibits 1,25(OH)2D3induced osteoclastogenesis (100). A recent paper by Atkins et
al. (11) describes ␥-carboxylation-dependent and -independent
effects of vitamin K promoting bone mineralization, osteoblast-to-osteocyte transition, and an anticatabolic bone phenotype.
In IBD patients, vitamin K status has not received as much
attention as vitamin D. Schoon et al. (91) demonstrated a
significant decrease in serum vitamin K levels and free (non␥-carboxylated) osteocalcin in a cohort of 32 stable CD patients with small intestinal involvement receiving prednisolone. In a more recent analysis in Japanese IBD patients,
similar vitamin K deficiency was documented and shown to be
more prominent in CD that in UC (59). Undercarboxylated
osteocalcin was also elevated in CD patients. There was no
significant difference in vitamin K intake between CD and UC;
therefore, malabsorption of vitamin K was postulated to be the
likely contributor (59). Although vitamin K absorption in
inflammatory conditions has not been studied, one case report
with a small bowel CD patient treated with warfarin for deep
vein thrombosis showed resistance to oral vitamin K for
reversal of overanticoagulation (37). Subsequent subcutaneous
administration of vitamin K was, however, effective, suggesting that indeed chronic inflammation may affect intestinal
vitamin K absorption. More systematic studies are needed to
evaluate the effects of inflammation and inflammatory mediators on intestinal vitamin K absorption and the role of vitamin
K deficiency in IBD-associated bone loss.
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IBD-ASSOCIATED BONE LOSS
Bone Formation vs. Resorption in IBD Patients
Biochemical markers that reflect the processes of bone
resorption and bone formation, and thus bone turnover, can be
measured in blood and urine (25). However, utilization of these
markers in IBD patients has not delivered consistent results.
Several studies have reported increased levels of markers of
bone resorption without a compensatory increase in markers of
formation (18, 19, 33, 84, 92, 94). Contrary data have also been
published demonstrating reduced levels of bone formation
markers and no change in resorptive markers in patients with
long-standing, quiescent CD (90), elevated levels of both types
AJP-Gastrointest Liver Physiol • VOL
of markers (9, 70), or no change between IBD patients and
healthy controls (67).
These differences highlight the difficulties in designing such
clinical studies. In most cases, the discrepancies can be explained by small population sample and many confounding
factors related to the heterogeneity of IBD: ethnicity, geographic region, CD vs. UC, duration of disease, disease activity
index, active disease vs. remission, gender, steroid use, small
intestinal involvement and/or resection, and age. These do not
even account for factors like seasonality of vitamin D status. In
a more uniform study, bone formation and bone resorption in
quiescent Irish IBD patients (CD and UC, separately) were
compared with age- and sex-matched healthy controls (39).
Both CD and UC patients had significantly higher serum under
carboxylated osteocalcin and bone-specific alkaline phosphatase concentrations, as well as significantly higher urinary type
I collagen cross-linked N-telopeptides (NTx). Total serum
osteocalcin was, however, significantly lower in CD patients
compared with healthy control subjects (39). Simultaneously
elevated markers of bone formation and resorption are a
hallmark of increased rate of bone turnover, which in older
adults contributes to faster bone loss and is recognized as a risk
factor for fracture. However, lower total osteocalcin level may
also suggest that the processes of bone resorption and bone
formation are not coupled in IBD patients and that the uncoupled bone metabolism can be a significant risk factor for
progressive loss of BMD in CD and UC patients, even during
remission.
Several studies investigated bone turnover markers in IBD
patients in the context of the RANK/RANKL/OPG pathway.
Turk et al. (103) recently demonstrated elevated serum levels
of free soluble RANKL, OPG, TNF, and IL-6 in both longstanding CD patients with low bone mass and in naive newly
diagnosed patients with osteopenia compared with healthy
controls. In general, osteocalcin levels were not different between CD and healthy patients, whereas CD patients had
elevated NTx, thus suggesting increased bone resorption,
which correlated well with serum cytokine levels. In a subset
of patients, 81% of which were on corticosteroid therapy, low
total osteocalcin level was accompanied by high NTx, thus
suggesting that steroids trigger the uncoupling mechanism and
result in simultaneous decrease in bone formation and increased bone resorption (103).
