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Pathophysiology of CKD-MBD

Article  in  Clinical Reviews in Bone and Mineral Metabolism · September 2011

DOI: 10.1007/s12018-011-9120-8

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Clinic Rev Bone Miner Metab
DOI 10.1007/s12018-011-9120-8
ORIGINAL PAPER
Pathophysiology of CKD-MBD
Grahame J. Elder
Ó Springer Science+Business Media, LLC 2011
Abstract To maintain calcium and phosphate balance
despite declining kidney function, adaptations must begin
in the earliest stages of chronic kidney disease (CKD).
These are generally successful in maintaining calcium and
phosphate levels within the normal range through CKD
stages 1–4. Although routine biochemistry is not informa-
tive about net gain of these minerals, newer markers such
as Klotho and fibroblast growth factor 23 may provide
clues to these early adjustments and be of prognostic value.
Bone cells play a central role in modulating responses to
CKD. This review describes that role and highlights the
dynamic communication between bone and the kidneys.
Keywords CKD-MBD
Bone
Osteocyte
Osteoblast

Osteoclast
Fibroblast growth factor 23
Klotho

Parathyroid hormone
Renal adaptation
Pathophysiology
Introduction
The laboratory, bone and vascular abnormalities that
develop with CKD have been recognized for many years,
so why create a new condition called CKD-mineral and
bone disorder (MBD)? The reason is connections.
Although the associations are not new, the defining feature
of CKD-MBD is the way that pathophysiology so inti-
mately links fracture risk, vascular calcification, morbidity
G. J. Elder (&)
Department of Renal Medicine, Westmead Hospital, Westmead,
Sydney, NSW 2145, Australia
e-mail: g.elder@garvan.unsw.edu.au
G. J. Elder
Osteoporosis and Bone Biology Program, Garvan Institute
of Medical Research, Sydney, NSW, Australia
and mortality to the biochemical and hormonal conse-
quences of kidney damage (Fig. 1). Defining a new dis-
order by consensus does not happen frequently in
medicine, so it is intriguing to reflect how greatly a new
appreciation of these connections has impacted on the
clinical management of CKD and integrated areas of lab-
oratory research that previously seemed more distantly
related.
Maintaining the body’s mineral balance is a matter of
matching input to output, which becomes more challenging
as kidney function declines. At the renal level, the ‘‘intact
nephron hypothesis’’ [1] describes how maintaining min-
eral homeostasis will demand greater work from fewer
nephrons. As this occurs, signal pathways that control
mineral homeostasis need to reset, and bone is compelled
to assume functions of mineral homeostasis that were
previously shared with the kidneys. These interconnections
are fascinating, but some understanding of bone cells and
how they work is necessary to savor their complexity—and
potential for failure. So, I will start by reviewing the rel-
evant bone biology and signals that affect it and then
proceed to changes that occur with progressive CKD.
Normal Bone; Basic Bone Biology
Normal bone turnover, mineralization and the maintenance
of bone volume are essential for a healthy skeleton that is
stable enough for locomotion, strong enough to resist
fractures and provides for the homeostasis and storage of
calcium and phosphate. To maintain strength and stability,
the skeleton must detect mechanical strain that indicates
where extra bone is required, repair microdamage that
occurs with daily activities and renew aging bone. Cortical
bone (as found in the shafts of long bones) is commonly
described as ‘‘compact’’; a misnomer if the extensive
123

Fig. 1 The term ‘‘chronic kidney disease mineral and bone disorder’’
unifies the important elements of this clinical cluster
cortical porosity of ageing or hyperparathyroidism is
present. Cortical bone contains yellow marrow and has
relatively slow turnover, particularly at peripheral sites [2].
By comparison, cancellous bone within vertebrae and at the
ends of long bones consists of bony plates or trabeculae,
which provides a large surface area. Cancellous and
endocortical bone surfaces near red marrow have higher
turnover because they are more active in haematopoiesis
and mineral homeostasis [2].
Bone Remodeling
Bone remodeling is the term used for the process of bone
turnover and renewal. The teams of cells involved are
termed bone multicellular units (BMUs) and consist of a
cutting cone of osteoclasts derived from hematopoietic
stem cells, followed by osteoblasts that are derived from
mesenchymal stem cells (MSC) that lay down matrix and
mineral [3]. Not surprisingly this is a tightly coupled pro-
cess, because as the BMU advances at 10–20 lm/day, any
imbalance between bone resorption and formation could
lead to unwanted changes in bone volume, microarchitec-
ture, strength or mineral homeostasis [4]. To avoid these
consequences, the cells of the BMU must be in close
communication with each other and with osteocytes; ter-
minally differentiated osteoblasts that are the most abun-
dant cells in bone and principal conductors of the ‘‘bone
orchestra.’’
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Clinic Rev Bone Miner Metab
Initiation of Remodeling
Bone regions share common (or stochastic) remodeling rates
that may vary with factors that include age and menopausal
status, and conditions such as thyroid disease or glucocorti-
coid use. Remodeling can also be pathological (or redundant)
when new bone is preferentially remodeled or targeted in
response to mechanical strain [4, 5]. Targeted remodeling
originates when loading leads to strain, or fatigue and micro
cracks that disrupt osteocyte canalicular networks [6]
(Fig. 2). When this is sensed by osteocytes, they initiate
remodeling by the generation of new BMUs (Fig. 2).
Affected osteocytes recruit haematopoietic stem cells from
nearby ‘stem cell niches’ [7] or circulating pre-osteoclasts,
and these differentiate into osteoclasts under the influence of
the receptor activator of nuclear factor jB-ligand (RANKL)
expressed by cells of the osteoblast lineage [8]. Next, the
osteoclasts resorb damaged bone over a period of 2–3 weeks;
during which time some will undergo apoptosis, but new
osteoclasts are recruited to continue the job. Osteoblast
teams then lay down a matrix of osteoid which they then
mineralize and finally the bone becomes quiescent while the
mineral crystals mature and the bone strengthens. In cortical
bone, the BMU forms a tunnel that is re-mineralized to form
a new osteon. In cancellous bone, the BMU excavates a
trench across the bone surface that is filled to form a hemi-
osteon. ‘‘Activation frequency’’ is a measure of the overall
intensity of bone turnover—although not necessarily the
birth of new BMUs.
Within the BMU, the distance between the cutting cone
that releases mineral and the osteoblast team that deposits
osteoid and mineral can be varied in a ‘‘concertina-like’’
fashion [9]. Consequently, the release of calcium and
phosphate can vary with BMU activity. In young animals,
this osteoclastic bone resorption appears to play a signifi-
cant role in calcium homeostasis, but in mature animals
calcium ion release or uptake from the osteocyte lacunar
and canalicular network seems most important for rapid
calcium adjustments [10–12] [Table 1].
Fig. 2 Microcrack disruption of the osteocyte canalicular system
initiates a remodeling cycle by the Bone Multicellular Unit:
osteoclasts remove damaged bone followed by osteoblast teams,
which produce matrix and re-mineralized bone, after which there is a
quiescent period
Clinic Rev Bone Miner Metab

