Bone growth during daily or intermittent calcitriol treatment during renal failure with advanced secondary hyperparathyroidism

original article

http://www.kidney-international.org & 2007 International Society of Nephrology

see commentary on page 531

Bone growth during daily or intermittent calcitriol treatment during renal failure with advanced secondary hyperparathyroidism CP Sanchez1 and YZ He1 1

Department of Pediatrics, University of Wisconsin, School of Medicine and Public Health, Madison, Wisconsin, USA

Calcitriol is a standard therapy for secondary hyperparathyroidism in chronic renal failure. We evaluated whether the effect of daily or intermittent calcitriol administration is more efficient in enhancing bone growth in renal failure with advanced secondary hyperparathyroidism in weanling 5/6 nephrectomized rats loaded with phosphorus to induce severe secondary hyperparathyroidism. The animals were treated daily or three times weekly with calcitriol for 4 weeks but the total weekly dose of calcitriol was the same. Although calcitriol increased the serum calcium, it did not lower parathyroid hormone (PTH) or improve tibia and body length. Animals with renal failure and advanced secondary hyperparathyroidism had decreased PTH/PTHrP, which was accompanied by an increase in the cyclin kinase inhibitor p57Kip2. Calcitriol treatment upregulated the PTH/PTHrP receptor but also increased inhibitors of cell proliferation such as p21Waf1/Cip1, IGFBP3, and FGFR3. Calcitriol also enhanced markers of chondrocyte differentiation, such as IGF1, Vitamin D receptor, FGF23, and bone morphogenetic protein-7. Receptor activator of nuclear factor-jb ligand levels improved with calcitriol treatment but without changes in osteoprotegerin suggesting an enhancement of osteo/chondroclastogenesis and mineralization. Overall, both daily and intermittent calcitriol had similar effects on endochondral bone growth in phosphorus-loaded rats with renal failure.

Calcitriol is a standard treatment for secondary hyperparathyroidism and renal bone disease. Whether daily calcitriol administration is more effective than intermittent therapy in improving growth in children with chronic renal failure and advanced secondary hyperparathyroidism remains to be defined. In pediatric patients with Stage 3–4 chronic renal failure with mild elevations of parathyroid hormone (PTH), daily calcitriol was equally effective as intermittent therapy in the reduction of PTH and enhancement of growth.1,2 A greater improvement of osteitis fibrosa was demonstrated during intermittent calcitriol therapy in children maintained on peritoneal dialysis with refractory secondary hyperparathyroidism, although 30% of the patients developed low turnover bone.3 Conversely, Mehls et al. reported that only daily calcitriol and not intermittent administration enhanced growth in rats with renal failure after 2 weeks of treatment.4 The divergence of growth findings in these studies suggests that daily and intermittent calcitriol may have different effects on endochondral bone growth. In the growth plate, calcitriol has dose-dependent inhibitory effects on chondrocyte proliferation and matrix synthesis.5,6 The aim of the current study is to assess the effects of daily or intermittent calcitriol on chondrocyte proliferation and differentiation in young rats with renal failure and advanced secondary hyperparathyroidism.

Kidney International (2007) 72, 582–591; doi:10.1038/sj.ki.5002375; published online 6 June 2007

RESULTS Anthromorphic measurements

KEYWORDS: growth plate; secondary hyperparathyroidism; bone growth; calcitriol

Weight gain did not differ in nephrectomized and control animals (Table 1). At the end of the study, body length was 16–19% less in Nx-Phos and in both calcitriol groups compared with Control (Table 1). Tibial length was 6–9% shorter in calcitriol and Nx-Phos groups (Table 1). Food efficiency ratio was used to evaluate growth under conditions of controlled food intake;7 the values did not differ in all groups (Table 1).

