Comparison of Active Vitamin D Compounds and a Calcimimetic in Mineral Homeostasis

BASIC RESEARCH www.jasn.org

Loan Nguyen-Yamamoto,* Isabel Bolivar,* Stephen A. Strugnell,† and David Goltzman*

*Department of Medicine, McGill University and McGill University Health Centre, Montreal, Quebec, Canada; and †Genzyme Corporation, Middleton, Wisconsin

ABSTRACT The differential effects between cinacalcet and active vitamin D compounds on parathyroid function, mineral metabolism, and skeletal function are incompletely understood. Here, we studied cinacalcet and active vitamin D compounds in mice expressing the null mutation for Cyp27b1, which encodes 25- ␣ hydroxyvitamin D-1 -hydroxylase, thereby lacking endogenous 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Vehicle-treated mice given high dietary calcium had hypocalcemia, hypophosphatemia, and marked secondary hyperparathyroidism. Doxercalciferol and 1,25(OH)2D3 each normalized these parameters and corrected both the abnormal growth plate architecture and the diminished longitudinal bone growth observed in these mice. In contrast, cinacalcet suppressed serum parathyroid hormone (PTH) cyclically and did not correct the skeletal abnormalities and hypocalcemia persisted. Vehicle-treated mice given a BASIC RESEARCH “rescue diet” (high calcium and phosphorus, 20% lactose) had normal serum calcium and PTH levels; cinacalcet induced transient hypocalcemia and mild hypercalciuria. The active vitamin D compounds and cinacalcet normalized the increased osteoblast activity observed in mice with secondary hyperparathyroidism; cinacalcet, however, increased the number and activity of osteoclasts. In conclusion, cinacalcet reduces PTH in a cyclical manner, does not eliminate hypocalcemia, and does not correct abnormalities of the growth plate. Doxercalciferol and 1,25(OH)2D3 reduce PTH in a sustained manner, normalize serum calcium, and improve skeletal abnormalities.

J Am Soc Nephrol 21: ●●●–●●●, 2010. doi: 10.1681/ASN.2009050531

2ϩ Vitamin D and extracellular calcium ([Ca ]e) play and/or serum fibroblast growth factor 23 (FGF-23), central roles in regulating systemic calcium ho- and in part because of loss of renal parenchyma. 2ϩ meostasis. [Ca ]e activates a cation-sensing Consequently, low levels of 1,25(OH)2D develop G-protein coupled receptor, the calcium-sensing in stage 3 CKD, leading to reduced calcium ab- receptor (CaSR), to modulate the concentrations of sorption.3 The resultant hypocalcemia and low circulating parathyroid hormone (PTH) and to reg- 1,25(OH)2D can lead to the development of sec- ulate renal calcium re-absorption.1 The activated ondary hyperparathyroidism, mainly because of form of vitamin D, 1,25-dihydroxyvitamin D the fact that extracellular ionized calcium 2ϩ [1,25(OH)2D] is synthesized via the action of the [Ca ]e and 1,25(OH)2D regulate PTH biosyn- enzyme 25-hydroxyvitamin D-1␣-hydroxylase ␣ [1 (OH)ase], which converts 25-hydroxyvitamin Received May 21, 2009. Accepted May 24, 2010. D to 1,25(OH)2D. This activated form of vitamin D Published online ahead of print. Publication date available at acts predominantly on a nuclear vitamin D receptor www.jasn.org. (VDR) to modulate parathyroid function, mineral Correspondence: 2 Dr. David Goltzman, Royal Victoria Hospital, metabolism, and skeletal function. 1,25(OH)2D Room H467, 687 Pine Avenue West, Montreal, Quebec, Canada synthesis is impaired in chronic kidney disease H3A 1A1. Phone: 514-843-1632; Fax: 514-843-1712; E-mail: (CKD), at least in part because of suppression of the [email protected] renal 1␣(OH)ase by elevated serum phosphorus Copyright © 2010 by the American Society of Nephrology

J Am Soc Nephrol 21: ●●●–●●●, 2010 ISSN : 1046-6673/2110-●●● 1 BASIC RESEARCH www.jasn.org

thesis, with 1,25(OH)2D predominantly suppressing PTH pocalcemic and hypophosphatemic and had markedly increased ϩ 4,5 2 gene transcription and [Ca ]e predominantly downregu- serum PTH (Figure 1), doxercalciferol normalized serum calcium 6,7 8 lating PTH mRNA translation. Both calcium and activated and serum phosphorus. 1,25(OH)2D3 similarly increased serum vitamin D9,10 coordinately regulate the growth of the parathy- calcium (Figure 1A) but increased serum phosphorus more 2ϩ roid gland. In addition, increased [Ca ]e, by activating the markedly than doxercalciferol (Figure 1C). Doxercalciferol 1 CaSR, potently suppresses PTH secretion. The ensuing sec- and 1,25(OH)2D3 each dramatically reduced circulating con- ondary hyperparathyroidism, with disordered skeletal remod- centrations of PTH (Figure 1D). With cinacalcet treatment, eling and mineralization defects, can contribute to the mani- serum calcium remained low at 2 and 24 hours after dose ad- festations of renal osteodystrophy. ministration (Figure 1A), and cyclic PTH fluctuations were

Active vitamin D compounds including 1,25(OH)2D3 (calcit- observed (Figure 1D): that is, PTH levels decreased at 2 hours 11 ␣ riol) and doxercalciferol [1 OHD2, Hec- 12 torol], a vitamin D2 analog that undergoes metabolic activation in vivo to form