Table 1 Principal cells found within bone

Osteoclasts are derived from hematopoietic stem cells that fuse to form large multinucleated bone resorbing cells. Osteoclast precursors
commit to the osteoclast lineage when receptor activator of nuclear factor jB (RANK) cell surface receptors bind RANK ligand (RANKL),
which is produced by osteoblasts, osteocytes and bone marrow stromal cells
Osteoblasts are bone forming cells derived from mesenchymal stem cells (MSC), which have potential to undergo differentiation to
chondrocytes, adipocytes and myocytes. MSCs commit to the osteoblast lineage and develop to mature osteoblasts under the influence of
numerous downstream factors that include Runx2 (also known as Cbfa1), Osterix and members of the Wnt signaling pathway [13]. They
express alkaline phosphatase (bone-specific alkaline phosphatase; BALP) and osteocalcin, secrete collagen and non-collagenous matrix
proteins and mineralize bone. Osteoblasts may flatten to become lining cells, be buried in the bone matrix aligned in the direction of bone
formation to become osteocytes, or undergo apoptosis
Osteocytes are terminally differentiated osteoblasts that have become embedded in the bone matrix. They maintain intimate connections with
each other and with mineralizing osteoblasts on the bone surface through networks of cell processes that traverse the bone canaliculi [14]
(Fig. 3). Osteocytes act as mechanosensors and regulate osteoid matrix formation and mineralization, responding to parathyroid hormone
(PTH) and c-terminal PTH fragments [15], estrogens, prostaglandins and 1,25-dihydroxyvitamin D [1,25(OH)2D]. Osteocytes act as ion
sensors and are likely to control calcium homeostasis by altering its concentration in the fluid bathing the lacunae and canaliculi. Osteocytes
have recently been reported to produce RANKL and they may in fact be the major cells controlling osteoclastic bone resorption in adults
[16, 17]

Fig. 4 An example of direct signaling to osteoclasts is by the

production of cell surface and soluble receptor activator of nuclear
factor jB-ligand (RANKL) and osteoprotegerin (OPG). Indirect
osteoclast to osteoblast signaling can occur by the release of cytokines
from the bone matrix

osteoclast activity and potentiate bone anabolic activity

Fig. 3 Fluorescence image of calvarial fragment demonstrating
osteocytes and their dense network of cell processes overlying
osteoblasts on the bone surface. Reproduced with permission from
[14]
Coupling Bone Cell Activity
The coupling of bone resorption and formation depends
upon direct and indirect signals between bone cells, as well
as ‘‘whole-body’’ controls such as metabolic or sympa-
thetic nervous system signals [18]. An example of direct
osteoblast to osteoclast signaling is the production by
osteoblasts of RANKL to facilitate osteoclast differentia-
tion and activity, or production of the RANKL decoy
receptor osteoprotegerin (OPG) to bind RANKL, reduce
(Fig. 4). An example of indirect osteoclast to osteoblast
signaling is the release of cytokines such as insulin-like
growth factor (IGF) and transforming growth factor (TGF)-
b from the bone matrix during osteoclastic resorption.
Upon release, these factors activate the osteoblast teams
and can also recruit MSCs to the site [19–21]. Direct,
bidirectional signaling can also occur between osteoblasts
and osteoclasts. An example is the proposed role for for-
ward signaling by Eph receptor-expressing osteoblasts to
stimulate bone formation and reverse signaling through
ephrin-expressed by osteoclasts to inhibit bone resorption
[22]. Additional osteoclast factors influencing bone turn-
over have recently been reported including the migration
and mineralization stimulating agents BMP-6 and sphin-
gosine 1-phosphate (S1P) [23], interferon regulatory fac-
tor-8 (IRF-8) [24], semaphorin 4d (Sema4D) [125] and
osteoclast-derived sclerostin [25], which may reduce
osteoblast anabolic activity with aging. Since direct
osteoclast to osteoblast contact does not occur in the