Analysis of bone loss in pediatric IBD patients presents with
its unique difficulties. Some of the more critical differences in
bone metabolism and bone mass assessment in pediatric and
adult IBD patients have been reviewed by Sylvester (97) and
Hill et al. (44). These difficulties begin with limitations of the
use of dual-energy X-ray absorptiometry (DXA). Clinical reports generated from DXA measurements compare patient
areal BMD (aBMD) with those of chronological age- and
sex-matched healthy control subjects, returning a calculated
aBMD Z-score. However, stunted growth seen in children with
IBD, delayed puberty, decreased muscle strength, and delayed
skeletal maturation makes BMD more a function of bone
mineral accrual than of the child’s chronological age. Therefore, low aBMD may not be an indication of a metabolic bone
disease, but rather a reflection of delayed bone development,
which makes clinical study design and interpretation so much
more difficult. Moreover, DXA-derived aBMD is influenced
by bone size and does not distinguish between osteomalacia
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face, mineralizing surface, bone formation rate, and with increased bone resorption as indicated by elevated osteoclast
number (41). Another recent study with DSS colitis also
suggested a decrease in osteoblast maturation and function as
the primary causes in the observed changes in bone density
(42). Our group’s findings related to osteoblast/osteocytespecific Phex gene also support at least partial contribution of
defective bone formation in colitis. Phex is a critical regulator
of systemic Pi homeostasis (52), with an intrinsic role in
osteoblast mineralization. Immortalized osteoblasts from Phexdeficient Hyp mice fail to mineralize under permissive conditions in vitro (109). We demonstrated that Phex expression is
significantly depressed in various models of colitis (TNBS,
adoptive T cell transfer, and IL-10⫺/⫺ mice) (64, 104). This
suppression can be reversed with anti-TNF antibody, and the
effect of TNF on osteoblast mineralization correlates with
decreased Phex protein and mRNA in vitro (104). Moreover,
consistent with the inhibitory role of NF-␬B on osteoblastic
bone formation (22), TNF repressed Phex gene transcription
via a concerted action of NF-␬B and the enzymatic activity of
poly(ADP-ribose)polymerase 1 (PARP-1). TNF failed to
downregulate Phex expression in PARP-1⫺/⫺ mice (64). This
finding opens a potential avenue for reducing IBD-associated
bone loss by modulating NF-␬B activity via PARP-1 inhibition, a concept already being explored in acute inflammation
and cancer (53, 86).
Adoptive transfer of naive CD4⫹CD45RBHi T cells to immunodeficient Rag or SCID mice results in colitis with similarities to human Crohn’s disease. Using this model, Byrne et
al. (21) demonstrated osteopenia which could be treated with
recombinant osteoprotegerin (OPG-Fc) in mice with established colitis. Mice transferred with CD4⫹CD45RBHi T cells
had a significant decrease in the number of osteoblasts and an
increase in the number of osteoclasts in their tibias compared
with mice transferred with CD4⫹CD45RBLo T cells. Colitis
mice had elevated serum tartrate-resistant acid phosphatase
(TRAP; osteoclast marker) and decreased serum alkaline phosphatase (ALP; osteoblast marker), suggesting both increased
resorption and decreased bone formation. Surprisingly,
OPG-Fc treatment resulted in a loss of detectable osteoclasts as
well as osteoblasts. Although the authors concluded that “osteopenia was induced by inflammatory cell infiltration and not
by malabsorption of calcium,” the presented data show that
OPG-Fc did not alter TNF-positive mononuclear infiltrate in
the bone while offering only measurement of serum Ca2⫹ and
Pi concentration, which are not reliable markers of moderate
changes in mineral (re)absorption (21).
Review
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IBD-ASSOCIATED BONE LOSS
SUMMARY
The simplistic view that skeletal abnormalities seen in IBD
patients are related to vitamin D3 and calcium deficiency is
challenged by the recent observations of the complex interplay
between inflammatory mediators and key players in mineral
homeostasis and their effect on the delicate balance of osteoblast-osteoclast activities. To date, neither patient nor the
preclinical data with animal models of IBD provide a uniform
explanation of the pathogenesis of bone loss in acute and
chronic intestinal inflammation. Although no single animal
model is a valid reflection of the complex and nonhomogenous
human IBD, more well-designed animal studies are needed to
understand the multifactorial nature of IBD-associated bone
loss. A systematic review of these preclinical studies and
studies with human IBD patients may help identify selected
molecular targets and responsive subsets of patients to guide
future therapies.
GRANTS
This research was support by National Institute of Diabetes and Digestive
and Kidney Diseases Grants 5R37DK033209 and 3R37DK033209-27S1 (to
F. K. Ghishan).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
REFERENCES
1. Abreu MT, Kantorovich V, Vasiliauskas EA, Gruntmanis U, Matuk
R, Daigle K, Chen S, Zehnder D, Lin YC, Yang H, Hewison M,
Adams JS. Measurement of vitamin D levels in inflammatory bowel
disease patients reveals a subset of Crohn’s disease patients with elevated
1,25-dihydroxyvitamin D and low bone mineral density. Gut 53: 1129 –
1136, 2004.
AJP-Gastrointest Liver Physiol • VOL
2. Airaksinen MS, Eilers J, Garaschuk O, Thoenen H, Konnerth A,
Meyer M. Ataxia and altered dendritic calcium signaling in mice
carrying a targeted null mutation of the calbindin D28k gene. Proc Natl
Acad Sci USA 94: 1488 –1493, 1997.