1,25(OH)2D2, are used clinically to manage elevated PTH in CKD. The calcimimetic cinacalcet, an allosteric modulator of the CaSR, requires the presence of calcium ions to be effective13 and is used clinically to manage secondary hyperparathyroidism.14 Cina- calcet has also been approved for use in primary hyperparathyroidism due to parathyroid carcinoma.15 Recently, calcimimetics have been proposed as a potential adjuvant treatment for familial hypophosphatemic rickets, which is complicated by secondary hyperparathyroidism.16 How- ever, the skeletal impact of calcimimetics is unclear. We therefore compared active vitamin D compounds and the calcimimetic, cinacalcet, in mice with targeted inactivation of Cyp27b1, the gene encoding the 1␣(OH)ase enzyme, that lack the capacity to synthesize endogenous 1,25(OH)2D. We Figure 1. Active vitamin D compounds and cinacalcet affect calcium, phosphorus, and exposed these mice [1␣(OH)ase null mice] parathyroid homeostasis differently. Serum and urine biochemistry and histomorphom- to two different environments: a high-cal- etry of the parathyroid glands are shown: in wild-type (WT) mice; in 1␣(OH)ase null mice ␣ Ϫ/Ϫ cium intake on which 1␣(OH)ase null mice [1 (OH)ase ] on a high-calcium intake (h) treated with vehicle, 1,25(OH)2D3, doxercal- ␣ are known to develop secondary hyperpara- ciferol, cinacalcet for 2 hours (hr), and cinacalcet for 24 hours; and in 1 (OH)ase null mice thyroidism, rickets, and osteomalacia17–19 on a rescue diet (r) treated with vehicle or cinacalcet for 24 hours. Each value is the mean Ϯ SEM. (A) Number of replicates for serum calcium from left to right are as follows: and a “rescue” diet18 containing high cal- 4 for WT, 6 for vehicle, 5 for 1,25(OH)2D3,7 for doxercalciferol, 3 for cinacalcet 2 hours, 3 cium, high phosphorus, and 20% lactose, for cinacalcet 24 hours, 7 for vehicle, 5 for cinacalcet 2 hours, and 5 for cinacalcet 24 which is known to normalize serum calcium hours. (B) Number of replicates for 24-hour urine calcium are as follows: 4 for vehicle and and phosphate in the absence of either active 4 for cinacalcet. (C) Number of replicates for serum phosphorus from left to right are as vitamin D or the vitamin D receptor. We follows: 3 for WT, 4 for vehicle, 4 for 1,25(OH)2D3, 6 for doxercalciferol, 3 for cinacalcet then determined the relative effects of active 2 hours, 3 for cinacalcet 24 hours, 6 for vehicle, 4 for cinacalcet 2 hours, and 5 for vitamin D compounds and calcimimetics cinacalcet 24 hours. (D) Number of replicates for serum PTH from left to right are as on parathyoid gland function, mineral ho- follows: 3 for WT, 7 for vehicle, 5 for 1,25(OH)2D3, 7 for doxercalciferol, 3 for cinacalcet meostasis, and skeletal function. 2 hours, 3 for cinacalcet 24 hours, 4 for vehicle, 4 for cinacalcet 2 hours, and 5 for cinacalcet 2 hours. (E) Number of replicates for parathyroid gland size done by histomor-

phometry from left to right are as follows: 3 for WT, 3 for vehicle, 3 for 1,25(OH)2D3, 3 for ϩ doxercalciferol, 3 for cinacalcet, 6 for vehicle, and 6 for cinacalcet. P Ͻ 0.05 compared RESULTS ϩϩ ϩϩϩ with WT mice; P Ͻ 0.01 compared with WT mice; P Ͻ 0.001 compared with WT mice; *P Ͻ 0.05 compared with vehicle-treated 1␣(OH)ase null mice on a high-calcium Serum Biochemistry and intake; **P Ͻ 0.01 compared with vehicle-treated 1␣(OH)ase null mice on a high-calcium Parathyroid Gland Histology intake; ***P Ͻ 0.001 compared with vehicle-treated 1␣(OH)ase null mice on a high- E Although vehicle-treated 1␣(OH)ase null calcium intake. P Ͻ 0.05 compared with vehicle-treated 1␣(OH)ase null mice on a rescue EE mice on the high-calcium intake were hy- diet; P Ͻ 0.01 compared with vehicle-treated 1␣(OH)ase null mice on a rescue diet.

2 Journal of the American Society of Nephrology J Am Soc Nephrol 21: ●●●–●●●, 2010 www.jasn.org BASIC RESEARCH after dose administration, but re- bounded by 24 hours, although not to vehicle-treated levels (Figure 1D).

