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5 Vitamin D Maintenance of Skeletal and Vascular Integrity Unrelated to the Attenuation of Secondary Hyperparathyroidism
insufficiency and low VDR in bone cells contributes to the wide range of bone disorders in CKD.
Initial studies in the VDR null mouse suggested that VDR was dispensable for
the ossification process, as non-visible abnormalities in bone mineral density were
observed if there was an adequate supply of calcium and phosphorus by the so
called rescue diet . However, comprehensive studies in the VDR null,
1α-hydroxylase null, and PTH null mice and multiple double knock-out combinations demonstrated calcitriol essential actions for a healthy bone. These include the
induction of osteoblastogenesis, skeletal anabolism and the appropriate coupling of
osteoblastic and osteoclastic activity [69, 70]. Indeed, the calcitriol/VDR complex
regulates the expression of genes that control both bone formation, mineralization
and remodeling (osteopontin, osteocalcin and, importantly, the Wnt receptor LRP5)
as well as osteoclastogenesis and bone resorption genes (RANK ligand and osteoprotegerin) through classical genomic actions (Reviewed in ).
In health, the prevalence of bone formation over resorption depends upon the
physiological or supraphysiological concentrations of serum calcitriol. Also, comparison of calcitriol and paricalcitol actions in bone, in mouse and rat CKD models,
have demonstrated that despite differences in osteoclastogenic potency, both compounds similarly maintained bone anabolism [71, 72]. Indeed, not only high calcitriol potently induces RANK ligand (RANKL) but it also suppresses the expression
of its decoy receptor osteoprotegerin (OPG) to amplify resorptive signals. In CKD,
defective calcitriol/VDR induction of osteopontin could not only adversely impact
ossification and remodeling but also osteoclast recruitment to resorb ectopic bone
(reviewed in ). Similarly, impaired induction of osteocalcin could negatively
affect bone strength and energy metabolism through osteocalcin-mediated insulin
release . Calcitriol actions in cells of the osteoblastic lineage also depend on
their stage of differentiation being anabolic and anticatabolic in more mature cells,
as demonstrated by overexpression of the VDR in mature osteoblasts in vivo .
The conflicts regarding net calcitriol actions in bone may result in part from the
coexistence of all of these distinct cell maturation stages.
The VDR also induces the LRP5 gene, involved in Wnt pathway activation, a
process critical for skeletal development and mineralization. Although LRP5 induction by the VDR appears to occur regardless of calcitriol binding, intracellular calcitriol levels determine cytosolic VDR content, as calcitriol binding protects the
VDR from proteosomal degradation .
CKD-induced defects in Wnt signaling in osteocytes and osteoblasts have been
the focus of intensive research due to the progressive accumulation of the Wnt
inhibitors sclerostin and Dkk1 with CKD progression  and, more significantly,
because of the strong association between impaired Wnt activity, bone loss and
increased vascular calcification .
Importantly, in CKD, impaired Wnt activation in bone occurs before elevations
in serum PTH, as demonstrated in a mouse model of polycystic kidney disease .
The early increases in bone sclerostin causing bone loss in these mice could be
prevented with an antibody against TGFβ , the most abundant cytokine in bone.
This suggests an early onset of CKD-induced increases in TGFβ signaling for Wnt
3 Molecular Biology of Vitamin D: Genomic and Nongenomic Actions of Vitamin D
inhibition. Studies in 7/8 nephrectomized (NX) rats fed either normal or high phosphorus demonstrated similar bone sclerostin levels at week 8 after NX despite
marked differences in serum P, PTH, FGF23 and renal damage between dietary
groups, thus corroborating the independence of this early increase in bone sclerostin
of the severity of SHPT . Furthermore, in both mouse and rat CKD models,
bone sclerostin decreased below the level of sham operated controls as PTH and
FGF23 increased, while elevations in bone levels of several Wnt inhibitors other
than sclerostin, including Dkk1, parallel the progressive loss of bone mass [77–79].