3. Akhter MP, Jung LK. Decreased bone strength in HLA-B27 transgenic
rat model of spondyloarthropathy. Rheumatology (Oxford) 46: 1258 –
1262, 2007.
4. Akhter S, Kutuzova GD, Christakos S, DeLuca HF. Calbindin D9k is
not required for 1,25-dihydroxyvitamin D3-mediated Ca2⫹ absorption in
small intestine. Arch Biochem Biophys 460: 227–232, 2007.
5. Alexander RT, Woudenberg-Vrenken TE, Buurman J, Dijkman H,
van der Eerden BC, van Leeuwen JP, Bindels RJ, Hoenderop JG.
Klotho prevents renal calcium loss. J Am Soc Nephrol 20: 2371–2379,
2009.
6. Ali T, Lam D, Bronze MS, Humphrey MB. Osteoporosis in inflammatory bowel disease. Am J Med 122: 599 –604, 2009.
7 American Gastroenterological Association medical position statement:
guidelines on osteoporosis in gastrointestinal diseases. Gastroenterology
124: 791–794, 2003.
9. Ardizzone S, Bollani S, Bettica P, Bevilacqua M, Molteni P, Bianchi
Porro G. Altered bone metabolism in inflammatory bowel disease: there
is a difference between Crohn’s disease and ulcerative colitis. J Intern
Med 247: 63–70, 2000.
10. Ashcroft AJ, Cruickshank SM, Croucher PI, Perry MJ, Rollinson S,
Lippitt JM, Child JA, Dunstan C, Felsburg PJ, Morgan GJ, Carding
SR. Colonic dendritic cells, intestinal inflammation, and T cell-mediated
bone destruction are modulated by recombinant osteoprotegerin. Immunity 19: 849 –861, 2003.
11. Atkins GJ, Welldon KJ, Wijenayaka AR, Bonewald LF, Findlay
DM. Vitamin K promotes mineralization, osteoblast-to-osteocyte transition, and an anticatabolic phenotype by ␥-carboxylation-dependent and
-independent mechanisms. Am J Physiol Cell Physiol 297: C1358 –
C1367, 2009.
12. Benchimol EI, Ward LM, Gallagher JC, Rauch F, Barrowman N,
Warren J, Beedle S, Mack DR. Effect of calcium and vitamin D
supplementation on bone mineral density in children with inflammatory
bowel disease. J Pediatr Gastroenterol Nutr 45: 538 –545, 2007.
13. Bernstein CN, Sargent M, Leslie WD. Serum osteoprotegerin is increased in Crohn’s disease: a population-based case control study.
Inflamm Bowel Dis 11: 325–330, 2005.
14. Bernstein CN, Seeger LL, Anton PA, Artinian L, Geffrey S, Goodman W, Belin TR, Shanahan F. A randomized, placebo-controlled trial
of calcium supplementation for decreased bone density in corticosteroidusing patients with inflammatory bowel disease: a pilot study. Aliment
Pharmacol Ther 10: 777–786, 1996.
15. Bianco SD, Peng JB, Takanaga H, Suzuki Y, Crescenzi A, Kos CH,
Zhuang L, Freeman MR, Gouveia CH, Wu J, Luo H, Mauro T,
Brown EM, Hediger MA. Marked disturbance of calcium homeostasis
in mice with targeted disruption of the Trpv6 calcium channel gene. J
Bone Miner Res 22: 274 –285, 2007.
16. Bindels RJ, Ramakers PL, Dempster JA, Hartog A, van Os CH. Role
of Na⫹/Ca2⫹ exchange in transcellular Ca2⫹ transport across primary
cultures of rabbit kidney collecting system. Pflügers Arch 420: 566 –572,
1992.
17. Binkley N. Is vitamin D the fountain of youth? Endocr Pract 15:
590 –596, 2009.
18. Bischoff SC, Herrmann A, Goke M, Manns MP, von zur Muhlen A,
Brabant G. Altered bone metabolism in inflammatory bowel disease. Am
J Gastroenterol 92: 1157–1163, 1997.
19. Bjarnason I, Macpherson A, Mackintosh C, Buxton-Thomas M,
Forgacs I, Moniz C. Reduced bone density in patients with inflammatory bowel disease. Gut 40: 228 –233, 1997.
20. Bronner F. Recent developments in intestinal calcium absorption. Nutr
Rev 67: 109 –113, 2009.
21. Byrne FR, Morony S, Warmington K, Geng Z, Brown HL, Flores
SA, Fiorino M, Yin SL, Hill D, Porkess V, Duryea D, Pretorius JK,
Adamu S, Manoukian R, Danilenko DM, Sarosi I, Lacey DL, Kostenuik PJ, Senaldi G. CD4⫹CD45RBHi T cell transfer induced colitis
in mice is accompanied by osteopenia which is treatable with recombinant human osteoprotegerin. Gut 54: 78 –86, 2005.