123

human BMU, details of these mechanisms need further
development.
Bone Turnover in Response to Mechanotransduction
The concept of mechanotransduction was 1st introduced by
Julius Wolff in 1892, when he published ‘‘The Law of
Transformation of Bone.’’. This work stated that ‘‘As a
consequence of primary shape variations and continuous
loading, or even due to loading alone, bone changes its inner
architecture according to mathematical rules and, as a sec-
ondary effect and governed by the same mathematical rules,
also changes its shape.’’ Now known as ‘‘Wolff’s Law’’ he
regarded this finding as a ‘‘brick to complete the building of
Charles Darwin’’ (http://jwi.charite.de/en/ accessed 25th
September 2011); a prescient statement, given the now
familiar concept of gene-environment interactions.
Osteocytes are the most numerous and long-lived bone
cells. The osteocyte networks are highly integrated and
ideally suited to being mechanical strain receptors and for
the conversion of strain into biochemical signals, or
‘‘mechanotransduction’’ [26]). Although the process is not
fully understood, mechanical stimulation of bone causes
motion and shear stress within the fluid or gel-filled can-
alicular network. The osteocyte processes in these tube-like
structures possess a central filament bundle that is tethered
to the canalicular wall by transverse tethering elements
[27]. When stimulated, these elements transmit a signal to
the actin cytoskeleton of the osteocyte process, causing a
biochemical response. Recent investigations have provided
direct evidence for this load-induced fluid flow [28], which
is also essential for maintaining bone elasticity and osteo-
cyte hydration and survival. The estrogen receptor (ER)-a
appears to participate in this signaling cascade, as dem-
onstrated by reduced bone formation following mechanical
loading in ER-a knockout mice compared to the wild type
[29]. Osteoblasts also respond to shear, stretch and
hydrostatic pressure by activation of mechanosensitive
channels, with downstream signaling possibly amplified by
the osteocyte network. It appears likely that ER-a receptors
interact with membrane IGF-receptors to sensitize osteo-
blasts to the anabolic effects of IGFs [30]. These findings
help to explain the action of selective ER modulators
(SERMS) such as raloxifene, which are ER-a agonists.
Humoral Bone Pathways
Sclerostin is a soluble protein encoded by the SOST gene
and produced predominantly by mature osteocytes. After
release, sclerostin is believed to reach osteoblasts via the
canalicular system [31] where it binds to LDL-receptor-
related protein (LRP) 5/6 and LRP4 and inhibits signaling
through the canonical (or stereotypical) Wnt signaling
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Clinic Rev Bone Miner Metab
Fig. 5 The Wnt signaling pathway is anabolic for bone. Wnts are
produced by stromal cells/osteoblast progenitors and bind to osteo-
blast LRP 5/6 and LRP 4 receptors. Sclerostin inhibits this binding.
SOST gene expression is itself inhibited by mechanical strain
pathway [32, 33] (Fig. 5). This pathway is anabolic for
bone and is described below (Fig. 5). SOST gene expres-
sion is influenced by a number of factors that influence
bone strength, particularly mechanical stimulation of bone,
which inhibits sclerostin production and increases osteo-
blast activity [34]. Illustrating this, osteocytes prone to
mechanical stimulation nearer the bone surface are more
often sclerostin negative than osteocytes further from the
surface and areas of remodeling [31]. One of the most
dramatic human examples of sclerostin suppression is
illustrated by the condition Van Buchem disease charac-
terized by a symmetrical high bone mass phenotype, gen-
erally by increased bone formation markers [35] and
caused by deletions downstream of the SOST gene [36].
As opposed to this pathological inhibition of SOST expres-
sion, sclerostin suppression occurs every day when PTH is
released and binds to osteocyte PTH/PTHrP receptors
(PPR). This circadian PTH pulse is imitated by the daily
injections of PTH [1–34] or PTH [1–84] that are used to
treat osteoporosis.
Dickkopf-1 (Dkk-1) is another potent, soluble inhibitor
of canonical Wnt signaling and bone formation, which is
highly expressed in osteocytes. Dkk-1 binds LRP5/6 and a
transmembrane protein, Kremen [37] (Fig. 5). The tertiary
complex that is formed is internalized, removing LRP5
from the cell surface and therefore preventing Wnt sig-
naling [38].
Wnt Signaling
The Wnts are a family of secreted glycoprotein growth
factors named after the WNT gene of Drosophila melano-
gaster that results in a wingless mutation, and the
integration 1 (INT) gene, which is activated in some mouse
Clinic Rev Bone Miner Metab
Fig. 6 The Wnt signaling pathway: Frizzled receptor protein (FRP),
low density lipoprotein-receptor-related protein (LRP), dishevelled
(Dvl), the ‘‘destruction complex’’ comprising Axin, adenomatous
polyposis coli (APC), glycogen synthase kinase (GSK), lymphoid
enhancer binding factor (LEF). Dickkopf-1 (Dkk-1) removes LRP
from the cell surface
tumors. Wnts bind to cell surface Frizzled receptor proteins
(FRP) and members of the transmembrane low density
lipoprotein-receptor-related protein (LRP) family, LRP5/6
[39] and LRP4 [40] (Fig. 6). Binding activates Dishevelled
(Dvl), with the presence of b arrestin required for full
activation [41]. Dvl activation inhibits a b-catenin
‘‘destruction complex’’ that would otherwise promote the
phosphorylation and proteosomal destruction of cytoplas-
mic b-catenin [42]. This destruction complex consists of
Axin and the proteins adenomatous polyposis coli (APC)
and glycogen synthase kinase-3 (GSK-3). When the com-
plex is inactivated, b-catenin is released, its levels rise, and
it is translocated to the nucleus. There it binds to T-cell
factor/lymphoid enhancer binding factor 1 (TCF/LEF1), a
transcriptional effector that increases OPG production and
promotes the expression of other target genes [43]. It now
appears that other potent regulators of osteoblast differen-
tiation such as BMP-2, Wnt3a and Runx2 may also influ-
ence osteoblast differentiation by affecting the balance
between LEF1 and alternative LEF1 isoforms that have
lower affinity for binding of b-catenin [44]. As osteoblasts
differentiate due to activation of the Wnt signaling path-
way, they produce progressively less RANKL and
increasingly more OPG [45]. Hence, in addition to stimu-
lating osteoblast activity, activation of the Wnt signaling
cascade alters the balance of OPG to RANKL, resulting in
a reduction of osteoclastic bone resorption.
Clearly many molecules influence Wnt/b-catenin sig-
naling, so that the activity of this pathway can be thought
of as a series of molecular switches that determine whether
mesenchymal precursors commit to differentiation along
the osteoblast lineage. Wnts inhibit mesenchymal precur-
sors from developing into adipocytes by blocking the
master adipogenic transcription factors [46] and the dif-
ferentiation of MSCs along the chondrocyte lineage [47].
Adaptations to CKD and Development of the Mineral
and Bone Disorder
The Intact Nephron Hypothesis
With this background in bone biology, we can now focus
on renal adaptations to CKD, described by Bricker et al. [1]
in 1960 (as the intact nephron hypothesis) and further
developed in 1972 as the trade-off hypothesis [48]. Both
hypotheses sought to explain the changes that occur as
glomerular filtration rate (GFR) falls and adaptive mecha-
nisms are triggered to maintain mineral homeostasis. The
authors saw ‘‘a common denominator’’ in the response of
disparate forms of renal disease to reductions in GFR. This
resulted in an increased demand on fewer residual neph-
rons to excrete increased quantities of solute; a concept that
relies upon most remaining nephrons being functionally
intact. To maintain homeostasis as GFR declines, sub-
stances such as creatinine that are filtered with little tubular
handling remain in balance because rising plasma levels
increase their filtration (and excretion) by each remaining
nephron. However, potassium, calcium and phosphate
undergo tubular handling (and would kill the patient if their
levels rose to maintain balance like creatinine does) and for
these solutes, balance is maintained by each intact nephron
excreting a greater fraction of its filtered load. For example,
while 10% of phosphate is excreted at normal GFR, 90%
needs to be excreted at lower levels of GFR to maintain
homeostasis. On the other hand, this adaptation is unnec-
essary if a reduction in GFR is accompanied by a simul-
taneous restriction of dietary phosphate [49].
Osteocytes Control Mineral Homeostasis and Adaptations
to CKD-MBD in Bone and Kidney
As mentioned earlier, by controlling BMU recruitment and
remodeling cycles, osteocytes and osteoblasts have an
ability to modulate homeostasis of calcium and phosphate.
But the enormous lacunar-canalicular network, which is
estimated to have a surface area more than 100-times that
of the trabecular bone surface [50], also provides osteo-
cytes an opportunity to influence ion levels by direct
interaction with their adjacent bony matrix [51]. This
process is termed ‘‘osteocytic osteolysis’’ and involves
osteocytic production of RANKL, downregulation of
OPG and promotion of osteoclast formation and activity
[16, 17].
123

