expression of fibroblast growth factor 23, vitamin d receptor, and sclerostin in bone tissue from...

5/20/2018 Expression of Fibroblast Growth Factor 23, Vitamin D Receptor, and Sclerostin in Bone Tissue from Hypercalciuric Stone Formers

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Article

Expression of Fibroblast Growth Factor 23, Vitamin D

Receptor, and Sclerostin in Bone Tissue fromHypercalciuric Stone FormersViviane Barcellos Menon,* Rosa Maria Affonso Moyses, Samirah Abreu Gomes,* Aluizio Barbosa de Carvalho,*Vanda Jorgetti, and Ita Pfeferman Heilberg*

AbstractBackground and objectivesIncreased bone resorption, low bone formation, and abnormal mineralizationhave been described in stone formers with idiopathic hypercalciuria. It has been previously shown that thereceptor activator of NF-kB ligand mediates bone resorption in idiopathic hypercalciuria (IH). The present studyaimed to determine the expression ofbroblast growth factor 23 (FGF-23), vitamin D receptor (VDR), andsclerostin in bone tissue from IH stone formers.

Design, setting, participants, & measurementsImmunohistochemical analysis was performed in undecalciedbone samples previously obtained for histomorphometry from 30 transiliac bone biopsies of idiopathichypercalciuria stone-forming patients between 1992 and 2002 and 33 healthy individuals (controls). Serumparameters were obtained from their medical records.

ResultsHistomorphometry disclosed 21 IH patients with high and 9 IH patients with normal bone resorption.Importantly, eroded surfaces (ES/BS) from IH patients but not controls were signicantly correlated withVDR immunostaining in osteoblasts (r=0.51;P=0.004), sclerostin immunostaining in osteocytes (r=0.41;P=0.02),and serum 1,25-dihydroxyvitamin D (r=0.55;P,0.01). Of note, both VDR and sclerostin immunostainingwere signicantly correlated with serum 1,25-dihydroxyvitamin D in IH patients (r=0.52;P=0.01 andr=0.53;P=0.02, respectively), althoughVDR and sclerostin expression did not differ between IH and controls. IH patientswith high bone resorption exhibited a signicantly stronger sclerostin immunostaining than IH patients with

normal bone resorption. FGF-23 expression in osteocytes from IH patients did not differ from controls and wasnot correlated with any histomorphometric parameter.

Conclusions These ndings suggest the contribution of VDR and sclerostin, as well as 1,25-dihydroxyvitamin D,to increase bone resorption in idiopathic hypercalciuria but do not implicate FGF-23 in the bone alterations seenin these patients.

Clin J Am Soc Nephrol9: 12631270, 2014. doi: 10.2215/CJN.10030913

IntroductionIdiopathic hypercalciuria (IH) represents a generalizeddysregulation of calcium homeostasis, which includesincreased gut calcium absorption, decreased renal cal-

cium reabsorption, and a tendency to lose calcium fromthe bone (1,2).

Abnormal bone remodeling among hypercalciuricstone formers has been disclosed by histomorphometryand characterized as increased bone resorption, low

bone formation, and a mineralization defect as pre-viously shown by our group (36) and others (7,8). In aprevious study, we found that the receptor activatorof NF-kB ligand (RANKL) mediated bone resorptionin IH patients (6).

Fibroblast growth factor 23 (FGF-23) is producedin bone and acts on kidney as a systemic phospha-turic factor. Although increased serum levels of FGF-

23 were found in hypophosphatemic nephrolithiasis

patients (9), FGF-23 overexpression in cultured cal-varias may suppress bone formation and matrixmineralization irrespective of the phosphate homeo-stasis (10).