22. Chang J, Wang Z, Tang E, Fan Z, McCauley L, Franceschi R, Guan
K, Krebsbach PH, Wang CY. Inhibition of osteoblastic bone formation
by nuclear factor-kappaB. Nat Med 15: 682–689, 2009.
300 • FEBRUARY 2011 •
www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.2 on June 17, 2017
(i.e., insufficient mineralization of bone matrix) and osteoporosis (insufficient amount of normally mineralized bone matrix). New more quantitative bone assessment methods relying
on objective and not relative measures of bone health such as
peripheral quantitative computed tomography provide a superior alternative.
Bone turnover marker data are also not easy to interpret in
children. Similar to DXA results, they can be influenced by
several confounding factors, such as growth rate, maturational
delay, low bone mass, and nutritional deficits. After adjusting
for several such confounders, Tuchman et al. (102) found that
young IBD patients had low bone formation and elevated bone
resorption markers. However, these were not newly diagnosed
patients and they had received treatment for IBD at the time of
study. A recent study with naive pediatric CD and UC patients
found that biochemical markers of both bone formation and
resorption were low compared with age- and sex-specific
reference ranges (107). Histomorphometric data showed that
bone formation activity was low on trabecular bone surfaces.
Osteoclast-covered surface was low, whereas eroded surface
was elevated, which might suggest increased bone resorption.
The authors reasoned, however, that eroded surface would also
be elevated when osteoblast activity is acutely suppressed.
Given the low bone formation parameters and low osteoclast
surface, it seems more likely that in children newly diagnosed
with IBD suppressed bone formation is the primary mechanism
leading to impaired bone accrual and low BMD.
Review
G200
IBD-ASSOCIATED BONE LOSS
AJP-Gastrointest Liver Physiol • VOL
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
phosphaturic substance acting as an autocrine enzyme in the renal
proximal tubule. FASEB J 24: 3438 –3450, 2010.
Huybers S, Apostolaki M, van der Eerden BC, Kollias G, Naber TH,
Bindels RJ, Hoenderop JG. Murine TNF⌬ARE Crohn’s disease model
displays diminished expression of intestinal Ca2⫹ transporters. Inflamm
Bowel Dis 14: 803–811, 2008.
Huybers S, Naber TH, Bindels RJ, Hoenderop JG. Prednisoloneinduced Ca2⫹ malabsorption is caused by diminished expression of the
epithelial Ca2⫹ channel TRPV6. Am J Physiol Gastrointest Liver Physiol
292: G92–G97, 2007.
Ichikawa T, Horie-Inoue K, Ikeda K, Blumberg B, Inoue S. Steroid
and xenobiotic receptor SXR mediates vitamin K2-activated transcription
of extracellular matrix-related genes and collagen accumulation in osteoblastic cells. J Biol Chem 281: 16927–16934, 2006.
Kanis JA, Melton LJ 3rd, Christiansen C, Johnston CC, Khaltaev N.
The diagnosis of osteoporosis. J Bone Miner Res 9: 1137–1141, 1994.
Khanal RC, Nemere I. Regulation of intestinal calcium transport. Annu
Rev Nutr 28: 179 –196, 2008.
Kiela PR, Ghishan FK. Recent advances in the renal-skeletal-gut axis
that controls phosphate homeostasis. Lab Invest 89: 7–14, 2009.
Kirkland JB. Poly ADP-ribose polymerase-1 and health. Exp Biol Med
(Maywood) 235: 561–568, 2010.
Kolek OI, Hines ER, Jones MD, LeSueur LK, Lipko MA, Kiela PR,
Collins JF, Haussler MR, Ghishan FK. 1␣,25-Dihydroxyvitamin D3
upregulates FGF23 gene expression in bone: the final link in a renalgastrointestinal-skeletal axis that controls phosphate transport. Am J
Physiol Gastrointest Liver Physiol 289: G1036 –G1042, 2005.
Koushik SV, Wang J, Rogers R, Moskophidis D, Lambert NA,
Creazzo TL, Conway SJ. Targeted inactivation of the sodium-calcium
exchanger (Ncx1) results in the lack of a heartbeat and abnormal
myofibrillar organization. FASEB J 15: 1209 –1211, 2001.
Kumari M, Khazai NB, Ziegler TR, Nanes MS, Abrams SA, Tangpricha V. Vitamin D-mediated calcium absorption in patients with
clinically stable Crohn’s disease: A pilot study. Mol Nutr Food Res 54:
1085–1091, 2010.
Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi
T, Ohyama Y, Kurabayashi M, Kaname T, Kume E, Iwasaki H, Iida
A, Shiraki-Iida T, Nishikawa S, Nagai R, Nabeshima YI. Mutation of
the mouse klotho gene leads to a syndrome resembling ageing. Nature
390: 45–51, 1997.