Correspondence: CP Sanchez, 3590 MSC/Pediatrics, University of Wisconsin School of Medicine and Public Health, 1300 University Avenue, Madison, Wisconsin 53706, USA. E-mail: [email protected] Received 29 November 2006; revised 21 March 2007; accepted 2 May 2007; published online 6 June 2007 582

Serum biochemical parameters

Bioactive PTH levels increased more than 40-fold in all nephrectomized animals and did not change after 4 weeks of daily or intermittent calcitriol (Table 2). Serum phosphorus, creatinine, and urea nitrogen were much higher in nephrecKidney International (2007) 72, 582–591

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CP Sanchez and YZ He: Calcitriol and bone elongation

tomized groups (Table 2). Calcitriol increased serum calcium in Nx-Phos animals comparable with Control (Table 2). There was a positive correlation between calcium and tibial length, R ¼ 0.4, Po0.03, but serum phosphorus and PTH negatively correlated with body and tibial length, R ¼ 0.7, Po0.007. Growth plate morphometry

The width of the growth plate was 20–30% shorter in phosphorus-loaded rats (Figure 1). There was no difference in the ratio between the proliferative zone and the growth plate, 0.570.005, P ¼ NS, and between the hypertrophic zone

and the total width in all groups, 0.470.05, P ¼ NS (Figure 1). The columnar architecture of the growth plate was nearly restored after 4 weeks of treatment with either daily or intermittent calcitriol (Figure 1). To evaluate whether daily or intermittent calcitriol has divergent effects on chondrocyte proliferation, several markers of cell proliferation were evaluated: PTH/PTHrP receptor, cyclin D1, histone-4, mTOR (mammalian target of rapamycin), Col2a1, and b1 integrin. In the growth plate, b1 integrin is critical in the maintenance of the columnar architecture, cell-to-extracellular matrix attachment and may

Table 1 | Anthropometric measurements and food efficiency ratio in all groups a

Change in body weight (g) Change in body lengtha (cm) Tibial lengthc (cm) Food efficiency ratioe

Nx-Phos N=8

Nx-Daily D N=9

Nx-Int D N=9

Control N=10

138734 12.171.1b 3.570.1d 3.371.2

139720 11.871.1b 3.670.1d 3.371.0

140733 12.171.0b 3.670.1d 3.370.8

151720 1471.1 3.870.09 3.070.3

a

Difference between final and baseline measurements. Po0.05 versus Control. Obtained at the time of killing. d Po0.01 versus Control. e Total food consumed/weight gain during the study period. b c

Table 2 | Serum biochemical measurements in all groups Nx-Phos N=8

Nx-Daily N=9

a

Bioactive 1-84PTH (ng/l) Calcium (mmol/l) Phosphorus (mmol/l) Urea nitrogen (mmol/l) Creatinine (mmol/l)

a

a

13627685 2.470.1 4.071.1a 29712a 97732a

11927403 2.070.3b 4.471.0a 2375a 88735a

Nx-Int D N=9 13477286 2.370.1 4.170.9a 27710a 88734a

Control N=10 29710 2.570.06 3.170.3 872 2779

a

Po0.0001 versus Control. Po0.0002 versus all groups.

b

500 Growth plate

Micrometer

400

300

aP<0.002

Proliferative zone

Hypertrophic zone

versus control a

a

a

200

100

0 Nx-Phos

Nx-Phos

Nx-Daily D

Nx-Daily D

Nx-Int D

Nx-Int D

Control

Control

Figure 1 | Measurements of the width of the growth plate, proliferative zone, and hypertrophic zone in all groups (upper panel) and the corresponding photomicrographs in the lower panel,  65. Note the disorganized growth plate architecture in the Nx-Phos group. Kidney International (2007) 72, 582–591