Serum 1,25-dihydroxyvitamin D [1,25(OH)2D3] lev-els in IH patients are either normal or high (1114),and vitamin D receptor (VDR) polymorphism has

been associated with hypercalciuria (15). In hypercal-ciuric patients, VDR concentration was normal in -

brobla sts (13) bu t 2-fold higher in PB MCs (1 4),despite normal serum 1,25(OH)2D3, suggesting an al-tered tissue vitamin D response. Genetic hypercalciuricstone-forming (GHS) rats exhibit increased VDR cou-pled with normal levels of 1,25(OH)2D3, resulting inincreased functional VDR1,25(OH)2D3 complexes(16). Finally, increased bone resorption has been ob-served after calcitriol exposure in GHS cultured cal-

varias (17).

*NephrologyDivision, FederalUniversity of SaoPaulo, Sao Paulo,Brazil; andNephrology DivisionUniversity of SaoPaulo, Sao Paulo,Brazil

Correspondence:Prof. Ita PfefermanHeilberg, NephrologyDivision,Universidade Federalde Sao Paulo, RuaBotucatu, 740VilaClementino, SaoPaulo, SP, Brazil04023-900. Email:[email protected]

www.cjasn.org Vol 9 July, 2014 Copyright 2014 by the American Society of Nephrology 126
mailto:[email protected]:[email protected]:[email protected]:[email protected]


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Another important regulator of bone metabolism pro-duced in osteocytes is sclerostin (Scl), which is codied bythe SOST gene and functions as a negative regulator of

bone mass and osteoblast differentiation (18,19), but it alsopromotes osteoclast formation and activity (19).

In the present study, we aimed to evaluate bone expression

of FGF-23, VDR, and Scl in IH stone formers and examinetheir relationship with bone remodeling alterations.

Materials and MethodsIn this retrospective study, undecalcied bone fragments

obtained from transiliac bone biopsies of 30 Caucasianstone-forming IH patients with normal renal function(previously performed from 1992 to 2002) were included.Only those patients with evidence of uni- or bilateral radio-opaque stones consistent with calcium composition wereselected. Exclusion criteria for bone biopsy were hyper-parathyroidism, renal tubular acidosis, sarcoidosis, diabe-tes mellitus, and use of corticosteroids, bisphosphonates,

citrate, or diuretics. IH was dened by normocalcemia and24-hour urinary calcium.250 mg/d (for women) and 300mg/d (for men) or .4 mg/kg per day in two nonconsec-utive samples under unrestricted diet. Histomorphometricparameters, serum levels of calcium, phosphate, creati-nine, 1,25(OH)2D3, and intact parathyroid hormone(PTH), obtained near the date of the bone biopsy wereretrieved from their medical records; 1,25(OH)2D3 had

been determined by radioimmunoassay (Diasorin; Stillwa-ter, MN/Nichols Institute Diagnostics, CA) and PTH bydifferent immunoradiometric assays according to theavailability of methods between 1992 and 2002 (35). TheEthical Committees of both institutions approved this

study, and all patients signed written consent by thetime that the biopsies were performed.

Bone HistomorphometryHistomorphometric parameters had been obtained by

transiliac bone biopsies as previously described (20). Thecontrol group for static parameters encompassed 33 healthyindividuals (18 men and 15 women; 25 Caucasian and 8non-Caucasian; 32.965.4 years) selected from our bone his-tomorphometry database (20) consisting of bone biopsiesobtained immediately after early death from victims of gun-shot or knife wounds, trauma, or trafc accidents who werenot known to have any disease or be users of anticonvulsant

drugs, corticosteroids, or any medication that interferedwith bone metabolism based on their medical records. Dy-namic parameters were compared with the controls from thework by Melsen and Mosekilde (21), which were obtainedfrom 41 volunteers with similar age: 12 men (mean age=32years) and 29 women (mean age=29 years).

ImmunohistochemistryImmunostaining for FGF-23, VDR, and Scl was per-

formed in IH patients and controls according to a techniquepreviously described (22). The primary antibodies usedwere monoclonal anti-human FGF-23 (provided by SusanC. Schiavi, Genzyme, Framingham, MA), rat monoclonalanti-human VDR (ABR-Afnity BioReagents, Inc. Golden,CO), and human monoclonal antibody anti-Scl (R&D Sys-tems, Minneapolis, MN). Counting was performed on 30

microscopicelds at a magnication of3200 for each bonesample. The area of immunopositivity was determined bythe number of positive points in the tissue compared withthe total number of points.