Kutuzova GD, Akhter S, Christakos S, Vanhooke J, Kimmel-Jehan
C, Deluca HF. Calbindin D (9k) knockout mice are indistinguishable
from wild-type mice in phenotype and serum calcium level. Proc Natl
Acad Sci USA 103: 12377–12381, 2006.
Kuwabara A, Tanaka K, Tsugawa N, Nakase H, Tsuji H, Shide K,
Kamao M, Chiba T, Inagaki N, Okano T, Kido S. High prevalence of
vitamin K and D deficiency and decreased BMD in inflammatory bowel
disease. Osteoporos Int 20: 935–942, 2009.
Larsen S, Bendtzen K, Nielsen OH. Extraintestinal manifestations of
inflammatory bowel disease: epidemiology, diagnosis, and management.
Ann Med 42: 97–114, 2010.
Leslie WD, Miller N, Rogala L, Bernstein CN. Vitamin D status and
bone density in recently diagnosed inflammatory bowel disease: the
Manitoba IBD Cohort Study. Am J Gastroenterol 103: 1451–1459, 2008.
Lichtenstein GR, Sands BE, Pazianas M. Prevention and treatment of
osteoporosis in inflammatory bowel disease. Inflamm Bowel Dis 12:
797–813, 2006.
Lu P, Boros S, Chang Q, Bindels RJ, Hoenderop JG. The betaglucuronidase klotho exclusively activates the epithelial Ca2⫹ channels
TRPV5 and TRPV6. Nephrol Dial Transplant 23: 3397–3402, 2008.
Majewski PM, Thurston RD, Ramalingam R, Kiela PR, Ghishan FK.
Cooperative role of NF-␬B and poly (ADP-ribose) polymerase 1
(PARP-1) in the TNF-induced inhibition of PHEX expression in osteoblasts. J Biol Chem 285: 34828 –34838, 2010.
Margolis DS, Kim D, Szivek JA, Lai LW, Lien YH. Functionally
improved bone in calbindin-D28k knockout mice. Bone 39: 477–484,
2006.
Marks J, Debnam ES, Unwin RJ. Phosphate homeostasis and the
renal-gastrointestinal axis. Am J Physiol Renal Physiol 299: F285–F296,
2010.
Martin A, Fries W, Luisetto G, Mussolin L, Plebani M, Giacomin D,
Naccarato R. Bone-density and calcium-metabolism in patients with
long-standing, quiescent Crohns-disease. Eur J Gastroenterol Hepatol 6:
611–616, 1994.
300 • FEBRUARY 2011 •
www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.2 on June 17, 2017
23. Chen H, Xu H, Dong J, Li J, Ghishan FK. Tumor necrosis factor-␣
impairs intestinal phosphate absorption in colitis. Am J Physiol Gastrointest Liver Physiol 296: G775–G781, 2009.
24. Christakos S, Ajibade DV, Dhawan P, Fechner AJ, Mady LJ. Vitamin D: metabolism. Endocrinol Metab Clin North Am 39: 243–253,
2010.
25. Civitelli R, Armamento-Villareal R, Napoli N. Bone turnover markers:
understanding their value in clinical trials and clinical practice. Osteoporos Int 20: 843–851, 2009.
26. Collins JF, Bai L, Ghishan FK. The SLC20 family of proteins: dual
functions as sodium-phosphate cotransporters and viral receptors.
Pflügers Arch 447: 647–652, 2004.
27. Corut A, Senyigit A, Ugur SA, Altin S, Ozcelik U, Calisir H, Yildirim
Z, Gocmen A, Tolun A. Mutations in SLC34A2 cause pulmonary
alveolar microlithiasis and are possibly associated with testicular microlithiasis. Am J Hum Genet 79: 650 –656, 2006.
28. Coutu DL, Wu JH, Monette A, Rivard GE, Blostein MD, Galipeau J.
Periostin, a member of a novel family of vitamin K-dependent proteins,
is expressed by mesenchymal stromal cells. J Biol Chem 283: 17991–
18001, 2008.
29. Cranenburg EC, Schurgers LJ, Vermeer C. Vitamin K: the coagulation vitamin that became omnipotent. Thromb Haemost 98: 120 –125,
2007.
30. De Groot T, Bindels RJ, Hoenderop JG. TRPV5: an ingeniously
controlled calcium channel. Kidney Int 74: 1241–1246, 2008.
31. Di Stefano M, Veneto G, Malservisi S, Cecchetti L, Minguzzi L,
Strocchi A, Corazza GR. Lactose malabsorption and intolerance and
peak bone mass. Gastroenterology 122: 1793–1799, 2002.
32. Dresner-Pollak R, Gelb N, Rachmilewitz D, Karmeli F, Weinreb M.
Interleukin 10-deficient mice develop osteopenia, decreased bone formation, and mechanical fragility of long bones. Gastroenterology 127:
792–801, 2004.