Osteocytes also influence systemic control of calcium,
phosphate and 1,25(OH)2D levels, through production of
fibroblast growth factor-23 (FGF23), which is a member of
the FGF ligand superfamily. FGF23 is a 251 amino acid
glycoprotein that undergoes post-translational modification
before being secreted as FGF23 (25-251). FGF23 gene
expression is upregulated by serum phosphate [52],
although whether this is a direct effect in healthy individ-
uals or those with CKD is still unclear, because a number
of studies have reported that FGF23 levels do not rise
acutely in response to phosphate loading [53–55]. As
opposed to the evidence for phosphate, there is good evi-
dence that FGF23 gene expression and FGF23 levels are
directly upregulated by 1,25(OJH)2D acting through oste-
ocyte/osteoblast vitamin D receptors (VDR) [56, 57] and
that PTH also increases FGF23 directly, in addition to
indirectly by conversion of 25(OH)D to 1,25(OH)2D [58,
59] (Fig. 7). Increased serum phosphate levels also stim-
ulate the post-translational O-glycosylation of FGF23,
which protects it from cleavage and increases its stability
[60]; the enzyme involved being coded by the polypeptide
N-acetylgalactosaminyltransferase 3 (GALNT3) gene.
Expression of FGF23 is suppressed by both the phosphate-
regulating gene with homologies to endopeptidases on the
X chromosome (PHEX) and dentin matrix protein 1
(DMP1), most likely by indirect mechanisms [61].
FGF23 together with FGF15 and FGF21 are designated
‘‘hormone-like’’ FGFs [62]. They differ from nonhormonal
Fig. 7 Serum phosphate, 1,25(OH)2D and PTH upregulate osteocyte
FGF23 gene expression. In turn, FGF23 reduces levels of phosphate
and 1,25(OH)2D by downregulating renal sodium-dependent phos-
phate co-transporter 2a (NPT 2a) expression and 1-alpha hydroxylase
activity, while upregulating 24-hydroxylase activity. FGF23 reduces
PTH by binding to the FGF receptor (FGFR)-Klotho complex.
Additionally, FGF23 increases parathyroid 1-alpha hydroxylase
activity and thereby local 1,25(OH)2D levels. Renal—parathyroid
pathways are indicated by the outer arrows
123
Clinic Rev Bone Miner Metab
FGFs by having a low affinity for heparan sulfate (HS),
which allows them to escape from HS-rich extracellular
matrices and to circulate systemically. However, it also
reduces their ability to bind to FGF receptors, because
efficient binding relies upon the affinity of FGFs for HS.
FGF23 therefore requires a separate cofactor in order to
bind to the FGF23 receptor 1 (FGFR1) involved with
phosphate control or FGFR3/FGFR4 involved with vitamin
D activation [63]. This binding factor was identified as
Klotho, a 130-kDa single-pass transmembrane protein
named after the 1st of the Moirae, who were the three
Greek goddesses controlling man’s fate. Klotho spun the
thread of life, so it was highly appropriate to assign her
name to a transgenic mouse gene that conferred longevity
above the wild type when over-expressed, but upon dele-
tion resulted in premature aging and a shortened life span
[64]. When it was realized that Klotho and FGF knockout
mice shared identical phenotypes, Klotho was discovered
to be the co-receptor for FGF23, compensating for
FGF23’s low HS binding affinity [65]–and emphasizing the
importance of phosphate homeostasis to survival!
In response to an increase in serum phosphate levels,
FGF23 is secreted and binds to the FGFR-Klotho complex
at a number of sites to restore phosphate homeostasis. In
the proximal tubule, FGF23 acts as a ‘‘phosphatonin’’ by
reducing the expression of proximal tubular sodium-
dependent phosphate co-transporter (NPT) 2a and increases
the fractional excretion of phosphate [66]. Increased
FGF23 also downregulates the activity of 1-alpha
hydroxylase (cytochrome P450 (CYP) 27B1) in the prox-
imal tubule, reducing the renal conversion of 25(OH)D to
1,25(OH) [67]. Additionally, FGF23 increases the activity
of the catabolic 24-hydroxylase enzyme (CYP 24A1),
which enhances the conversion of 1,25(OH)2D to 1,24,25
(OH)3D and reduces the availability of substrate to the
1-alpha hydroxylase enzyme by converting 25(OH)D to
24,25(OH)2D [67] (Fig. 7).
Klotho FGF23 Interactions
Because Klotho is required for hormonal FGF23 binding,
FGF23 activity is targeted to tissues where Klotho
expression occurs. These are predominantly the distal
convoluted tubule, to a lesser extent the proximal tubule,
the parathyroid and the choroid plexus and to minor
degrees other endocrine organs [68]. So, it remains a
conundrum that Klotho expression is principally located in
distal tubular cells, while the phosphaturic target of FGF23
appears to be in the proximal tubule. One explanation is