Statistical Analyses

Normalityof distribution was assessed by the KolmogorovSmirnov test. Accordingly, data are expressed as mean6SD

or median (25th, 75th percentiles). Differences betweengroups were analyzed using MannWhitney or unpairedt tests depending on the distribution of variables. Correla-tions between immunohistochemistry and biochemical orhistomorphometric parameters were assessed by Pearsonor Spearman correlation testing as appropriate, and con-dence intervals are presented. The level of signicancewas set up asP,0.05. Prism software version 4.0 (GraphPadSoftware Inc., San Diego, CA) was used for statisticalanalyses.

ResultsIH patients with calcium stones (18 men and 12 pre-

menopausal women; 36.369.3 years) presented mean uri-nary calcium of 340.8670.6 mg/24 h, eGFR of 81.1612.2ml/min per 1.73 m2, and serum creatinine, calcium, andphosphorus of 0.9260.12, 9.660.5, and 3.260.5 mg/dl, re-spectively. Two patients were hypophosphatemic, andtwo patients presented 1,25(OH)2D3 values slightly abovenormal ranges. All had serum PTH within normal limits:median values for intact PTH were 33.5 (21.5, 49.5) and20.3 (12.5, 37.5) pg/ml and median values for 1,25(OH)2D3were 40.7617.1 and 50.1616.1 pg/ml according to themethod used.

Bone HistomorphometryAs shown in Table 1, IH patients presented signicantly

lower mean bone volume (BV/TV) and trabecular number(Tb.N) and signicantly higher trabecular separation (Tb.Sp). Mineralization lag time (MLT) was signicantlyhigher, and mineralizing surface (MS/BS) was signi-cantly lower. Both eroded surfaces (ES/BS) and osteoclastsurfaces (Oc.S/BS) were signicantly higher. ES/BS fromIH patients did signicantly correlate with serum 1,25(OH)2D3 (r=0.55; P,0.01).

Immunohistochemistry

Compared with controls, IH patients did not presentstatistical differences for bone immunostaining of FGF-23in osteocytes (6.865.9% versus 6.065.0%; P=0.49), VDRimmunostaining in osteoblasts (21.4617.8% versus28.7620.1%; P=0.14), VDR in osteocytes (13.869.6% ver-sus 12.068.5%; P=0.52), VDR in the total of both cells(16.4611.5% versus 18.9611.1%; P=0.35); VDR immuno-staining in osteoclasts was negligible, and Scl bone immu-nostaining in osteocytes (9.1611.1% versus 6.165.5%;P=0.22; data not shown in tables).

As seen inFigure 1, mean bone FGF-23 immunostainingin IH patients classied as having high bone resorption (ES/BS=1 SD above mean,n=21) did not differ from normal boneresorption (ES/BS,1 SD, n=9). Mean VDR bone immuno-staining in the sum of all stained cells was numerically butnot statistically higher in patients with high versus normal

1264 Clinical Journal of the American Society of Nephrology
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ES/BS. Mean Scl immunostaining in osteocytes was signif-

icantly stronger in high versus normal bone resorption IHpatients. The median (25th, 75th percentiles) estimated du-ration of disease for patients with high versus normal ES/BS(7.0 [5, 17] versus 2.0 [1.5, 10] years) presented a marginaltrend to signicance (P=0.05; data not shown).

Figure 2 illustrates immunoreactivity localization ofFGF-23 in osteocytes, VDR in osteoblasts, and Scl in osteo-cytes in bone tissue.

Table 2 shows signicant correlations between serum1,25(OH)2D3 and bone expression of VDR in osteoblastsand Scl in osteocytes.