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affect chondrocyte proliferation.8 In the current study, b1 integrin localized to the upper hypertrophic chondrocytes and declined in Nx-Phos but increased twofold with calcitriol (Figure 2a). Likewise, Col2a1 (type II collagen, Figure 2b) in the extracellular matrix and the proteoglycan aggrecan (Figure 2c) improved with calcitriol. In agreement with our previous studies, PTH/PTHrP receptor, an essential regulator of chondrocyte proliferation, declined in phosphorus loaded animals and improved with calcitriol;9 3673% in Nx-Phos, 7479% in Nx-Daily D, 7271% in Nx-Int D, and 8072% in Control, Po0.02. Cyclin D1 is essential in the progression through the G1 phase of the cell cycle and highly responsive to stimulation by growth factors, including PTH/PTHrP. Our findings showed that cyclin D1 localized to the proliferating chondrocytes and it was twofold lower in the Nx-Phos group. With an increase in PTH/PTHrP receptor, there was a concomitant enhancement of cyclin D1 in both calcitriol groups (Figure 3a). Histone-4, which also localized to the proliferative zone, declined by 80% in the Nx-Phos and increased three- to fourfold with calcitriol but remained lower than Control (Figure 3b). The mTOR is an important factor in cell cycle progression, which can enhance the translation of cyclin D1. The protein expression of mTOR was restricted to the proliferating chondrocytes and the protein expression declined by 35% in Nx-Phos and increased by 20% with

calcitriol (Figure 3c). Our current findings suggest that calcitriol may directly increase PTH/PTHrP receptor with subsequent enhancement of other cell cycle proteins although, the expression remained lower in all nephrectomized groups compared with animals with normal renal function. The decline in PTH/PTHrP receptor was accompanied by an upregulation of the cyclin-dependent kinase inhibitor p57Kip2 by 40% in the hypertrophic chondrocytes of nephrectomized animals independent of calcitriol; this change may promote chondrocyte hypertrophy and hinder cell proliferation (Figure 4a). Calcitriol has been demonstrated to enhance mineralization and increase chondrocyte differentiation. To assess the effects of daily and intermittent calcitriol on chondrocyte maturation, the following proteins associated with cell differentiation were used, including CaR, IGF-I, IGFBP-3, p57Kip2, p21Waf1/Cip1, FGF23, FGFR3, VDR, and BMP-7 expression. As previously discussed, daily or intermittent calcitriol did not affect p57Kip2 but increased another cyclindependent kinase inhibitor p21Cip/Waf, which also localized to the hypertrophic chondrocytes (Figure 4b). Likewise, bone morphogenetic protein-7 (BMP-7) that has been associated with chondrocyte maturation was localized to the hypertrophic chondrocytes and the expression of BMP-7 was downregulated in phosphorus-loaded rats and increased in both calcitriol groups (Figure 4c). 140

a Labeling index

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Figure 2 | Upregulation of b1 integrin, Col2a1, and aggrecan expression during calcitriol therapy in chondrocytes. The protein expression of b1 integrin (a) located in the hypertrophic chondrocytes as denoted by arrows (brown color, left panel,  65) and the corresponding quantification (right panel). Col2a1 (type II collagen) protein expression (b) in the extracellular matrix of the growth plate (brown color,  25) in all groups; lighter staining was evident in Nx-Phos group. Aggrecan protein expression (c) localized intracellularly and along the cell membrane of the hypertrophic chondrocytes (denoted by arrows, left panel,  65) and the corresponding Labeling index (right panel). 584

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120

a Labeling index

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aP< 0.001 bP< 0.05

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Figure 3 | Markers of chondrocyte proliferation, cyclin D1, histone-4, and mTOR, increased during treatment with calcitriol. (a) Cyclin D1, (b) histone-4, and (c) mTOR protein expression localized to the proliferating chondrocytes, denoted by arrows (left panel,  65) and corresponding Labeling index in the right panel.