1,25(OH)2D3 and doxercalciferol both decreased the size of the parathyroid glands below that seen in the vehicle- treated 1␣(OH)ase null mice on a high- calcium diet (Figure 1E); parathyroid glands remained enlarged however in the cinacalcet-treated mice (Figure 1, D and E). On the rescue diet, serum calcium, phosphorus, and PTH were normalized in null mice (Figure 1, A, C, and D, re- spectively). Cinacalcet treatment significantly reduced serum calcium after 2 hours [although not to the levels seen in Figure 2. Active vitamin D compounds but not cinacalcet normalize an unmineralized the vehicle-treated 1␣(OH)ase null growth plate and unmineralized bone. Representative von Kossa stains of (A) the growth ␣ mice on the high-calcium intake], but plates and (B) the metaphyses are shown: of the tibiae of wild-type mice; of 1 (OH)ase ␣ Ϫ/Ϫ null mice [1 (OH)ase ] on a high-calcium intake (h) treated with vehicle, 1,25(OH)2D3, not after 24 hours (Figure 1A). Cinacal- Ϫ Ϫ doxercalciferol, and cinacalcet; and of 1␣(OH)ase null mice [1␣(OH)ase / ] on a rescue cet also increased 24-hour urine calcium diet (r) treated with vehicle, and cinacalcet. Black staining denotes mineralization; purple (Figure 1B). Serum phosphorus levels staining depicts the collagen in the growth plates. The white arrows on the black were higher than those in the vehicle- mineralized trabeculae denote the absence or presence of unmineralized bone, which treated controls (Figure 1C) 2 hours af- stains light blue. Red arrows denote osteoclasts lining the bone of cinacalcet-treated null ter cinacalcet administration but were mice on a rescue diet. Magnification: ϫ100 in A; ϫ200 in B. not significantly elevated by 24 hours. Serum PTH levels fell at 2 hours, but were not significantly similar to that observed in wild-type mice (Figure 3A), and the different from vehicle-treated levels after 24 hours (Figure 1D). mineral apposition rate (MAR) (Figure 3C) and bone formation Parathyroid glands also remained slightly increased in the ci- rate (BFR/BS) (Figure 3C) in these groups was not significantly nacalcet-treated mice on a rescue diet (Figure 1E). different from those of wild-type mice (Figure 3B). In null mice on a rescue diet, the MAR was markedly improved in the vehicle- Mineralization of Bone and Cartilage treated and cincalcet-treated mice (Figure 3B) and the BFR/BS On the high-calcium intake, vehicle-treated 1␣(OH)ase null was not significantly different between the vehicle-treated and ci- mice had widened and distorted growth plates which were nacalcet-treated mice (Figure 3C). poorly mineralized (Figure 2A). Excess osteoid was also seen in the bone of the metaphysis (Figure 2B). Both 1,25(OH)2D3 Assessment of Bone Turnover and doxercalciferol normalized the size, structure, and miner- Osteoblast surfaces of bone and serum osteocalcin levels were in- alization of the growth plate, and reduced osteoid was seen creased in vehicle-treated null mice on a high-calcium intake, (Figure 2). In contrast, widened and poorly mineralized compared with those in wild-type mice (Figure 4, A and B). Os- growth plates were seen after cinacalcet treatment along with teoblast surfaces and osteoblast activity as indicated by serum abundant osteoid lining trabeculae (Figure 2). On the rescue osteocalcin were reduced to normal in all treated animals after diet, the width of the growth plate in the 1␣(OH)ase null mice secondary hyperparathyroidism was reduced or eliminated was largely restored in vehicle-treated mice and mineralization (Figure 4, A and B). In null mice on a rescue diet, elimination of bone was normalized (Figure 2). After cinacalcet treatment, of the secondary hyperparathyroidism also resulted in a nor- the growth plate remained widened; however, mineralization malized osteoblast surface, and osteocalcin levels were not dif- of bone markedly improved (Figure 2). ferent from those in wild-type mice (Figure 4, A and B). Compared with the double labeling seen in wild-type mice, In 1␣(OH)ase null mice on a high-calcium intake, osteoclast altered deposition of calcein, a fluorochrome that binds newly numbers were increased after cinacalcet treatment relative to calcified matrix, was seen in the bone of vehicle-treated wild-type mice and to the vehicle-treated control. Serum TRAP5b 1␣(OH)ase null mice on a high-calcium intake compared with levels generally paralleled these changes (Figure 4, C and D). On a the double labeling seen in wild-type mice (Figure 3A). Calcein rescue diet, cinacalcet treatment also increased osteoclast num- labeling was similarly but somewhat less markedly abnormal in bers significantly compared with vehicle-treated 1␣(OH)ase null cinacalcet-treated null animals (Figure 3A). In contrast, discrete mice, and compared with wild-type mice. TRAP5b levels were double labels were easily evident in trabecular bone of increased in parallel with the increases in osteoclasts in the cina- ␣ 1,25(OH)2D3 and doxercalciferol-treated 1 (OH)ase null mice, calcet-treated animals on a rescue diet.

J Am Soc Nephrol 21: ●●●–●●●, 2010 Vitamin D and a Calcimimetic 3 BASIC RESEARCH www.jasn.org