FGF-23 immunostaining did not correlate with any histo-morphometric parameter, which is seen in Table 3, nor in thecontrol group (data not shown). ES/BS signicantly corre-lated with VDR bone immunostaining in osteoblasts and thesum of all stained cells from IH but not in the controls (data

not shown). Tb.N was positively correlated with VDR boneimmunostaining in osteoblasts from IH patients but nega-tively correlated with VDR in osteoblasts and the sum of allcells from controls (r=20.46;P=0.008 andr=20.49;P=0.004,respectively; data not shown). Scl immunostaining in osteo-cytes was signicantly correlated with ES/BS in IH patients

but not the control group. In the control group, Scl immu-nostaining correlated with BV/TV (r=0.47; P,0.01), Tb.N(r=0.48; P,0.01), and Tb.Sp (r=20.56; P,0.001; data notshown).

DiscussionDecreased bone mineral density (BMD) is commonly

encountered in IH patients (23), and an increased preva-lence of bone fractures has been reported for nephrolithia-sis patients (24,25). GHS rats also exhibited reduced BMDand more brittle and fracture-prone bones (26) because oflower trabecular volume and mineralized volume and

thickness. Human histomorphometric studies have showneither normal or low bone formation but a unanimous

bone mineralization defect (3,5,7,8,27,28).The exact pathophysiological underlying mechanisms

for altered bone remodeling in stone formers remainunknown, but increased production of cytokines by pe-ripheral monocytes of hypercalciuric patients has beenreported (29,30). At a bone level, we have previouslyshown a lower expression of TGF-bin bone immunostainsfrom hypercalciuric stone formers (6), which might havecontributed to the lower bone formation and delayed bonemineralization (31).

To address if a putative excess of FGF-23 could nega-tively regulate bone mineralization (10), the rst purposeof the present study was to evaluate its bone expression.Bone FGF-23 from IH patients was not different from

Table 1. Histomorphometric parameters

Variables IH Patients (n=30) Controls (n=33) PValue

StructureBV/TV (%) 18.7867.54 25.4266.52 ,0.001

Tb.N (mm) 1.65 (1.25, 1.84) 1.90 (1.75, 2.30) 0.002Tb.Sp (mm) 481 (394, 676) 359 (319, 439) ,0.001Tb.Th (mm) 125.13630.75 126.58624.41 0.97

FormationOV/BV (%) 1.62 (0.88, 2.82) 1.60 (0.98, 2.75) 0.95OS/BS (%) 10.20 (5.60, 16.98) 9.3 (6.65, 15.60) 0.93Ob.S/BS (%) 1.13 (0.28, 2.92) 0.70 (0.30, 1.56) 0.49MLT (d) 48.40632.20 23.0062.40 ,0.001O.Th (mm) 12.3265.08 10.7562.81 0.42MAR (mm/d) 0.6960.23 0.6560.12 0.40MS/BS (%) 6.4865.13 13.7567.25 ,0.001BFR/BS (mm3 mm2/d) 0.0760.16 0.0960.05 0.52

ResorptionES/BS (%) 7.29 (3.17, 14.00) 2.05 (1.55, 2.90) ,0.001Oc.S/BS (%) 0.30 (0, 0.46) 0 (0, 0.02) ,0.001

Mean6SD or median (25th, 75th percentiles). IH, idiopathic hypercalciuria; BV/TV, bone volume; Tb.N, trabecular number; Tb.Sp,trabecular separation; Tb.Th, trabecular thickness; OV/BV, osteoid volume; OS/BS, osteoid surface; Ob.S/BS, osteoblastic surface;MLT, mineralization lag time; O.Th, osteoid thickness; MAR, mineral apposition rate; MS/BS, mineralizing surface; BFR/BS, boneformation rate; ES/BS, eroded surface; Oc.S/BS, osteoclast surface.

Figure 1. | Bone immunostaining quantification (%) of fibroblastgrowth factor 23 (FGF-23), vitamin D receptor (VDR), andsclerostin(Scl). Idiopathic hypercalciuria patients with high (black bars; n=21)or normal (white bars;n=9) bone resorption defined by eroded surface(*P=0.03 versus white bars). The quantification of immunostaining wasdetermined by the ratio of positive to total number of stained cells inbone tissue.