140 120 Labeling index

a4

aP< 0.0001

versus control

100 80

a a

a

60 40 20 0

Nx-Phos

Nx-Daily D

Nx-Int D

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Control 100

b

aP< 0.05

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Control

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80 Labeling index

Nx-Daily D

a

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60 40 20

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Nx-Daily D

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0

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Nx-Phos 120

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Control

aP< 0.002 bP< 0.05

versus all groups versus control

80 60

Nx-Int D

a

b

Nx-Phos

Nx-Daily D

b

40 20

Nx-Phos

Nx-Daily D Kip2

Nx-Int D

0

Control

Nx-Int D

Control

Cip/Waf

Figure 4 | Enhancement of p57 in renal failure and increase in p21 and BMP-7 during calcitriol therapy. (a) Cyclin-dependent kinase inhibitor p57Kip2 localized in the hypertrophic chondrocytes, (b) p21Cip/Waf in the terminal chondrocytes, and (c) BMP-7 localized in the hypertrophic chondrocytes, denoted by arrows and brown color (left panel,  65). Labeling index is illustrated in the corresponding panels on the right. Kidney International (2007) 72, 582–591

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CaR is a G-protein-coupled receptor essential in both chondrocyte proliferation and chondrocyte maturation.10 Our results showed that CaR, which localized to the lower proliferating and upper hypertrophic chondrocytes, declined by 40% in Nx-Phos and was restored with calcitriol (Figure 5a). In contrast to our previous studies in rats with renal failure and mild secondary hyperparathyroidism, IGF-I expression increased with both daily and intermittent calcitriol but remained 15–20% lower than Control (Figure 5b). Calcitriol has been reported to promote cell differentiation and inhibit proliferation by upregulating IGFBP-3; this maybe related to the enhancement of the differentiation process through an IGF-Idependent or -independent pathway.5 IGFBP-3, a well documented inhibitor of cell proliferation,11 declined in phosphorusloaded animals and increased with daily and intermittent calcitriol by approximately 35% (Figure 5c). Vitamin D receptor (VDR) expression was most intense in the hypertrophic chondrocytes. Calcitriol whether given daily or intermittently significantly upregulated its own receptor (Figure 6a). FGF23 is an important regulator of serum phosphorus. It is well documented that phosphorus is important in chondrocyte differentiation and mineralization.12,13 Currently, there are no published studies to demonstrate the localization and role of FGF23 in the growth plate cartilage in renal failure. Our results show that FGF23 protein localized to the hypertrophic chondrocytes and may play a role in chondrocyte maturation

(Figure 6b). FGF23 was 35% lower in Nx-Phos compared with Control and substantially increased by 1.5 times with calcitriol without any changes in serum phosphorus (Figure 6b). FGF23 expression did not correlate with serum phosphorus or PTH. FGFR3, a negative regulator of chondrogenesis, declined in Nx-Phos and increased with calcitriol (Figure 6c). Upregulation of FGFR3 as demonstrated in achondroplasia may induce premature exit of proliferating chondrocytes into hypertrophy leading to shorter bone growth.14 Osteo/chondroclastic resorption is a necessary step towards mineralization and bone formation. The regulation of osteoclast formation and function are tightly regulated by the receptor activator of nuclear factor-kb ligand (RANKL), which induces osteoclastogenesis and its decoy receptor osteoprotegerin (OPG). Osteoclastogenesis can also be determined by the relative ratio between RANKL and OPG; an increase in RANKL enhances bone resorption, increases proteolytic enzymes, and its expression is much higher at the onset of mineralization.15–17 In the current experiments, RANKL protein localized to the hypertrophic chondrocytes and increased by 30% with daily and intermittent calcitriol (Figure 7a). OPG expression that also localized to the same layer of hypertrophic chondrocytes did not change with calcitriol (Figure 7b). There was good correlation between RANKL and OPG, R ¼ 0.87, Po0.0001. The increase in RANKL expression in the present study suggests that

140

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Figure 5 | CaR, IGF-I, and IGBP3 protein expression in chondrocytes increased with calcitriol. (a) Calcium receptor, (b) IGF-I, and (c) IGFBP3 protein expression in the growth plate of all groups in the left panel as denoted by arrows,  65, with the quantification of protein response in the corresponding right panel. Calcium receptor is localized to the lower proliferating and upper hypertrophic chondrocytes, IGF-I in the hypertrophic chondrocytes and IGFBP3 in the proliferative zone. 586