that despite lower levels of Klotho in the proximal tubule,
FGF23 may still be able to signal through the proximal
FGFR-Klotho complex to regulate phosphate absorption.
Another possibility, made more feasible by the close
Clinic Rev Bone Miner Metab
anatomical proximity of distal and proximal tubules, is that
when FGF23 binds to receptors in the distal tubule a sec-
ond signal is produced that influences proximal tubular
phosphate transport [69, 70]. In addition to targeting the
action of FGF23, Klotho is independently active, being
shed from cellular membranes in the distal tubule and
cleaved to circulate in soluble, lower molecular weight
forms with enzymatic activity [71, 72]. This soluble Klotho
has recently been reported to enter the proximal tubule and
inhibit phosphate transport, independent of FGF23 activity
[73]. Secreted Klotho also co-localize with calcium chan-
nels (transient receptor potential ion channel 5; TRPV5) on
distal tubular plasma membranes, which are responsible for
approximately 15% of overall calcium reabsorption [74].
Klotho stabilizes these channels, or prevents their degra-
dation, and in this way appears responsible for controlling
levels of urinary calcium [75].
Hormonal Adaptations to CKD; FGF, Klotho
and Phosphate Excretion
FGF23 levels rise early in the course of CKD, probably in
response to incremental rises of serum phosphate and
possibly in response to even earlier reductions in Klotho
expression [76]. Initially, the increased plasma FGF23 is an
appropriate, compensatory response and the resultant
increase in fractional excretion of phosphate can maintain
serum phosphate levels within the normal range through to
CKD stage 3 or 4 in up to 90% of patients [77]. However,
even extreme FGF23 levels will not prevent hyperphos-
phatemia if nephron loss continues. At this point, FGF23
levels may equal or exceed those seen in conditions of
severe FGF-23 excess such as hypophosphatemic rickets
and tumor-induced osteomalacia (TIO) [78] (Fig. 8) and
have been associated with increased mortality and cardio-
vascular disease, although a causative relationship remains
to be established [79]. This rise in FGF23 also downreg-
ulates production while increasing catabolism of
1,25(OH)2D, and initially this might also be regarded as
appropriate. Lower 1,25(OH)2D levels reduce dietary
phosphate absorption and de-repress PTH production,
increasing phosphaturia, but the risk for this adaptation is
that over time severe hyperparathyroidism may ensue.
Fig. 8 Levels of FGF23 in
CKD greatly exceed those seen
in most other conditions of
FGF23 excess. Reproduced with
permission from [78]
FGF23, Klotho and PTH in Health
An interesting recent finding highlights the interdepen-
dence of FGF23, Klotho and PTH. In a rat model, renal
Klotho expression was reduced in the absence of PTH,
making the proximal tubule refractory to even high levels
of FGF23 [126]. However, when the animals received PTH
supplementation, renal Klotho levels were restored and
FGF23 could induce phosphaturia and normalize serum
phosphate. These experiments demonstrate ‘‘two-way reg-
ulation’’ between FGF23 and PTH when renal function is
normal, with PTH stimulating renal Klotho and osteocyte
FGF23 expression. Parathyroid glands express both FGFR1
and Klotho and as demonstrated in experimental animal
studies, the feedback loop is completed by the rise in
FGF23 binding to the FGFR-Klotho complex and sup-
pressing PTH [80]. In addition to this direct feedback,
FGF23 may also suppress PTH release by stimulation of
parathyroid 1-a hydroxylase activity, increasing the con-
version of 25(OH)D to 1,25(OH)2D to in turn suppress
PTH [81] (Fig. 7).
Changes with CKD
Despite these elegant feedback loops, as renal function
declines FGF23 fails to inhibit PTH (possibly due to
reduced parathyroid Klotho-FGFR1) or to maintain renal
phosphate excretion [82], a circumstance not dissimilar to
FGF or Klotho knockout mouse models, except that these
animals retain the capacity to produce calcitriol. Their
phenotypes bear a striking resemblance to that of severe
CKD, comprising hyperphosphatemia, growth retardation,
skin and muscle atrophy, hypogonadism, vascular and soft
tissue calcification, reduced bone density, premature aging
and a reduced lifespan [83].
PTH Responses to Altered Mineral Metabolism
In 1849 and 1850, Sir Richard Owen dissected a Great
Indian Rhinoceros, which till its death had been the prop-
erty of the London Zoological Society. He discovered the
Glands of Owen, later named the parathyroids and by 1925
the role of PTH in calcium regulation had been recognized
123