On a rescue diet, radiolucency (Fig- ure 5A) and length of bones improved in the vehicle-treated null mice but the femurs remained slightly shorter than the femurs of wild-type mice (Figure 5B). Cinacalcet treatment resulted in more radiolucent and shorter bones (Figure 5, A and B). Bones of cinacalcet-treated 1␣(OH)ase null mice on a rescue diet also displayed lower BMD than the vehicle-treated mice and the wild-type mice (Figure 5C), but this appeared to reflect primarily increased bone resorption. MicroCT images confirmed the reduced mineralization of secondary ossification centers in the epiphyses and the reduction of mineralized trabeculae in the metaphyses of femurs of vehicle-treated 1␣(OH)ase null mice on a high-calcium diet (Figure 6). Reduced mineralization of the epiphyses was also seen with cinacalcet but a modest increase in mineralized trabeculae was observed (Figure 6). In con- Figure 3. Treatment of osteomalacic null mice with active vitamin D compounds but not with cinacalcet normalizes MAR and BFR/BS. Dynamic histomorphometry results are trast, both 1,25(OH)2D3 and doxercalcif- shown. (A) Photomicrographs of fluorescence double calcein labeling of bone: in wild- erol produced marked improvement in Ϫ Ϫ type (WT) mice; in 1␣(OH)ase null mice [1␣(OH)ase / ] on a high-calcium intake (h) mineralization in the epiphyses and an ␣ treated with vehicle, 1,25(OH)2D3, doxercalciferol, and cinacalcet; in 1 (OH)ase null mice increase in mineralized trabeculae on a rescue diet (r) treated with vehicle, and cinacalcet. Magnification, ϫ200. (B) MARs, (Figure 6). calculated from double calcein labeling as described in Concise Methods: in wild-type In the vehicle-treated and cinacalcet- ␣ Ϫ/Ϫ mice; in 1 (OH)ase null mice on a high-calcium intake (hours) treated with 1,25(OH)2D3 treated 1␣(OH)ase mice on a rescue and doxercalciferol; in 1␣(OH)ase null mice on a rescue diet treated with vehicle, and diet, mineralization of the epiphyses Ϯ cinacalcet. Each value is the mean SEM. (C) BFR/BS, calculated as described in Concise also improved compared with the ve- ␣ Methods: in WT mice; in 1 (OH)ase null mice on a high-calcium intake (hours) treated with hicle-treated 1␣(OH)aseϪ/Ϫ mice on a 1,25(OH) D and doxercalciferol; in 1␣(OH)ase null mice on a rescue diet treated with 2 3 high-calcium intake, but there were vehicle, and cinacalcet. Each value is the mean Ϯ SEM. Number of replicates for MAR and reduced trabeculae in cinacalcet- BFR/BS from left to right are as follows: 3 for WT; 4 for 1,25(OH)2D3, and 5 for doxercal- Ϫ Ϫ ciferol for 1␣(OH)ase / mice on a high-calcium intake; 8 for vehicle, and 8 for cinacalcet treated animals on a rescue diet, re- on a rescue diet. MAR and BFR could not be calculated for vehicle-treated and cinacalcet- flecting the increased bone resorption treated 1␣(OH)ase null mice on a high-calcium intake (h) in view of the indistinct calcein (Figure 6). labeling in the osteomalacic bone of these mice, which precluded calculation of interlabel width. Nevertheless, the results of these treatment arms are denoted in Figure 3, B and C, as zero. DISCUSSION

Growth and Mineral Density of Long Bones In the 1␣(OH)ase null mice on a high-calcium intake, both ␣ On a high-calcium intake, femurs of vehicle-treated 1 (OH)ase 1,25(OH)2D3 and doxercalciferol normalized serum calcium null mice showed increased radiolucency and reduced length and dramatically reduced circulating PTH levels and parathy- compared with femurs of wild-type mice (Figure 5, A and B). roid gland size. Both compounds increased serum phosphorus Both the radiolucency and length of the femurs normalized levels on this normal phosphorus, 0% lactose diet, but at the after 1,25(OH)2D3 and doxercalciferol treatment (Figure 5A). dose used only 1,25(OH)2D3 induced hyperphosphatemia. Femurs of cinacalcet-treated mice remained more radiolucent Whether this observation represents a systematic difference and shorter than those of wild-type mice (Figure 5, A and B). between the two active vitamin D compounds is unclear and Bone mineral density (BMD) testing confirmed the normaliza- whether there are differential effects on phosphate absorption tion of mineralization of the bones after 1,25(OH)2D3 and and/or differential potency of these two sterols in their capacity doxercalciferol treatment, but BMD was still reduced after ci- to increase the phosphaturic hormone FGF-23 will require fur- nacalcet treatment associated with the decreased mineraliza- ther study. tion (Figure 5C). The effects of 1,25(OH)2D3 and of doxercalciferol on the