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CP Sanchez and YZ He: Calcitriol and bone elongation

calcitriol enhances mineralization, which has been described in other studies.18 DISCUSSION

The current findings demonstrate that treatment with either daily or intermittent calcitriol has similar effects on body and tibial length, serum calcium, circulating PTH, and on

chondrocyte proliferation and hypertrophy in phosphorusloaded animals with renal failure. Our results are in contrast to the findings of Mehls et al. that daily and not intermittent calcitriol improved growth in animals with renal failure;4 these differences may be due to the higher PTH and phosphorus levels, younger age of the animals, and longer duration of renal failure in the current study. Although the 120 100 Labeling index

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Figure 6 | Calcitriol enhanced VDR, FGF23, and FGFR3, all markers of chondrocyte differentiation. Markers of chondrocyte differentiation (a) VDR, (b) FGF23, and (c) FGFR3 in the hypertrophic chondrocytes in all groups marked by arrows (right panel,  65) and the corresponding Labeling index (right panel). VDR and FGF23 are localized to the hypertrophic chondrocytes and FGFR3 staining is located both in the proliferating and upper hypertrophic chondrocytes. 80

a

aP< 0.006

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versus all groups versus control b

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versus Nx-Phos, control

60 40 20 0

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Figure 7 | RANKL and OPG expression increased in the growth plate during calcitriol therapy in renal failure. (a) RANKL and (b) OPG protein expression in the growth plate of all experimental groups (denoted by arrows,  65). The corresponding quantification of protein response is shown in the right panel. Both RANKL and OPG are confined to the upper hypertrophic chondrocytes. Kidney International (2007) 72, 582–591

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parathyroid gland was not evaluated in the present experiment, the higher PTH and phosphorus levels may suggest the presence of nodular hyperplasia, which may have contributed to calcitriol resistance, poor growth, and absence of PTH reduction. The growth plate is a highly organized tissue with various layers of chondrocytes at different stages of differentiation. Chondrocyte proliferation is primarily regulated by the PTHrP-Indian hedgehog-TGF-b feedback loop. We have previously shown that phosphorus-loaded rats with renal failure had smaller growth plate with significant reduction in chondrocyte proliferation most likely owing to a decrease in PTH/PTHrP receptor expression.19 PTH/PTHrP delays chondrocyte differentiation and maintain proliferation by downregulating the transcription factor Runx2, activating protein kinase A and decreasing p57Kip2.20,21 With considerable decrease in PTH/PTHrP receptor, our results showed an upregulation of p57Kip2 in phosphorus-loaded rats independent of calcitriol. It is unclear whether the decline in PTH/ PTHrP receptor occurred first in the course of renal failure with subsequent increase in the cyclin kinase inhibitor p57Kip2 that may account for poor growth in advanced secondary hyperparathyroidism. Further studies are needed to assess the sequence of events that eventually leads to reduction of chondrocyte proliferation. The members of the cyclindependent kinase inhibitors Cip/Kip family, including p57Kip2 and p21Waf1/Cip1, inhibit phosphorylation by binding to the proliferating nuclear cell antigen preventing DNA replication.22,23 Our findings suggest that phosphorus loading per se in renal failure can lead to alterations in the downstream signaling pathways of PTH/PTHrP and upregulation of p57Kip2, which may contribute to inhibition of chondrocyte proliferation leading to suboptimal growth. Calcitriol treatment in the current study enhanced PTH/ PTHrP receptor with subsequent increase in cyclin D1, mTOR, and histone-4. PTHrP using the transcription factor CREB stimulates cyclin D1 promoter activity via the CRE promoter element.24 Although calcitriol increased PTH/PTHrP receptor, the expression of the cell cycle inhibitor p57Kip2 remained elevated with concomitant upregulation of p21Waf1/Cip1; the upregulation of both cell cycle inhibitors may promote chondrocyte differentiation and hinder cell proliferation, and may explain the lack of growth in the phosphorus-loaded rats as demonstrated in the current study. Calcitriol is well known to have dose-dependent effect on cell proliferation, including chondrocytes. Inhibition of chondrocyte proliferation occurred with calcitriol greater than 1010 M and stimulation at concentration lower than 1012 M.25 In higher doses, calcitriol inhibited the progression of cell cycle from G1 to S phase by decreasing the targets of E2F transcription factors leading to accumulation of cells in the G1 phase;26 this downregulation of DNA replication genes only occurred in cells with functional VDR.26 The inhibitory effects of calcitriol on cell proliferation may also be due to the increase in IGBP-3 and increase in the cyclin kinase inhibitor p21Waf1/Cip1 through its actions on the vitamin D response 588