[84]. However, the intricate mechanisms by which calcium,
phosphate, 1,25(OH)2D, FGF23 and Klotho influence PTH
transcription and/or post-transcriptional regulation con-
tinue to be illuminated.
Fluctuations in ionized calcium rapidly alter PTH
secretion, keeping ionized calcium levels within a narrow
range. Reduction of extracellular calcium levels below the
‘‘calcium set point’’; the calcium level corresponding to the
midpoint of the steep, reverse sigmoidal PTH response
curve, activates calcium sensing receptor (CaR) signaling
via an inhibitory G-coupled protein to release preformed
PTH from stored secretory granules. On the other hand,
PTH secretion is suppressed when elevated levels of
extracellular calcium acting through an alternative G-cou-
pled protein cause the release of calcium from endoplasmic
reticulum.
Even when calcium levels are strikingly elevated, PTH
secretion cannot be completely suppressed. However, the
parathyroid can respond to elevation of calcium levels by
increasing the degradation of preformed PTH to PTH
fragments and constituent amino acids within parathyroid
cells. This results in the secretion of a higher proportion of
PTH carboxyl-terminal fragments, which may lack the
calcaemic activity of PTH 1-84. The best characterized of
these is PTH [7–84], which appears to act as an antagonist
of the PPR and to exert biological effects through a novel
c-terminal receptor expressed solely by osteocytes [15, 85].
This receptor is possibly part of a larger receptor complex
and may require levels of PTH above the normal upper
range for activity.
Transcriptional Regulation of PTH
In contrast to FGF23, PTH increases renal Cyp27B1
activity and the conversion of 25(OH)D to 1,25(OH)2D
(Fig. 7). This provides direct negative feedback by down-
regulation of PTH mRNA transcription, as well as indirect
feedback by activation of osteocyte FGF23 release.
Increased levels of 1,25(OH)2D also increase CaR
expression, sensitizing parathyroid cells to inhibition of
PTH secretion by calcium [86]. Compared to the rapid
effects of calcium on PTH secretion, these transcriptional
pathways influence PTH over hours, with much longer
periods required for changes to occur in cellular
proliferation.
Post-Transcriptional PTH Regulation
Phosphate levels directly influence PTH (Fig. 7) by alter-
ing the stability of PTH mRNA [87], with a rise in the
serum phosphate increasing PTH mRNA stability and a
reduction in dietary phosphate reducing PTH mRNA sta-
bility [88]. These changes occur independently of calcium
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Clinic Rev Bone Miner Metab
or vitamin D levels and the underlying mechanisms have
recently been clarified. Adenine uridine-rich binding factor
1 (AUF1) is the protein that stabilizes PTH mRNA while
the destabilizing protein K-homology-type splicing regu-
latory protein (KSRP) recruits degradation machinery that
reduces PTH mRNA stability [89]. These parathyroid
proteins bind to the 3’ untranslated region (UTR) of PTH
mRNA. The KSRP destabilizing protein is in turn con-
trolled by the peptidyl-prolyl cis/trans isomerase Pin1. In
hypocalcaemia and experimental CKD, Pin1 activity is
reduced, resulting in KSRP phosphorylation and inactiva-
tion. This allows increased binding of the stabilizing pro-
tein AUF1 to PTH mRNA. On the other hand, in phosphate
depletion experiments KSRP activity is increased, leading
to a reduction in AUF1 binding and to reduced PTH
mRNA stability [90].
Parathyroid Hyperplasia
As secondary hyperparathyroidism develops, parathyroid
cells undergo mitosis causing parathyroid enlargement.
This hyperplasia is a consequence of hypocalcaemia,
hyperphosphatemia and reduced levels of 1,25-di-
hydroxyvitamin D [91] and can be prevented by phosphate
restriction or therapy with 1,25(OH)2D [87, 92].
1,25(OH)2D exerts its anti-proliferative effects through
induction of p21 gene expression [93]. Similarly, reducing
dietary phosphate induces p21, independent of changes in
1,25(OH)2D [94]. On the other hand, increased cellular
proliferation cannot be ascribed solely to downregulation
of p21 expression.
A further mechanism has recently been identified.
Reduced levels of 1,25(OH)2D or high levels of dietary
phosphate induce translocation to the cell surface of a
tumor necrosis factor-a-converting enzyme (TACE), which
results in release of the growth promoter TGF-a [95, 96]. In
turn, TGF-a binds to and activates the epidermal growth
factor receptor (EGFR). Activated EGFR then translocates
to the nucleus, where it functions as an autocrine signal for
cell growth. With phosphate restriction parathyroid TGF-a
returns to normal levels rapidly, which suggests that low-
ering dietary phosphate may counteract uremia-induced
parathyroid hyperplasia by preventing parathyroid TGF-a
enhancement in addition to inducing expression of p21.
In order to impede the development of secondary
hyperparathyroidism and parathyroid hyperplasia, treat-
ment with calcitriol or its analogs is often initiated in the in
the early stages of CKD. Given that osteocytes respond to
1,25(OH)2D by increasing the secretion of FGF23, this
approach has been questioned [56]. Nevertheless, some
studies suggest that the additional effect of calcitriol ther-
apy on already elevated FGF23 levels may be relatively
modest [97]. Moreover, treatment with calcitriol or its
Clinic Rev Bone Miner Metab
analogs has been associated with reduced or stable mor-
tality [98, 99] and in animal studies, with increased renal
expression of Klotho [100]. Optimal treatment should in