Accordingly, bone biopsies in CKD patients corroborated Wnt inhibition despite the
lower number of osteocytes positive for sclerostin ). Undoubtedly, these findings challenge the accuracy of serum sclerostin to reflect the degree of bone Wnt
inhibition or even sclerostin levels in bone. More importantly, they provide a previously unrecognized mechanism for the abnormalities in the vitamin D endocrine
system in CKD to affect the bone-vasculature axis from early CKD stages: Impaired
activation of Wnt signals in bone. Indeed, even disregarding the induction of LRP5
by unliganded VDR, a normal vitamin D status could attenuate the adverse TGFβ/
Smad signaling on bone. In fact, VDR signaling antagonizes a range ofTGFβ/Smaddependent transcriptional activation of profibrotic genes through the recruitment of
VDR to loci on these genes that prevent/attenuate Smad3 binding .
Importantly, vitamin D regulation of Wnt signaling is tissue specific. In contrast
to bone, in the kidney and the vasculature, vitamin D inhibits Wnt signals [81, 82]
through VDR binding to β-catenin in the cytosol to prevent its translocation to the
nucleus  thereby attenuating the adverse impact on Wnt activation on the progression of renal damage and vascular calcification.
In addition, calcitriol/VDR transactivation of the FGF23 gene in osteocytes and
osteoblasts is an essential pro-survival action, as the dominant role of FGF23 is the
renal elimination of phosphorus to prevent hyperphosphatemia and its pro-aging
consequences. Indeed, the main features of the FGF23-null mouse are hyperphosphatemia, high circulating calcitriol, ectopic calcifications, premature aging, arteriosclerosis, osteoporosis , a phenotype that can be rescued by dietary
phosphorus restriction [84–86]. Furthermore, double knockouts of FGF23 and
either the VDR  or CYP27B1 also rescue the adverse pro-aging features of
the FGF23 null mice by preventing hyperphosphatemia. Since FGF23 suppresses
CYP27B1 and induces CYP24A1 in the kidney [83, 89], it is clear that the capability of FGF23 to simultaneously get rid of excessive phosphorus while tightly preventing elevations in serum calcitriol is essential for its pro-survival effects.
However, although renal mRNA levels for CYP27B1 are reduced, 25(OH)D supplementation to hemodialysis patients can normalize serum calcitriol . However,
the contribution of extrarenal calcitriol production cannot be fully disregarded as
FGF23 increases rather than decrease parathyroid CYP27B1 expression .
Similarly, despite the increased CYP24A1 mRNA, serum levels of
24,25-dihydroxyvitamin D in non-supplemented or supplemented patients were
persistently lower than normal [92, 93] Clearly, in advanced CKD, the activity of
either enzyme fails to reflect FGF23 control of the respective genes, that is, the damaged kidney fails to respond to FGF23 tight control of renal calcitriol production.
There are several putative VDREs for VDR/RXR binding in the FGF23 promoter. Interestingly, FGF23 gene transactivation by calcitriol decreases from
80-fold to four-fold in the presence of inhibitors of new protein synthesis indicating that osteoblasts’ full response to calcitriol induction of the FGF23 gene is
The low levels of FGF23 in the VDR null mice and CYP27B1 mice  suggest
that impaired induction of FGF23 during vitamin D deficiency could contribute to
accelerate pro-aging features and mortality. Furthermore, although phosphorus,
PTH, calcium and the calcium X phosphorus product are recognized stimulators of
circulating FGF23 levels [95–97], in vitro studies have demonstrated that only calcitriol regulates FGF23 gene transcription . Importantly, calcitriol fails to transactivate the FGF23 gene if high calcitriol and hypophosphatemia occur
simultaneously, as demonstrated in a transgenic mouse with an ablation in the gene
for the phosphorus transporter NPT2a . This supports the prevalent role of phosphorus over calcitriol in the upregulation of FGF23. The COSMOS study has also
reported the optimal range for serum phosphorus associated to the lowest risk of
mortality in CKD-5D patients and the benefits of correcting serum phosphorus to
achieve optimal range .