Clin J Am Soc Nephrol 9: 12631270, July, 2014 FGF-23, VDR, and Sclerostin in Idiopathic Hypercalciuria, Menon et al. 1265
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controls and did not correlate with histomorphometric pa-rameters (nor in the control group). These ndings differfrom the ndings of Pereira et al.(32), who observed bone

FGF-23 expression to be inversely correlated with skeletalmineralization in CKD patients. However, given that renalfunction was not compromised in the present series, the

Figure 2. | Immunohistochemistry for FGF-23, VDR, and Scl in bone tissue (positive immunostaining is shown in red). No differences be-tween thepatterns of FGF-23 immunostaining in osteocytes (arrows) in (A) a patient with high bone resorption, (D) a patient with normal boneresorption, and (G) a control patient are observed. Examples of higher expression of VDR in osteoblasts (arrows) and Scl immunostaining inosteocytes (arrows) from an idiopathic hypercalciuria patient with high boneresorption are given in B and C, respectively, contrastingwith thefewer stained cells in an idiopathic hypercalciuria patient with normal bone resorption (E and F for VDR and Scl, respectively) and a controlindividual (H and I, respectively). Original magnification, 3200.

Table 2. Correlation between bone immunostaining of fibroblast growth factor 23, vitamin D receptor, and sclerostin with serumparameters in IH patients

Parameters FGF-23 (OC)VDR

Scl (OC)

OB OC

sCa 0.14 (20.49 to 0.26) 20.03 (20.42 to 0.36) 20.02 (20.41 to 0.36) 20.28 (20.60 to 0.14)sP 0.28 (20.12 to 0.60) 0.12 (20.30 to 0.50) 0.12 (20.30 to 0.50) 0.12 (20.32 to 0.50)PTH 0.19 (20.55 to 0.24) 20.18 (20.54 to 0.23) 0.15 (20.26 to 0.51) 20.11 (20.51 to 0.29)1,25(OH)2D3 20.33 (20.67 to 0.09) 0.52 (0.13 to 0.77)

a20.02 (20.44 to 0.39) 0.53 (0.16 to 0.79)b

Data are presented asr(95% condence interval). FGF-23, broblast growth factor 23; OC, osteocyte; VDR, vitamin D receptor; Scl,sclerostin; OB, osteoblast; sCa, serum calcium; sP, serum phosphate; PTH, intact parathyroid hormone; 1,25(OH) 2D3, 1,25 dihidrox-yvitamin D3.aP=0.01.bP=0.02.



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Table3.

Correlationsbetweenboneimmuno

stainingofFGF-2

3,

VDR,

andSclwithhistomorphometricparametersin30IH

patients

Parameters

FGF-23(OC)

VDR

Scl(OC)

OB

OC

Total

BV/

TV(%)

20.0

1(20.5

1to0.2

4)

20.0

3(20.4

0to0.3

4)

0.0

4(20.3

3to0.4

0)

20.2

9(20.6

1to0.0

9)

0.09(20.3

1to0.4

3)

Tb.N(mm)

20.0

1(20.8

3to0.2

6)

0.4

4(0.0

3to0.73

)a

20.1

5(20.5

3to0.2

9)

0.1

0(20.2

2to0.6

1)

0.15(20.2

9to0.5

4)

Tb.Sp(mm)

0.1

1(20.2

6to0.5

8)

20.4

2(20.7

2to20

.00)

0.0

5(20.3

8to0.4

6)

20.2

5(20.6

5to0.1

2)

20.18(20.5

6to0.2

6)

Tb.Th(mm)

20.2

0(20.4

6to0.4

5)

0.2

2(20.2

3to0.5

8)

0.1

4(20.3

0to0.5

3)

0.2

7(20.1

6to0.6

3)

20.01(20.4

3to0.4

2)

OV/BV(%)

20.2

2(20.6

0to0.1

1)

0.3

2(20.0

5to0.6

1)

20.0

9(20.4

4to0.2

8)

0.1

7(20.2

3to0.5

0)

20.08(20.4

3to0.2

9)

OS/

BS(%)

20.1

4(20.5

7to0.1

5)

0.1

9(20.1

9to0.5

1)

20.2

0(20.5

2to0.1

8)

0.1

9(20.2

6to0.4

7)

20.18(20.5

0to0.2

0)

Ob.