fact be low dose or ‘physiological’ replacement, aimed to
avoid hypercalcemia, hyperphosphatemia and the pro-
gression of vascular calcification that can be aggravated by
aggressive therapy. We should also avoid overzealous
suppression of PTH levels toward the ‘‘normal’’ range in
patients with CKD stages 5 or 5D, which can result in
exposure of a low bone turnover phenotype [101].
PTH and Stem Cell Niches
Mesenchymal and hematopoietic stem cells survive and
regenerate in a complex and protected bone micro-envi-
ronment, influenced by surrounding cells including osteo-
blasts, osteocytes and sympathetic neurones [102, 103].
PTH, prostaglandins and beta-adrenergic stimuli all appear
to play important roles by signaling through osteoblast
lineage cells to maintain stem cell survival and activity
[104], osteogenesis and erythropoiesis. PTH has been
shown to increase the proliferation rate of mesenchymal
stem cells (MSC) with a diminution of senescence and
apoptosis [105], a putative protective action thought to
reduce MSC stress and preserve genome integrity. This
ability was highlighted in mice following lethal irradiation
and marrow transplantation; animals treated with PTH
survived, while those treated with mock injection had less
than 50% survival at 10 days [106].
Low bone turnover is a common form of renal osteo-
dystrophy estimated to be present in over 60% of white
patients on dialysis [107]. It has been suggested that when
Wnt signaling and osteoblast activity is inhibited early in
CKD (for which low turnover, ‘‘hypodynamic’’ or ady-
namic bone might be the histomorphometric phenotype),
the development of secondary hyperparathyroidism may be
a compensatory mechanism to maintain or ‘‘rescue’’ stem
cell niches [108]. Of course, with progressive CKD such
‘‘compensatory’’ PTH responses becomes inappropriate
and sustained elevation of PTH can result in bone changes
of hyperparathyroidism including the proliferation of
fibroblast-like cells in the bone marrow, termed osteitis
fibrosa. These cells are now considered to be pre-osteo-
blasts that have proliferated in the presence of elevated
PTH levels and will fail to mature into osteoblasts unless
normal PTH levels are restored [109].
Understanding changes that may lead to reduced
osteoblast activity and bone turnover in early CKD is still
in the developmental stage. However, abnormalities of
bone morphogenic protein (BMP) levels and of Wnt sig-
naling are likely contributors. BMP-7 is an important
promoter of osteoblast differentiation that is produced
primarily by the kidneys, so that levels are reduced in CKD
[110]. BMP-7 has been used successfully to treat the
adynamic bone disorder that developed in adult mice after
the induction of renal impairment [111]. Because BMP-7
upregulates the expression of osteoblast PTH receptors,
higher levels of PTH may be required for regulation of
stem cell niches when levels of BMP-7 are reduced by
renal damage [108]. Alterations to Wnt signaling and levels
of Wnt inhibitory proteins have also been recently descri-
bed in animal models of CKD [112, 113] and in patients
undergoing dialysis [114]. In studies using the Jck mouse
model of polycystic kidney disease, an early rise in FGF23
was reported, with a later rise in levels of PTH, phosphate
and the expression of genes associated with osteoblast
function [112]. The authors proposed that an early increase
in SOST expression reduced Wnt signaling and conse-
quently the OPG to RANKL ratio. In turn, this would
increase osteoclast activity and predispose to osteitis fib-
rosa, which was the pattern described on bone biopsy.
A later rise in PTH levels sustained high bone turnover and
reduced SOST gene expression, but elevated levels of
secreted frizzled related protein 4 (sFRP4) were considered
sufficient to maintain suppression of Wnt and osteoblast
gene expression. In a rat model of renal failure, PTH and
phosphate levels were elevated after 20 weeks and the Wnt
inhibiting proteins sFRP and Dkk-1 were over-expressed
[113]. In a recent report, which included 630 bone biopsies
from patients undergoing dialysis, 62% of white and 32%
of black subjects were diagnosed to have low bone turn-
over [107]. In 60 patients, sclerostin levels were assessed
and found to be approximately 5 times greater than pre-
menopausal women and twice those of post menopausal
women [114]. After adjustment for age, gender, dialysis
duration, diabetes and treatment with vitamin D, sclerostin
levels correlated negatively with activation frequency
(p \ 0.003) and bone formation rate/bone surface
(p = 0.007). However, there were no associations between
levels of Dkk-1 and these bone parameters.
Whether low (or absent) bone remodeling increases
bone micro damage and fracture risk remains the subject of
a longstanding debate, as does the use of the term ‘‘ady-
namic bone disease’’ to describe this condition [115]. At
the other extreme, poorly mineralized and/or high turnover
‘‘woven’’ bone undoubtedly reduces bone’s structural
integrity. However, the extremes of turnover have impli-
cations for more than bone health. Bone’s exchangeable
calcium pool is located at least in part in the hydrated
osteoid covering the bone surfaces [9]. With low turnover,
quiescent, mineralized bone surfaces increase at the
expense of osteoid, so that the surface available for calcium
exchange diminishes. In the context of CKD and a reduced
renal excretory capacity for calcium, loss of this bone
‘‘sink’’ for calcium sequestration may increase the risk of
vascular calcification, particularly when calcium balance is
123