4 Journal of the American Society of Nephrology J Am Soc Nephrol 21: ●●●–●●●, 2010 www.jasn.org BASIC RESEARCH

itory action of active vitamin D. A recent communication of the reduced effectiveness of cinacalcet relative to active vitamin D in other rodent models sup- ports the current observations.21 Calci- mimetics have been reported to reduce parathyroid hyperplasia,22 but parathyroid gland size was not significantly decreased by cinacalcet in the 1␣(OH)ase null mice on a high-calcium intake, in contrast to the effects produced by the active vitamin D compounds. Further- more, the significant hypocalcemia observed in the vehicle-treated 1␣(OH)ase null mice was not improved by the use of cinacalcet, suggesting that the CaSR, although expressed in the gastrointestinal tract,23 is not capable of substituting for vitamin D-mediated intestinal calcium transport. When 1␣(OH)ase null mice were maintained on a rescue diet, animals be- came normocalcemic and normophos- Figure 4. Active vitamin D compounds and cinacalcet normalize osteoblast activity but phatemic and had normal PTH levels. The cinacalcet, and not active vitamin D compounds, increases osteoclasts. Bone turnover is Ϫ Ϫ normalization of these blood analytes shown: in wild-type (WT) mice; in 1␣(OH)ase null mice [1␣(OH)ase / ] on a high-calcium ␣ likely reflects the capacity of the 20% lac- intake (h) treated with vehicle, 1,25(OH)2D3, doxercalciferol, and cinacalcet; in 1 (OH)ase null mice on a rescue diet (r) treated with vehicle, and cinacalcet. (A) Osteoblast surface tose in the rescue diet to facilitate vitamin relative to bone surface (OB.S/BS); (B) serum osteocalcin concentrations; (C) osteoclast D–independent calcium and phosphorus numbers relative to bone perimeter [N.Oc/bone perimeter (mm)]; (D) serum concentra- absorption in the gut.18 Treatment of tions of TRAP5b. Each value is the mean Ϯ SEM. Number of replicates for OB.S/BS from these animals with cinacalcet induced left to right are as follows: 4 for WT, 5 for vehicle, 5 for 1,25(OH)2D3, 5 for doxercalciferol, modest hypocalcemia and hypercalci- 5 for cinacalcet, 7 for vehicle, and 6 for cinacalcet. Number of replicates for osteocalcin uria, reflecting the reduced renal cal- from left to right are as follows: 5 for WT, 3 for vehicle, 4 for 1,25(OH)2D3, 5 for cium re-absorption consequent to stim- doxercalciferol, 3 for cinacalcet, 6 for vehicle, and 5 for cinacalcet. Number of replicates ulation of the renal CaSR. Although for N.Oc/bone perimeter from left to right are as follows: 3 for WT, 4 for vehicle, 7 for serum PTH was decreased at 2 hours, at 1,25(OH)2D3, 5 for doxercalciferol, 5 for cinacalcet, 8 for vehicle, and 12 for cinacalcet. 24 hours PTH was not significantly dif- Number of replicates for serum TRAP5b from left to right are as follows: 4 for WT, 3 for ferent than vehicle-treated levels. The el- vehicle, 3 for 1,25(OH)2D3, 3 for doxercalciferol, 3 for cinacalcet, 3 for vehicle, and 4 for ϩ ϩϩ ϩϩϩ cinacalcet. P Ͻ 0.05 relative to WT mice; P Ͻ 0.01 relative to WT mice; P Ͻ 0.001 evated serum phosphorus in these ani- relative to WT mice; *P Ͻ 0.05 relative to vehicle-treated 1␣(OH)ase null mice on a mals at 2 hours but not at 24 hours likely high-calcium intake; **P Ͻ 0.01 relative to vehicle-treated 1␣(OH)ase null mice on a reflects the induction by the calcimi- high-calcium intake; ***P Ͻ 0.001 relative to vehicle-treated 1␣(OH)ase null mice on a metic of a mild hypoparathyroid state E high-calcium intake; P Ͻ 0.05 relative to vehicle-treated 1␣(OH)ase null mice on a rescue over a portion of the 24-hour period. In EEE FF diet. P Ͻ 0.001 relative to vehicle-treated 1␣(OH)ase null mice on a rescue diet. P Ͻ recent studies we have reported an in- 0.01 relative to cinacalcet-treated 1␣(OH)ase null mice on a high-calcium intake. verse relationship between bone formation and serum FGF-23 levels in the parathyroid glands likely stemmed from both direct inhibitory 1␣(OH)ase null mice.24 It will be of importance in future stud- actions of these compounds on parathyroid growth9,10 and ies to measure FGF-23 levels in response to cinacalcet to deter- PTH production,4,5 as well as from indirect effects due to in- mine if this hormone may be modified directly by the calcimi- creased serum calcium activating the CaSR. In contrast, al- metic. though cinacalcet significantly reduced circulating PTH con- Osteoblast numbers and activity were increased in bone of centrations, the results were biphasic20 and less pronounced vehicle-treated null animals on a high-calcium intake, consis- than the reductions produced by 1,25(OH)2D3 or doxercalcif- tent with the skeletal anabolic effect of the high circulating erol. The lesser effectiveness of cinacalcet in reducing PTH in concentrations of PTH.18 Osteoblast activity was reduced by this model likely reflects both the potency and the pharmaco- 1,25(OH)2D3 and doxercalciferol as PTH fell, and this likely kinetics of cinacalcet as well as the absence of any direct inhib- reflected reduction of excess osteoblast stimulation by the su-

J Am Soc Nephrol 21: ●●●–●●●, 2010 Vitamin D and a Calcimimetic 5 BASIC RESEARCH www.jasn.org

putative positive effects of cinacalcet on osteoblasts due to stimulation of the CaSR27 or other potential cation-sensing receptors29 were not clearly evident in the absence of the skeletal anabolic effect of high circulating PTH. In contrast to osteoblast levels, osteoclast numbers and activity were increased by cinacalcet in mutant mice on both a high-calcium intake and a rescue diet. The induction of osteoclast resorption by cinacalcet in our studies is consistent with in vitro evidence demon- strating the presence of CaSR in monocyte-macrophages,30 and osteoclasts,31,32 and with in situ hybridiza- tion showing the presence of CaSR tran- scripts in bone marrow cells and osteoclasts.28 In addition, in vivo studies have shown that transgenic mice with a constitutively active mutant CaSR targeted to mature osteoblasts demon- strated osteopenia because of enhanced bone resorption, apparently through the stimulation of RANK ligand produc- Figure 5. Active vitamin D compounds, but not cinacalcet, normalize femoral length and tion,33 and a recent communication has BMD. Representative contact radiographs (A), quantitation of femur lengths (B), and BMD reported that rats with CKD treated with of femurs (C) are shown: from wild-type (WT) mice; from 1␣(OH)ase null mice cinacalcet also demonstrate increased ␣ Ϫ/Ϫ [1 (OH)ase ] on a high-calcium intake (h) treated with vehicle, 1,25(OH)2D3, doxercal- bone resorption and reduced bone vol- ␣ ciferol, and cinacalcet; from 1 (OH)ase null mice on a rescue diet (r) treated with vehicle, ume.21 Finally, when the CaSR is genet- Ϯ and cinacalcet. Each femur length value is the mean SEM. Number of replicates for ically disrupted in the same 1␣(OH)ase femur length from left to right are as follows: 6 for WT, 5 for vehicle, 5 for 1,25(OH) D , 2 3 null mouse model, osteoclastic bone re- 7 for doxercalciferol, 6 for cinacalcet, 5 for vehicle, and 5 for cinacalcet. Each BMD value is the mean Ϯ SEM. Number of replicates for BMD from left to right are as follows: 4 for sorption, even in the face of normocal- cemia, is reduced.34 Consequently, it WT, 8 for vehicle, 5 for 1,25(OH)2D3, 7 for doxercalciferol, 6 for cinacalcet, 6 for vehicle, ϩ ϩϩ and 6 for cinacalcet. P Ͻ 0.05 relative to WT mice; P Ͻ 0.01 relative to WT mice; would appear that the increased oste- ϩϩϩ P Ͻ 0.001 relative to WT mice; **P Ͻ 0.01 relative to vehicle-treated 1␣(OH)ase null oclastic bone resorption stimulated by mice on a high-calcium intake; ***P Ͻ 0.001 relative to vehicle-treated 1␣(OH)ase null cinacalcet is occurring via activation of E mice on a high-calcium intake. P Ͻ 0.05 relative to vehicle-treated 1␣(OH)ase null mice the skeletal CaSR. Further studies will be EE on a rescue diet; P Ͻ 0.01 relative to vehicle-treated 1␣(OH)ase null mice on a rescue required to determine whether this in- diet. creased bone resorption occurs via an indirect action in osteoblasts or a direct praphysiologic serum PTH levels. Although cinacalcet cyclically effect on osteoclast precursors or mature osteoclasts, or both. inhibited PTH in this model, the transient peaks in circulating Interestingly, this increase in osteoclastic activity occurred in PTH produced by cinacalcet were insufficient to maintain osteo- the presence of hypocalcemia, suggesting that the effect of ci- blast numbers at the elevated levels seen in vehicle-treated null nacalcet on the osseous calcium receptor superseded the effect animals on a high-calcium intake. Furthermore, reductions in of low calcium, or that cinacalcet rendered the osseous CaSR 2ϩ osteoblast numbers were greater with cinacalcet than with the more sensitive to activation by [Ca ]e. vitamin D sterols, possibly because of intrinsic bone anabolic A previous study described the effects of therapy with calci- activity of active forms of vitamin D.18,19,25 On a rescue diet, the mimetics on bone histology in patients treated with hemodial- cinacalcet-treated null mice also maintained osteoblast num- ysis35 and noted that a few patients developed adynamic bone, bers and activity at a level significantly lower than in the se- although the sample size was small and the patients all received verely hyperparathyroid vehicle-treated mutant mice on a concurrent vitamin D and/or phosphate binder therapy. Over- high-calcium intake. CaSR has been reported to function in all, our studies in the current model are not consistent with vitro in a variety of skeletal cells including cells of the osteoblast suppression of bone turnover by cinacalcet. lineage.26–28 In our studies in these adult mice, however, any We and others have previously noted that in models ex-