33. Duggan P, O’Brien M, Kiely M, McCarthy J, Shanahan F, Cashman
KD. Vitamin K status in patients with Crohn’s disease and relationship
to bone turnover. Am J Gastroenterol 99: 2178 –2185, 2004.
34. El-Matary W, Sikora S, Spady D. Bone mineral density, vitamin D, and
disease activity in children newly diagnosed with inflammatory bowel
disease. Dig Dis Sci 2010. Aug 20. [Epub ahead of print].
35. Eto N, Tomita M, Hayashi M. NaPi-mediated transcellular permeation
is the dominant route in intestinal inorganic phosphate absorption in rats.
Drug Metab Pharmacokinet 21: 217–221, 2006.
36. Fries W, Giacomin D, Plebani M, Martin A. Effect of experimental
colitis on bone metabolism in the rat. Digestion 55: 229 –233, 1994.
37. Fugate SE, Ramsey AM. Resistance to oral vitamin K for reversal of
overanticoagulation during Crohn’s disease relapse. J Thromb Thrombolysis 17: 219 –223, 2004.
38. Gattineni J, Bates C, Twombley K, Dwarakanath V, Robinson ML,
Goetz R, Mohammadi M, Baum M. FGF23 decreases renal NaPi-2a
and NaPi-2c expression and induces hypophosphatemia in vivo predominantly via FGF receptor 1. Am J Physiol Renal Physiol 297: F282–F291,
2009.
39. Gilman J, Shanahan F, Cashman KD. Altered levels of biochemical
indices of bone turnover and bone-related vitamins in patients with
Crohn’s disease and ulcerative colitis. Aliment Pharmacol Ther 23:
1007–1016, 2006.
40. Giral H, Caldas Y, Sutherland E, Wilson P, Breusegem S, Barry N,
Blaine J, Jiang T, Wang XX, Levi M. Regulation of rat intestinal
Na-dependent phosphate transporters by dietary phosphate. Am J Physiol
Renal Physiol 297: F1466 –F1475, 2009.
41. Hamdani G, Gabet Y, Rachmilewitz D, Karmeli F, Bab I, DresnerPollak R. Dextran sodium sulfate-induced colitis causes rapid bone loss
in mice. Bone 43: 945–950, 2008.
42. Harris L, Senagore P, Young VB, McCabe LR. Inflammatory bowel disease
causes reversible suppression of osteoblast and chondrocyte function in mice.
Am J Physiol Gastrointest Liver Physiol 296: G1020–G1029, 2009.
43. Hewison M. Vitamin D and the immune system: new perspectives on an
old theme. Endocrinol Metab Clin North Am 39: 365–379, 2010.
44. Hill RJ, Brookes DS, Davies PS. Bones in pediatric Crohn’s disease: a
review of fracture risk in children and adults. Inflamm Bowel Dis 2010.
Sep 7. [Epub ahead of print].
45. Hoenderop JG, Nilius B, Bindels RJ. Calcium absorption across
epithelia. Physiol Rev 85: 373–422, 2005.
46. Hu MC, Shi M, Zhang J, Pastor J, Nakatani T, Lanske B, Razzaque
MS, Rosenblatt KP, Baum MG, Kuro-o M, Moe OW. Klotho: a novel
Review
G201
IBD-ASSOCIATED BONE LOSS
AJP-Gastrointest Liver Physiol • VOL
standing Crohn’s disease in remission. Aliment Pharmacol Ther 15:
783–792, 2001.
91. Schoon EJ, Muller MC, Vermeer C, Schurgers LJ, Brummer RJ,
Stockbrugger RW. Low serum and bone vitamin K status in patients
with longstanding Crohn’s disease: another pathogenetic factor of osteoporosis in Crohn’s disease? Gut 48: 473–477, 2001.
92. Schulte C, Dignass AU, Mann K, Goebell H. Reduced bone mineral
density and unbalanced bone metabolism in patients with inflammatory
bowel disease. Inflamm Bowel Dis 4: 268 –275, 1998.
93. Shikhare G, Kugathasan S. Inflammatory bowel disease in children:
current trends. J Gastroenterol 45: 673–682, 2010.
94. Silvennoinen J, Risteli L, Karttunen T, Risteli J. Increased degradation of type I collagen in patients with inflammatory bowel disease. Gut
38: 223–228, 1996.
95. St-Arnaud R, Arabian A, Travers R, Barletta F, Raval-Pandya M,
Chapin K, Depovere J, Mathieu C, Christakos S, Demay MB,
Glorieux FH. Deficient mineralization of intramembranous bone in
vitamin D-24-hydroxylase-ablated mice is due to elevated 1,25-dihydroxyvitamin D and not to the absence of 24,25-dihydroxyvitamin D.
Endocrinology 141: 2658 –2666, 2000.
96. Suzuki Y, Landowski CP, Hediger MA. Mechanisms and regulation of
epithelial Ca2⫹ absorption in health and disease. Annu Rev Physiol 70:
257–271, 2008.