S/BS(%)

20.2

0(20.5

3to0.1

9)

0.1

8(20.1

9to0.5

1)

20.0

5(20.4

1to0.3

1)

20.0

5(20.2

4to0.4

9)

20.15(20.4

8to0.2

3)

MLT(d)

20.3

0(20.5

6to0.2

1)

0.3

6(20.0

2to0.6

5)

20.1

6(20.5

1to0.2

4)

0.3

7(20.1

5to0.5

7)

0.26(20.3

7to0.3

9)

MS/BS(%)

20.3

5(20.6

7to0.1

4)

0.1

3(20.3

2to0.5

3)

20.2

1(20.5

9to0.2

5)

20.2

8(20.6

6to0.1

4)

20.39(20.5

0to0.2

4)

BFR

/BS(mm

3

mm

2/d)

20.0

8(20.4

5to0.2

9)

0.2

3(20.1

4to0.5

5)

0.0

4(20.3

4to0.4

0)

0.1

1(20.2

9to0.4

6)

20.04(20.3

8to0.3

6)

ES/

BS(%)

20.0

7(20.4

7to0.2

8)

0.5

1(0.1

9to0.74

)b

0.1

5(20.2

2to0.4

8)

0.4

7(0.1

2to0.7

1)c

0.41(0.0

7to0.6

8)d

Oc.S/BS(%)

20.0

8(20.5

2to0.2

2)

20.0

9(20.4

4to0.2

8)

20.0

5(20.4

0to0.3

2)

20.2

2(20.5

0to0.2

0)

20.12(20.4

7to0.2

4)

Dataarepresentedasr(95%condenceinterval).O

C,osteocyte;OB,osteoblast;Total,sumofallstainedcells;BV/TV,

bonevolu

me;Tb.N,trabecularnumber;Tb.Sp,trab

ecularseparation;

Tb.T

h,trabecularthickness;OV/BV,osteoidvolume;OS/BS,osteoidsurface;Ob.S

/BS,osteoblasticsurface;MLT,m

ineralizationlagtime;MS/BS,m

ineralizingsurface;BFR/BS,boneformation

rate;ES/BS,erodedsurface;Oc.S/BS,osteocla

stsurface.

aP=0.0

4.

bP=0.0

04.

cP=0.0

1.

dP=0.0

2.



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comparison may be not suitable. Although a signicantinverse association between serum phosphate and FGF-23 was reported in recurrent stone formers (9), the samplewas not exclusively composed of hypercalciuric individu-als. In our study, there were no signicant correlations

between FGF-23 bone immunostaining and serum phos-

phate or between PTH and vitamin D. The small numberof hypophosphatemic patients in the current series mighthave accounted for the lack of such correlation.

Among the presumed mechanisms responsible for im-paired bone formation in IH patients, elevated serum 1,25(OH)2D3 levels may play a role (1114,33,34) through de-creasing bone collagen synthesis (31) or increasing boneresorption (35). A greater increase in urinary calcium with1,25D in GHS rats fed a low calcium diet has suggested thatthe additional urinary calcium must result from increased

bone resorption because of the increased number of VDR intheir bone cells (35). With a few exceptions (27,28), most ofthe previous histomorphometric studies have shown in-creased bone resorption among hypercalciuric patients

(3,5,7,8). Vitamin D-induced osteoclastogenesis seems tobe accomplishe d through a VDR-mediated increas e inRANKL expression in osteoblasts (36).