Because FGF23 requires membrane klotho as a co-receptor for its phosphaturic
actions , and calcitriol also induces the klotho gene , it is clear that the
maintenance of a normal bone-kidney FGF23/klotho axis is crucial for survival. The
next sections examine calcitriol control of renal klotho and its abnormalities in
Vitamin D Induction of Bone FGF23 Production
and Renal Klotho Content to Prevent/Attenuate
For decades, the most critical action of the calcitriol/VDR complex in the kidney
has been the induction of CYP24A1 to maintain serum calcitriol within normal
limits by degrading excessive circulating calcitriol and/or 25(OH)D to prevent
hypercalcemia and hyperphosphatemia. Accordingly, CYP24A1 has a 25-fold
higher affinity for calcitriol than for 25(OH)D. Induction of CYP24A1 is a classical
genomic action of the calcitriol/VDR complex mainly on two proximal VDREs on
this gene promoter . The pathophysiological relevance of calcitriol induction of
CYP24A1 in almost every vitamin D responsive tissue was conclusively demonstrated by the severe hypercalcemia and nephrocalcinosis of the CYP24A1 null
mouse  and in children and adults with a loss of function mutation of this gene
At present, calcitriol/VDR induction of the mRNA levels of the longevity gene
α-klotho, and the identification of a VDRE in the human klotho promoter 
provide a potential causal link for the epidemiological association between vitamin
3 Molecular Biology of Vitamin D: Genomic and Nongenomic Actions of Vitamin D
D deficiency and higher risk of all-cause mortality in the general population, a risk
markedly aggravated in CKD patients. Indeed, while klotho disruption confers a
premature aging like syndrome , its overexpression is sufficient to extend lifespan in mice .
Klotho is expressed in the kidney, the parathyroid gland and the choroid plexus
 where it acts as a high affinity receptor for circulating FGF23. In fact, appropriate levels of renal and parathyroid klotho are required for FGF23 phosphaturic
and PTH suppressive actions, respectively. Therefore, calcitriol induction of renal
klotho should attenuate the pro-aging features of hyperphosphatemia. Accordingly,
progressive reductions of renal klotho in CKD patients stages 3–4 were associated
with an impaired response to FGF23, reduced fractional excretion of phosphate and
with a fourfold higher propensity for abdominal aortic calcification, measured by
the Kaupilla index , thus supporting the potentiation of FGF23 protective
actions in cells harboring klotho.
However, klotho also exists in a soluble form, generated by proteolytic cleavage
of the transmembrane klotho, which is found in blood, urine and cerebrospinal fluid
[108, 109]. FGF23-independent endocrine actions of soluble klotho (sklotho)
include the modulation of the activity of membrane channels, co-transporters and
signaling pathways not fully characterized that contribute to its potent survival benefits. Indeed, the systemic administration of recombinant klotho rescues the phenotype of the klotho null mice , and the renal and cardiovascular damage
associated to acute or chronic renal injury [111–113]. Therefore, maintenance of
renal and/or circulating klotho has become a priority in nephrology and so was the
need of accurate measurements of sklotho as a biomarker of the severity of CKD
and the risk for cardiovascular mortality.
The demonstration that the specific ablation of renal klotho resulted in an 80 %
reduction in circulating sklotho supported the main contribution of the kidney to
serum sklotho . Furthermore, since the phenotypic features of the mouse with a
renal specific klotho ablation recapitulated all of those in the klotho null mice ,
serum sklotho was considered a valuable biomarker of renal klotho content and of
mortality risks. Indeed, serum sklotho decreases with age , hypertension ,
and systemic inflammation , all recognized determinants of renal damage
and cardiovascular disease. However, the recent report that the kidney is the main
organ for the clearance of circulating klotho into the urine, through a process of
transcytosis through tubular cells , raised numerous concerns regarding the
accuracy of circulating sklotho to reflect renal klotho content and its pro-survival
benefits. Indeed, sklotho accumulation in the blood due to an impaired transcytosis by the damage kidney will mask the actual renal klotho reduction. Therefore,
upon improvement of currently available assays for serum klotho, it will be important to establish the optimal range for serum sklotho associated with the lowest
mortality risk in the course of CKD. It will be also important to identify the optimal range for urinary sklotho, as is in the apical site of the renal tubule where
sklotho acts to induce phosphaturia, urinary K excretion and calcium reabsorption
(Reviewed in ).