97. Sylvester FA. IBD and skeletal health: children are not small adults!
Inflamm Bowel Dis 11: 1020 –1023, 2005.
98. Sylvester FA, Wyzga N, Hyams JS, Davis PM, Lerer T, Vance K,
Hawker G, Griffiths AM. Natural history of bone metabolism and bone
mineral density in children with inflammatory bowel disease. Inflamm
Bowel Dis 13: 42–50, 2007.
99. Tabb MM, Sun A, Zhou C, Grun F, Errandi J, Romero K, Pham H,
Inoue S, Mallick S, Lin M, Forman BM, Blumberg B. vitamin K2
regulation of bone homeostasis is mediated by the steroid and xenobiotic
receptor SXR. J Biol Chem 278: 43919 –43927, 2003.
100. Takeuchi Y, Suzawa M, Fukumoto S, Fujita T. Vitamin K(2) inhibits
adipogenesis, osteoclastogenesis, and ODF/RANK ligand expression in
murine bone marrow cell cultures. Bone 27: 769 –776, 2000.
101. Thurston RD, Larmonier CB, Majewski PM, Ramalingam R, Midura-Kiela M, Laubitz D, Vandewalle A, Besselsen DG, Muhlbauer
M, Jobin C, Kiela PR, Ghishan FK. Tumor necrosis factor and
interferon-gamma down-regulate Klotho in mice with colitis. Gastroenterology 138: 1384 –1394, 1394.e1–2, 2010.
102. Tuchman S, Thayu M, Shults J, Zemel BS, Burnham JM, Leonard
MB. Interpretation of biomarkers of bone metabolism in children: impact
of growth velocity and body size in healthy children and chronic disease.
J Pediatr 153: 484 –490, 2008.
103. Turk N, Cukovic-Cavka S, Korsic M, Turk Z, Vucelic B. Proinflammatory cytokines and receptor activator of nuclear factor kappaB-ligand/
osteoprotegerin associated with bone deterioration in patients with
Crohn’s disease. Eur J Gastroenterol Hepatol 21: 159 –166, 2009.
104. Uno JK, Kolek OI, Hines ER, Xu H, Timmermann BN, Kiela PR,
Ghishan FK. The role of tumor necrosis factor alpha in down-regulation
of osteoblast Phex gene expression in experimental murine colitis.
Gastroenterology 131: 497–509, 2006.
105. Urakawa I, Yamazaki Y, Shimada T, Iijima K, Hasegawa H, Okawa K,
Fujita T, Fukumoto S, Yamashita T. Klotho converts canonical FGF
receptor into a specific receptor for FGF23. Nature 444: 770 –774, 2006.
106. Villa-Bellosta R, Ravera S, Sorribas V, Stange G, Levi M, Murer H, Biber
J, Forster IC. The Na⫹-Pi cotransporter PiT-2 (SLC20A2) is expressed in
the apical membrane of rat renal proximal tubules and regulated by dietary
Pi. Am J Physiol Renal Physiol 296: F691–F699, 2009.
106a vitamin K2. Monograph. Altern Med Rev 14: 284 –293, 2009.
107. Ward LM, Rauch F, Matzinger MA, Benchimol EI, Boland M, Mack
DR. Iliac bone histomorphometry in children with newly diagnosed
inflammatory bowel disease. Osteoporos Int 21: 331–337, 2010.
108. Williams KB, DeLuca HF. Characterization of intestinal phosphate
absorption using a novel in vivo method. Am J Physiol Endocrinol Metab
292: E1917–E1921, 2007.
109. Xiao ZS, Crenshaw M, Guo R, Nesbitt T, Drezner MK, Quarles LD.
Intrinsic mineralization defect in Hyp mouse osteoblasts. Am J Physiol
Endocrinol Metab 275: E700 –E708, 1998.
110. Zarrabeitia MT, Hernandez JL, Valero C, Zarrabeitia AL, Ortiz F,
Gonzalez-Macias J, Riancho JA. Klotho gene polymorphism and male
bone mass. Calcif Tissue Int 80: 10 –14, 2007.
300 • FEBRUARY 2011 •
www.ajpgi.org
Downloaded from http://ajpgi.physiology.org/ by 10.220.33.2 on June 17, 2017
68. Mascarenhas MR, Thayu M. Pediatric inflammatory bowel disease and
bone health. Nutr Clin Pract 25: 347–352, 2010.
69. Miheller P, Muzes G, Racz K, Blazovits A, Lakatos P, Herszenyi L,
Tulassay Z. Changes of OPG and RANKL concentrations in Crohn’s
disease after infliximab therapy. Inflamm Bowel Dis 13: 1379 –1384,
2007.
70. Miheller P, Toth M, Molnar E, Zagoni T, Racz K, Tulassay Z. [Serum
bone marker measurements in bone metabolism disorders associated with
inflammatory bowel diseases]. Orv Hetil 142: 1557–1560, 2001.