CP Sanchez and YZ He: Calcitriol and bone elongation

element in the p21Waf1/Cip1 promoter leading to the activation of the TGF-b signaling pathway.27–29 In this study, there was a greater increase in p21Waf1/Cip1, IGF-I, IGFBP3, BMP-7, and FGFR3 during treatment with calcitriol, all markers that promote chondrocyte maturation. The maintenance of the columnar architecture is vital in cell-to-cell contact and cell-to-matrix communication in the growth plate. Distortions of the growth plate matrix as shown in our phosphorus-loaded rats may lower chondrocyte proliferation, alter cell enlargement and delay cartilage mineralization. Our current results show that the expression of the matrix protein aggrecan, collagenbinding protein b1 integrin, and extracellular Col2a1 expression were much lower in the growth plate of the phosphorusloaded rats and calcitriol treatment restored the architecture of the growth plate and increased the expression of the matrix proteins. b1 integrins, which are highly expressed in chondrocytes, are cell surface receptors important in cell adhesion, migration, and matrix assembly.30 Deletion of b1 integrin gene was associated with chondrodysplasia, abnormal shape of the chondrocytes, failure to form columns in the growth plate, decreased collagen fibril density in the cartilage matrix, and loss of adhesion to the Col2a1 matrix.8 Although our previous studies have not shown differences in intracellular Col2a1 and Col10a1, our results showed the downregulation of b1 integrin and aggrecan in the growth plate of phosphorus-loaded animals. The problems with communication between chondrocytes and the pericellular matrix may contribute to the overall decline of chondrocyte activity in renal failure. Our previous experiments did not show alterations in the growth plate architecture of rats with renal failure and mild secondary hyperparathyroidism;19 however, the columnar organization in the phosphorus-loaded uremic rats is markedly abnormal, which was restored by calcitriol. The CaR is crucial in the regulation of PTH secretion and studies suggest that CaR may play a role in chondocyte maturation.10 Our experiments show that CaR substantially declined in the growth plate of phosphorus-loaded animals with renal failure and restored by calcitriol. The increase in serum calcium during calcitriol therapy may contribute at least in part to the upregulation of the CaR, as calcium is critical to cell growth and enhances chondrocyte differentiation. Rodriguez et al.31 have reported that increasing the extracellular calcium even in CaR (/) chondrocytes enhanced chondrocyte differentiation associated with proteoglycan accumulation, mineral deposition, and matrix gene expression. We have previously reported that calcium loading in renal failure lead to hypercalcemia with inhibition of cell proliferation and reduction in circulating PTH.32 Although serum calcium increased with calcitriol in the current study, the increment was much lower than our calcium-loaded rats and hence may not be effective in the inhibition of PTH secretion in phosphorus-loaded animals. FGF23, a novel circulating factor essential in phosphorus homeostasis, is elevated in hypophosphatemic rickets and in Kidney International (2007) 72, 582–591