positive. High bone turnover with a mismatch of formation
to resorption may also increase the net efflux of calcium
and phosphate from bone and similarly predispose to vas-
cular calcification.
Markers of Early CKD
Because rises in FGF23 occur as early as CKD stage 1 and
2 when the estimated GFR is still 60–90 ml/min per
1.73 m2 [116, 117] (Fig. 8), FGF23 measurement may
provide a sensitive marker of early disturbances in phos-
phate metabolism, progressive kidney failure and risk of
mortality. This could be used as a guide to commencing
dietary phosphate restriction or other interventions to
reduce dietary phosphate absorption, and to commencing
other measures aimed to reduce progression of kidney
damage [118]. As well as being a prognostic marker, the
rise in levels of FGF23 that occurs with progressive CKD
may be of independent clinical significance, having been
associated with disorders of myocardial mass and perfor-
mance, increased risk of vascular calcification, CKD pro-
gression and mortality [119–121]. Urinary levels of
secreted Klotho might also be utilized for the detection of
nascent renal damage, because declines have been reported
as early as CKD stage 1 [122, 123]. Intervention studies are
currently underway, which will help to define the utility of
estimating and treating these early markers by dietary and
other means [124]. It is hoped these strategies might reduce
CKD progression and complications.
Summary
The development of CKD produces compensatory altera-
tions to bone and mineral metabolism that progress as renal
function declines, often resulting in end-organ damage.
The complexity of these interactions is only now being
appreciated, with intricate feedback pathways demon-
strated between bone and the kidneys, parathyroids, the
brain and sympathetic nervous system, the pancreas, gas-
trointestinal tract, adipose tissues and of course the vas-
cular tree. Many factors in addition to those highlighted
here influence the balance between these systems, ranging
from reactive oxygen species to bone marrow temperature
regulation by brown fat! All of these are likely to be
influenced by the development of CKD.
The response of bone turnover to these physiological
changes forms a continuum, ranging from adynamic or
hypodynamic, with implications for stem cell niches,
hematopoiesis and osteogenesis, to severe hyperparathy-
roidism and osteitis fibrosa. I have described some of the
contributing mechanisms, such as altered Wnt signaling
and reduction in levels of BMP-7, but have not addressed
123
Clinic Rev Bone Miner Metab
others, such as associations between low turnover and
diabetes that may be mediated by advanced glycation end-
products, neurological or circulatory effects. Together with
other articles in this edition, I hope this ‘‘nephrologists
guide to bone biology’’ might highlight the beautiful
complexity of this fascinating area for clinical and labo-
ratory research. Certainly, it is an area that seems to
acquire momentum every day.
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