6 Journal of the American Society of Nephrology J Am Soc Nephrol 21: ●●●–●●●, 2010 www.jasn.org BASIC RESEARCH

growth of vehicle-treated normocalcemic and normophosphatemic mutant mice on the rescue diet was not com- pletely normalized, direct vitamin D action on the growth plate may be required and this requirement was not met in the 1␣(OH)ase null mice treated with cinacalcet alone. In this study, treatment of null mice on a high-calcium in-

take with doxercalciferol or 1,25(OH)2D3 not only reversed rickets but also, by normalizing ambient calcium and phosphorus,18 repaired osseous mineralization. In contrast, cinacalcet only modestly reversed osteomalacia in null mice on a high-calcium intake. Recently, it was reported that mice with osteoblast-specific deletion of CaSR exhibited severely under- mineralized skeletons,38 suggesting an important role for the CaSR in osteoblast-mediated bone mineralization, yet in our studies cinacalcet only partially improved bone mineraliza- Figure 6. Active vitamin D compounds, but not cinacalcet, im- tion. The persistent hypocalcemia occurring with cinacalcet prove mineralization of the epiphyses and increase mineralized trabeculae. Representative longitudinal sections of three-dimen- treatment may therefore have limited matrix mineralization sional reconstructed distal ends of the femurs are shown: from despite any putative activation of the osseous CaSR. On a res- wild-type mice; from 1␣(OH)ase null mice on a high-calcium in- cue diet, mineralization was normalized with cinacalcet treat- take (h) treated with vehicle, 1,25(OH)2D3, doxercalciferol, and ment despite the fact that transient mild hypocalcemia was cinacalcet; from 1␣(OH)ase null mice on a rescue diet (r) treated present. The marked improvement in serum calcium in the with vehicle, and cinacalcet. Two-dimensional images of the fem- null mice on the rescue diet even after cinacalcet treatment, ora were obtained by micro-CT to generate three-dimensional coupled with the transient increase in serum phosphorus in reconstructions from 20 adjacent images as described in Concise null mice on the rescue diet after cinacalcet treatment, may Methods. White arrows denote mineralization in the secondary therefore have improved osseous mineralization. ossification sites in the epiphyses and grey arrows denote miner- In summary, our studies show that when 1␣(OH)ase null alized bony trabeculae in the metaphyses. mice are exposed to an environment in which they exhibit persistent hypocalcemia, hypophosphatemia, secondary hy- pressing the null mutation of either the 1␣(OH)ase gene18,36 or perparathyroidism, rickets, and osteomalacia, doxercalciferol 37 the VDR gene, osteoclast activity is either only modestly in- or 1,25(OH)2D3 treatment can normalize serum calcium and creased or not increased despite markedly elevated circulating PTH, parathyroid gland size, and bone parameters, although

PTH. This observation has been interpreted as evidence that 1,25(OH)2D3 may cause significant hyperphosphatemia. In active vitamin D and the VDR are required for optimal PTH contrast, cinacalcet treatment can improve hyperparathyroid- stimulated bone resorption, and active vitamin D forms may in ism in this model, but does not normalize the accompanying fact be more potent osteoclast activators than PTH. The mineral and skeletal abnormalities. When 1␣(OH)ase null present studies suggest that the mechanism for this effect of mice are exposed to an environment in which they become vitamin D may lie, in part, in correcting the hypocalcemia and normocalcemic and normophosphatemic, cinacalcet treat- thereby enhancing the stimulation of osteoclastic bone resorp- ment may perturb mineral homeostasis by causing transient tion via CaSR. hypocalcemia and hyperphosphatemia. Furthermore, our data