CP Sanchez and YZ He: Calcitriol and bone elongation

chronic renal failure.33 Our current results show that FGF23 protein is localized to the hypertrophic chondrocytes and may participate in chondrocyte maturation. In this study, phosphorus loading lowered FGF23 in the chondrocytes and calcitriol enhanced the expression of both FGF23 and one of its receptor, FGFR3, a well-known inhibitor of bone growth and chondrocyte proliferation. Calcitriol may affect FGF23 expression through its actions on the vitamin D response element in the FGF23 promoter or via a VDR-independent pathway.34,35 We did not measure circulating FGF23, so it is unclear whether serum levels correlate with FGF23 expression in the growth plate. As FGF23 is important in phosphorus regulation, it may play an essential role in cartilage mineralization. The upregulation of FGFR3 by calcitriol may contribute in part to the inhibition of bone growth as demonstrated in the current study. Osteo/chondroclastic resorption is an important step in endochondral bone growth. Our previous studies demonstrated upregulation of gelatinase B/MMP-9 expression and increase TRAP staining in the chondro-osseous junction of phosphorus-loaded animals with renal failure. In this study, we evaluated the expression of RANKL and OPG, which are essential in osteo/chondroclast activation and function. RANK and its ligand RANKL are members of the tumor necrosis factor receptor family that participate in osteo/ chondroclastogenesis by binding to osteoclast/chondroclast precursors. OPG is a decoy receptor for RANKL and interferes with the activation of RANK. Both OPG and RANKL were localized to the same layer of hypertrophic chondrocytes, which may suggest coupled actions in osteo/ chondroclastogenesis. In agreement with other studies, our current paper shows that calcitriol increased RANKL without any changes in OPG expression.36 Several investigators have demonstrated that RANKL/OPG ratio may be an indicator of osteoclast activation with an increase at the onset of mineralization.36,37 The present findings suggest that calcitriol may enhance osteo/chondroclastogenesis in rats with severe secondary hyperparathyroidism by targeting the vitamin D response element in the RANKL promoter region.38,39 Calcitriol is a standard form of treatment for secondary hyperparathyroidism in children with chronic renal failure. Our findings suggest that there is an overall delay in chondrocyte proliferation and chondrocyte differentiation in the growth plate of animals with renal failure and advanced secondary hyperparathyroidism. Calcitriol, whether given daily or intermittently, is equally effective in the enhancement of chondrocyte maturation and restoration of the growth plate architecture. Although calcitriol increased PTH/PTHrP receptor and associated cell cycle proteins, there was a greater increase in the expression of the cyclin kinase inhibitors p57Kip2 and p21Waf1/Cip1 associated with enhancement of the bone growth inhibitor FGFR3; such findings may contribute to the poor growth demonstrated during calcitriol therapy in animals with renal failure and advanced secondary hyperparathyroidism. Kidney International (2007) 72, 582–591