In the present study, we did not observe an increase inbone expression of VDR in the total of stained bone cellsfrom IH patients compared with controls. However, whenwe compared the patients who exhibited high versusnormal bone resorption, VDR immunostaining was nu-merically higher but not signicantly different in theformer group (Figure 1). Moreover, a strong and signif-icant correlation between bone expression of VDR andES/BS was disclosed in the IH group as a whole, which wasattributed to the marked VDR staining in osteoblasts, that

was not evidenced in control group. Although we recog-nize that osteoclasts also express VDR (37), the reasonswhy we did not observe substantial VDR immunostainingin these cells remain unclear. When we tried to integratepresent VDR bone expression with that of RANKL, osteo-protegerin, and TGF-b evidenced in our previous study(6), we found no correlation. However, it is noteworthythat, in the current sample, we could not retrieve sufcientamounts of tissue from six bone specimens derived fromthe material in the work by Gomes et al. (6), probably

because of multiple cuts on the microtome and exposureto chemical reagents in prior immunohistochemical anal-ysis. Unfortunately, all these specimens had originated

from patients with high ES/BS, and therefore, it mighthave compromised the disclosure of a possible correlationbetween these parameters. In the present series, serum1,25(OH)2D3 correlated with VDR immunostaining in os-teoblasts from IH patients. These ndings agree well withexperimental data showing a 4-fold increase in the level ofVDR in GHS calvarias versus controls and a greater sen-sitivity to 1,25(OH)2D3 compared with bone from controlrats (17,35). The direct correlation that we have found be-tween VDR and Tb.N in osteoblasts in IH further rein-forces th e contr ibu tion of V DR to in cr eas e boneresorption. Conversely, Tb.N was negatively correlatedwith VDR expression in controls. It has already been spec-ulated that even small elevations of serum 1,25(OH)2D3seem to be sufcient to increase bone resorption whendietary calcium intake is low, indicating that 1,25(OH)2D3

may upregulate its own receptor (14,38). A previous evalu-ation of stone-forming patients in our laboratory (39)revealed a very low mean calcium intake (around 500mg/d), and most of the bone tissue samples presently ana-lyzed had been retrieved from patients with up to 60% ex-hibiting low calcium intake (3,5). In the present study, we

observed a trend for a direct association between VDR boneexpression and mineralization lag time (r=0.36; P=0.06).Therefore, the increase in bone expression of VDR mighthave helped to further compromise bone mineralization inour IH patients, because the low calcium intake probablycontributed to a negative calcium balance. Indeed, an exper-imental study by Lieben et al. (40) has shown that, whenintestinal calcium absorption decreases, calcium mobiliza-tion from bone is elicited and bone matrix mineralizationis suppressed because of decreased calcium incorporationin bone by osteoblastic VDR signaling that stimulates min-eralization inhibitors.

The signaling pathways through which Scl exerts itseffects on bone metabolism are still incompletely under-

stood. Although Scl has emerged as a stimulator ofosteoblasts apoptosis (18) and a potent inhibitor of Wntb-catenin signaling (hence, decreasing bone formation)(41), an experimental model in which the Wnt pathwaywas specically inhibited in osteocytes has shown normal

bone formation but a signicant increase in osteoclasticbone resorption instead (42). The reduction of the antios-teoclastogenic factor osteoprotegerin (OPG) and the con-comitant increase in the osteocytic RANKL/OPG ratiomight have contributed to the increased number of osteo-clasts and resorption in this model. Additional studiesshowing a severe osteopetrotic phenotype in mice lackingRANKL specically in osteocytes (43) reinforced that os-

teocytes are the major source of RANKL in bone remodel-ingin vivo. The mechanism whereby Scl controls osteoclastformation and activity seems to occur through a direct in-crease in mRNA expression of RANKL in osteocytes to agreater extent than the decrease in OPG (19). It has beenhypothesized that, in response to catabolic stimulus, Sclexpressed by mature mineral embedded, or even apoptos-ing osteocytes, may act in an autocrine manner, increasinglocal expression of RANKL relative to OPG, as well as aparacrine manner, inducing such expression in immatureosteocytes immediately below the osteoblastic lining cells(44).