Calcium, phosphorus and 1,25(OH)2D have all been impli- suggest that cinacalcet may exert direct effects on the skeletal cated in the normal development and mineralization of the calcium receptor that may independently increase bone re- growth plate. However, the role of the calcium receptor in sorption and modify skeletal homeostasis. Therefore, in the growth plate development remains unclear. In the present setting of reduced active vitamin D, calcimimetics do not ap- study, treatment of 1␣(OH)ase null mice on a high-calcium pear optimal for sole treatment of secondary hyperparathy- intake with active vitamin D compounds improved both the roidism and skeletal abnormalities, especially in the presence architecture and mineralization of the growth plate and of rickets and osteomalacia. normalized long bone growth. However, our studies in 1␣(OH)ase null mice failed to show any positive effect of cinacalcet alone on growth plate architecture, or longitudi- CONCISE METHODS nal bone growth. There are several potential explanations for this observation. It is possible that calcimimetic-induced Mice and Materials modulation of calcium signaling at the CaSR differs in the The derivation of the 1␣(OH)ase homozygous null mice [1␣(OH)aseϪ/Ϫ], chondrocyte, as compared with the parathyroid and osseous by homologous recombination in embryonic stem cells, was previ- cells, or that calcium functions independently of the CaSR ously described by Panda et al.17 Briefly, a neomycin resistance gene in the growth plate. Finally, in view of the fact that the replaced exons VI, VII, and VIII of the mouse 1␣(OH)ase gene, re-

J Am Soc Nephrol 21: ●●●–●●●, 2010 Vitamin D and a Calcimimetic 7 BASIC RESEARCH www.jasn.org

moving both the ligand-binding and the heme-binding domains.17 measurement of serum osteocalcin levels according to the manufac- RT-PCR of renal RNA from homozygous 1␣(OH)aseϪ/Ϫ mice con- turer’s specifications. A mouse TRAP5b assay (IDS, Fountain Hills, firmed the lack of 1␣(OH)ase expression. Mice were generated AZ) was used according to the manufacturer’s specifications to deter- through breeding of heterozygous mice on a C57BL/6J genetic back- mine osteoclast-derived TRAP5b in mouse serum samples. ground and identified by Southern blots and/or PCR with tail genomic DNA as the template as described previously;17,18 wild-type Histology littermates were used as controls in all of the experiments. To enhance Femurs, tibias, and thyroparathyroidal tissue were removed and fixed in fertility of females, all breeders were maintained on a high-calcium PLP fixative (2% paraformaldehyde containing 0.075 M lysine and 0.01 diet containing 1.5% calcium in the drinking water and autoclaved M sodium periodate) overnight at 4°C and processed histologically as chow containing 1% calcium, 0.85% phosphorus, 0% lactose, and 2.2 described previously.40 The bones were decalcified in EDTA glycerol so- units per gram of vitamin D (Ralston Purina Co., St. Louis, MO). At lution for 14 days at 4°C. Decalcified bones and thyroparathyroidal tissue 3 ␮ approximately 21 days of age, mice were weaned and maintained on were dehydrated and embedded in paraffin, after which 5- m sections drinking water containing 1.5% calcium gluconate and either a nor- were cut on a rotary microtome. The sections were stained with hema- mal diet of autoclaved chow containing 1% calcium, 0.85% phospho- toxylin and eosin (H&E) or histochemically for TRAP activity41 and alkaline phosphatase (ALP) activity as described below. Alternatively, un- rus, 0% lactose, and 2.2 units per gram of vitamin D3 (Ralston Purina Co.) or a rescue diet18 of ␥-irradiated chow containing 20% lactose, decalcified bones were embedded in LR White acrylic resin (London Resin Co., London), and 1-␮m sections were cut on an ultramicrotome. 2% calcium, 1.25% phosphorus, and 2.2 units per gram of vitamin D2 (TD96348; Harlan Teklad, Madison, WI). No significant differences These sections were stained for mineral with the von Kossa staining pro- in any parameter determined were observed in wild-type mice on a cedure and counterstained with toluidine blue. Histomorphometric in- high-calcium intake or a rescue diet.18 Consequently, control data dices were determined as suggested by the ASBMR Histomorphometry are shown for the wild-type mice on the high-calcium intake. Ci- Nomenclature Committee.42 nacalcet (Amgen, Thousand Oaks, CA) was obtained via the local pharmacy. Doxercalciferol and 1,25(OH) D were provided by Histochemical Staining for TRAP 2 3 Enzyme histochemistry for TRAP staining was performed as de- Genzyme Corporation, Middleton, WI. scribed previously.41 De-waxed sections were preincubated for 20 minutes in buffer containing 50 mM sodium acetate and 40 mM In Vivo Experiments sodium tartrate at pH 5.0. Sections were then incubated for 15 min- All animal experiments were carried out in compliance with and ap- utes at room temperature in the same buffer containing 2.5 mg/ml proval by the Institutional Animal Care and Use Committee. Mice naphthol AS-MX phosphate (Sigma-Aldrich, St. Louis) in dimethyl- were maintained in a virus- and parasite-free barrier facility and ex- formamide as substrate and 0.5 mg/ml fast garnet GBC (Sigma-Al- posed to a 12-hour/12-hour light/dark cycle. From weaning, animals drich) as a color indicator for the reaction product. After the product received, every 2 days by subcutaneous injection, either of the follow- was washed with distilled water, the sections were counterstained with ing: vehicle (50% propylene glycol, 40% saline, 10% ethanol); doxer- methyl green and mounted in Kaiser’s glycerol. ALP staining was calciferol, 150 pg/g; 1,25(OH) D , 50 pg/g; or cinacalcet, 30 ␮g/g daily 2 3 performed on methyl methacrylate (MMA)-embedded undecalcified by oral gavage. The doses of doxercalciferol and 1,25(OH) D used 2 3 bone samples using a vector red alkaline phosphatase substrate kit were based on preliminary studies in the null mice on a high-calcium (Vector Laboratories43). intake; the doses selected for each compound were those that reduced serum PTH to the normal range without causing hypercalcemia. The Double Calcein Labeling dose of cinacalcet used was based on previous studies in mice and Double calcein labeling was performed by intraperitoneal injection of 19,21,39 rats and on preliminary studies in the null mice on a high- mice with 10 ␮g of calcein per gram of body weight (C-0875; Sigma- ␮ calcium intake in which doses of 10, 20, 30, and 50 g/g were em- Aldrich) at 10 and 3 days before the mice were killed. Bones were ␮ ployed; no further PTH suppression was seen at 50 g/g than at 30 harvested and embedded in LR White acrylic resin as described above. ␮ g/g, so the latter dose was selected. At about 3 and one-half months Serial sections were cut, and the freshly cut surface of each section was of age, the animals were killed, and blood and urine samples were viewed and imaged using fluorescence microscopy. The double cal- collected for analysis. Serum was obtained 2 and 24 hours after the cein interlabel width was measured using Northern Eclipse image final cinacalcet administration and 24 hours after the final doxercal- analysis software version 6.0 (Empix Imaging Inc., Mississauga, ON, ciferol and 1,25(OH)2D3 injections. Tibias, femurs, and thyropar- Canada), and the mineral apposition rate (MAR; MAR ϭ interlabel athyoidal tissues were also obtained, at the time of death, for histology width/labeling period) was calculated. The bone formation rate was and for histomorphometric analysis. calculated according to the following formula: BFR ϭ MAR ϫ (MS/ BS), where MS is the mineralizing surface and BS is the bone surface.41 Biochemical and Hormone Analyses Calculations were made on a minimum of duplicate specimens from Serum calcium and phosphorus and urine calcium were determined replicate mice of each group. by autoanalyzer (Beckman Synchron 67, Beckman Instruments). Se- rum intact PTH was measured by a two-site immunoradiometric as- Histomorphometry say (Immutopics, San Clemente, CA). A mouse osteocalcin two-site After H&E staining or immunohistochemical staining, histomorpho- immunoradiometric assay (IRMA; Immutopics) was used for the metric measurements were performed as described previously42 in the