original article

MATERIALS AND METHODS Thirty-six male weanling Sprague–Dawley rats, 6273 g and 2370.6 cm (Harlan Laboratories, Indianapolis, IN, USA), were housed in individual cages at constant temperature. After 24 h of acclimatization, 26 animals underwent the first stage of a two-stage 5/6 nephrectomy (Nx) under ketamine and xylazine anesthesia as described previously.9 After 1 week, the Nx animals underwent total nephrectomy of the right kidney. Ten rats underwent sham nephrectomy (Control) in two stages corresponding to the 5/6 nephrectomy. To induce advanced secondary hyperparathyroidism, all nephrectomized animals were fed high phosphorus diet (NxPhos, 1.2% phosphorus) for the entire duration of the 5 weeks study period. All Control animals were fed the standard rodent diet. To ensure equivalent caloric intake, the Control animals were pair-fed with Nx-Phos animals by providing the amount of food each day to Control rats that had been consumed the previous day by Nx-Phos group. All procedures were reviewed and approved by the Research Animal Resource Center at the University of Wisconsin-Madison and conducted in accordance with the accepted standard of humane animal care. All animals were weighed daily and body length was measured weekly under anesthesia. Calcitriol was started 24 h after the secondstage surgery, nine animals received daily calcitriol at a dose of 50 ng/kg/day (Nx-Daily D) and nine rats were given calcitriol three times weekly at 350 ng/kg/week (Nx-Int D). The last dose of intermittent calcitriol was given 48 h before killing. Both Nx-Phos (N ¼ 8) and Control (N ¼ 10) received saline injections. All injections were given by intraperitoneal route, using equivalent volume and at the same time of the day for a total of 4 weeks. The dose of calcitriol was adapted from our earlier study that showed changes in the growth plate after 10 days of treatment.9 After 4 weeks of calcitriol, the animals underwent trans-cardiac perfusion using 4% paraformaldehyde in phosphate-buffered saline (PBS). At the time of killing, blood samples were obtained and stored at 801C until biochemical assays for serum calcium, creatinine, phosphorus, urea nitrogen, and PTH. Blood was collected 48 h after the last dose of intermittent calcitriol and 24 h after the last dose of daily calcitriol. The proximal tibiae were collected, decalcified in 15% ethylenediaminetetraacetic acid in PBS for 2 weeks and embedded in paraffin.32 Tibial length was measured between the proximal and distal articular surfaces using a caliper. Five micrometer sections of the proximal tibia were collected for morphometric analysis and immunohistochemistry studies as previously described.32 Serum biochemical determinations Serum urea nitrogen, creatinine, calcium, and phosphorus levels were measured using standard laboratory methods. PTH levels were measured using the Rat BioActive PTH ELISA assay kit, which detects the full length biologically active form (Immutopics Inc., San Clemente, CA, USA). Growth plate morphometry For morphometric analysis, three 5-mm sections of bone were obtained, stained with hematoxylin and eosin and viewed under light microscopy at  25. The total width of the growth plate at the proximal end of each tibia was measured at equally spaced intervals using an image analysis software as described previously (Kontron Instruments Ltd Elektronik 200, Hallbergmoos, Germany).9 The widths of the zones occupied by hypertrophic chondrocytes and proliferative chondrocytes were measured by similar method. 589

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Immunohistochemistry experiments Immunohistochemistry experiments were performed using methods described previously.40 All primary antibodies were obtained from (Santa Cruz Biotechnology, Santa Cruz, CA, USA) unless indicated. The following primary antibodies were selected to evaluate chondrocyte proliferation: (a) cyclin D1 at 2 mg/ml, (b) histone-4 at 5 mg/ml, (c) b1integrin at 5 mg/ml, (d) mTOR (mammalian target of rapamycin) at 8 mg/ml, and (e) PTH/PTHrP receptor at 4.4 mg/ml (Upstate Biotechnology, Upstate USA Inc., Charlottesville, VA, USA). Chondrocyte maturation was assessed using: (a) CaSR at 5 mg/ml (b), IGF-I at 10 mg/ml (Upstate Biotechnology), (c) IGFBP3 at 10 mg/ml, (d) p57Kip2 at 4 mg/ml, (e) p21Waf1/Cip1 at 8 mg/ml, (f) FGF23 at 4 mg/ml, (g) FGFR3 at 5 mg/ml, (h) VDR at 5 mg/ml, and (i) BMP-7 at 5 mg/ml. Osteo/chondroclastic activity was evaluated using RANKL at 6 mg/ml and OPG at 5 mg/ml. Aggrecan was assessed in the hypertrophic chondrocytes and Col2a1 protein in the extracellular matrix after incubation with trypsin. Slides were viewed at  65 by bright field microscopy, images were captured using a charge coupled device (CCD) camera control unit and displayed on a computer monitor. Approximately, 50–60 cell profiles were assessed in each growth plate. For quantification of the protein expression, the number of cells expressing the protein was counted and expressed as percentage of the labeled cells over the total number of cells where the expression is localized (Labeling Index).40

CP Sanchez and YZ He: Calcitriol and bone elongation

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Statistical analysis

All results are expressed as mean values71 s.d. Data were evaluated by one-way analysis of variance and comparisons among groups were carried out using Bonferroni/DUNN post hoc tests using the StatViews statistical software (SAS Institute, Cary, NC, USA). The Pearson product moment correlation coefficient was used to evaluate the relationship between two numerical variables. For all statistical tests, probability values less than 5% were considered to be significant.

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ACKNOWLEDGMENTS

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