In the current study, mean Scl bone immunostaining in

the IH group did not differ from control but was signif-icantly higher in patients with high versus normal boneresorption. Moreover, there has been a signicantly pos-itive correlation of Scl bone expression with ES/BS in theentire IH group, which was not observed in the controls.Interestingly, a signicant negative correlation of Sclimmunostaining with the mineralization surface was evi-denced in the subgroup with high bone resorption(r=20.63; P=0.02) but not in the group with normal boneresorption (r=0.50; P=0.18; data not shown), further sug-gesting that, within the IH group, the higher bone resorp-tion and compromised mineralization exhibited by most ofthe patients are associated with bone expression of Scl.These ndings agree well with a report about a more sig-nicant rise in bone resorption (CrossLaps) than formation(b-AP) in patients with immobilization-induced bone loss

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(45). Additionally, in a very recent randomized, placebo-controlled trial using a neutralizing antibody to Scl(Romosozumab) in postmenopausal women, the levels ofserumb-isomer of the C-terminal telopeptide of collagen, a

bone resorption marker, presented a signicant and sus-tained reduction after 12 months, whereas the increase in

procollagen N-terminal propeptide, a bone formationmarker, was only transitory (46). We did not nd an asso-ciation between bone expression of Scl and RANKL fromour previous study (6), but of note, the immunostaininghad not been performed in osteocytes (6). At present, wedid detect a signicant positive correlation between Sclimmunostaining and serum 1,25(OH)2D3 in IH patients,which is in accordance with literature data showing that1,25(OH)2D3 may increase Scl expression in human osteo-

blastic cells (47). These ndings led us to hypothesize thatthe increased bone resorption seen in some IH patientscould be mediated by an increase in vitamin D signaling,stimulating Scl synthesis and leading to an imbalance inthe OPG/RANKL ratio.

In the control group, Scl immunostaining correlatedpositively with BV/TV and Tb.N and negatively with Tb.Sp. Once again, given that Scl inhibits osteoblastic activity, anegative correlation would be expected. However, somestudies have also found direct correlations between serum Scland BMD (48,49) or BV/TV in healthy populations (50).

A limitation of the present study is that, given itsretrospective design and the lack of available bloodsamples, serum levels of Scl (and FGF-23) could not bedetermined. Nevertheless, Durosier et al. (50) have shownthat Scl levels are markedly different according to the im-munoassay used, jeopardizing its interpretation. Althoughthe current number of patients is relatively low, this study

comprises one of the largest and substantial series in theliterature for a bone biopsy study.

In summary, although other investigators had foundincreased VDR in monocytes (14), no other study in hu-mans has hitherto evaluated VDR expression in bone tis-sue from IH patients and its relation with bone resorption.It is possible that a longer duration of disease might havecontributed to increase bone resorption and inuenced theimmunohistochemical pattern presently disclosed.

In conclusion, present ndings suggest the contributionof VDR and Scl to increase bone resorption in IH but do notimplicate FGF-23 in the bone alterations seen in these pa-tients. Additional studies are warranted to determine whether

antiresorptive therapy will correct these bone abnormalities.Targeting VDR and Scl may be a new therapeutic approach toprevent bone loss in IH.

AcknowledgmentsWe thankSusan Schiavi forprovidingbroblast growth factor 23

antibody; Rosimeire A.B. Costa, Rita Martin, Luciene M. dos Reis,

Fabiana G. Graciolli, and Wagner V. Dominguez for technical as-

sistance; Maria Aparecida da Glria and Maria Teresa de Seixas

Alves for invaluable suggestions on immunohistochemistry; and

Ana M. Misael and Fellype C. Barreto for histomorphometric data.

This study was supportedby Fundao de Amparo Pesquisa do

Estado de So Paulo (FAPESP) Grant 08/10515-0.

Portions of thisstudy were presented at the 50th European Dialysis

and Transplant Association Meeting, May 1821, 2013, in Istanbul,

Turkey.

Disclosures

None.

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Received:October 7, 2013Accepted:March 10, 2014

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expression of fibroblast growth factor 23, vitamin d receptor, and sclerostin in bone tissue from...

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