71. Moe SM. Disorders involving calcium, phosphorus, and magnesium.
Prim Care 35: 215–237, v–vi, 2008.
72. Moschen AR, Kaser A, Enrich B, Ludwiczek O, Gabriel M, Obrist P, Wolf
AM, Tilg H. The RANKL/OPG system is activated in inflammatory
bowel disease and relates to the state of bone loss. Gut 54: 479 –487,
2005.
73. Nakashima T, Takayanagi H. Osteoimmunology: crosstalk between the
immune and bone systems. J Clin Immunol 29: 555–567, 2009.
74. Ohnishi M, Nakatani T, Lanske B, Razzaque MS. Reversal of mineral
ion homeostasis and soft-tissue calcification of klotho knockout mice by
deletion of vitamin D 1alpha-hydroxylase. Kidney Int 75: 1166 –1172,
2009.
75. Okunade GW, Miller ML, Pyne GJ, Sutliff RL, O’Connor KT,
Neumann JC, Andringa A, Miller DA, Prasad V, Doetschman T,
Paul RJ, Shull GE. Targeted ablation of plasma membrane Ca2⫹ATPase (PMCA) 1 and 4 indicates a major housekeeping function for
PMCA1 and a critical role in hyperactivated sperm motility and male
fertility for PMCA4. J Biol Chem 279: 33742–33750, 2004.
76. Papet I, El Yousfi M, Godin JP, Mermoud AF, Davicco MJ, Coxam
V, Breuille D, Obled C. HLA-B27 rats develop osteopaenia through
increased bone resorption without any change in bone formation. J
Musculoskelet Neuronal Interact 8: 251–256, 2008.
77. Poser JW, Esch FS, Ling NC, Price PA. Isolation and sequence of the
vitamin K-dependent protein from human bone. Undercarboxylation of
the first glutamic acid residue. J Biol Chem 255: 8685–8691, 1980.
78. Price PA, Williamson MK. Primary structure of bovine matrix Gla
protein, a new vitamin K-dependent bone protein. J Biol Chem 260:
14971–14975, 1985.
79. Raggatt LJ, Partridge NC. Cellular and molecular mechanisms of bone
remodeling. J Biol Chem 285: 25103–25108, 2010.
80. Rauner M, Stupphann D, Haas M, Fert I, Glatigny S, Sipos W,
Breban M, Pietschmann P. The HLA-B27 transgenic rat, a model of
spondyloarthritis, has decreased bone mineral density and increased
RANKL to osteoprotegerin mRNA ratio. J Rheumatol 36: 120 –126,
2009.
81. Renkema KY, Alexander RT, Bindels RJ, Hoenderop JG. Calcium
and phosphate homeostasis: concerted interplay of new regulators. Ann
Med 40: 82–91, 2008.
82. Renkema KY, Nijenhuis T, van der Eerden BC, van der Kemp AW,
Weinans H, van Leeuwen JP, Bindels RJ, Hoenderop JG. Hypervitaminosis D mediates compensatory Ca2⫹ hyperabsorption in TRPV5
knockout mice. J Am Soc Nephrol 16: 3188 –3195, 2005.
83. Riancho JA, Valero C, Hernandez JL, Ortiz F, Zarrabeitia A, Alonso
MA, Pena N, Pascual MA, Gonzalez-Macias J, Zarrabeitia MT.
Association of the F352V variant of the Klotho gene with bone mineral
density. Biogerontology 8: 121–127, 2007.
84. Robinson RJ, Iqbal SJ, Abrams K, Al-Azzawi F, Mayberry JF.
Increased bone resorption in patients with Crohn’s disease. Aliment
Pharmacol Ther 12: 699 –705, 1998.
85. Romas E. Clinical applications of RANK-ligand inhibition. Intern Med
J 39: 110 –116, 2009.
86. Rouleau M, Patel A, Hendzel MJ, Kaufmann SH, Poirier GG. PARP
inhibition: PARP1 and beyond. Nat Rev Cancer 10: 293–301, 2010.
87. Rudnicki M, Frolich A, Transbol I. Inappropriate hypercalcitriolemia
in ileum-resected patients with Crohn’s disease. Miner Electrolyte Metab
18: 52–55, 1992.
88. Sabbagh Y, O’Brien SP, Song W, Boulanger JH, Stockmann A,
Arbeeny C, Schiavi SC. Intestinal npt2b plays a major role in phosphate
absorption and homeostasis. J Am Soc Nephrol 20: 2348 –2358, 2009.
89. Schoeber JP, Hoenderop JG, Bindels RJ. Concerted action of associated proteins in the regulation of TRPV5 and TRPV6. Biochem Soc
Trans 35: 115–119, 2007.
90. Schoon EJ, Geerling BG, Van Dooren IM, Schurgers LJ, Vermeer C,
Brummer RJ, Stockbrugger RW. Abnormal bone turnover in long-