8 Journal of the American Society of Nephrology J Am Soc Nephrol 21: ●●●–●●●, 2010 www.jasn.org BASIC RESEARCH secondary spongiosa in the metaphyseal area (0.5 mm below the traced areas of parathyroid glands were recorded automatically by growth plate) at the distal end of femur. The parameters obtained for Northern Eclipse image. bone formation were the osteoblast surface per bone surface (Ob.S/ BS; percentage), and the MAR (micrometers per day) and the BFR/ Statistical Analysis BS(␮m3/␮m2 per day). The parameter measured for bone resorption Statistical comparisons, using Graph-Pad Prism (GraphPad Software was the number of osteoclasts per bone perimeter [N.Oc./bone pe- Inc., San Diego) analysis software were made using t test or ANOVA, rimeter (mm)]. After H&E staining or histochemical staining of sec- followed by a Bonferroni adjustment or a Neuman-Keuls multiple tions from replicate mice of each group, images of fields were photo- comparison test. P Ͻ 0.05 was considered significant. graphed with a digital camera. Images of micrographs from single sections were digitally recorded using a rectangular template, and recordings were processed and analyzed using Northern Eclipse im- ACKNOWLEDGMENTS age analysis software as described.44–46 This work was supported by an investigator-initiated grant to D.G. from Genzyme Corporation and by a grant to D.G. from the Cana- BMD Analysis Densitometry was performed by PIXImus on the right femur under dian Institutes of Health Research. anesthesia as described previously.46 In brief, mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (0.1 mg/kg) in DISCLOSURES PBS and placed prone on the platform of a PIXImus densitometer D.G. has served as a consultant for Amgen, Genzyme, Eli Lilly, Merck, No- (software version 1.46.007; Lunar Corp., Madison, WI) for BMD vartis, Proctor and Gamble/Aventis, and Servier. S.S. is an employee of Gen- measurements according to the manufacturer’s instruction. In some zyme. experiments, the variability in measurements was examined by re- peating scans after repositioning the animals. Percent coefficient of variation of BMD for the repeated scans was 1 to 3%. REFERENCES

1. Brown EM: Calcium receptor and regulation of parathyroid hormone Skeletal Radiography secretion. Rev Endocr Metab Disord 1: 307–315, 2000 After removal of soft tissue, radiographs were taken using a Faxitron 2. Jurutka PW, Bartik L, Whitfield GK, Mathern DR, Barthel TK, Gurevich model 805 radiographic inspection system (Faxitron Contact, Fax- M, Hsieh JC, Kaczmarska M, Haussler CA, Haussler MR: Vitamin D itron, 22-kV voltage, and 4-minute exposure time). X-Omat TL film receptor: Key roles in bone mineral pathophysiology, molecular mech- (Eastman Kodak, Rochester, NY) was used and processed routinely. anism of action, and novel nutritional ligands. J Bone Miner Res 22 Suppl 2: V2–V10, 2007 3. Andress DL, Coyne DW, Kalantar-Zadeh K, Molitch ME, Zangeneh F, Microcomputed Tomography Sprague SM: Management of secondary hyperparathyroidism in Femurs obtained from mice were dissected free of soft tissue, fixed stages 3 and 4 chronic kidney disease. Endocr Pract 14: 18–27, 2008 4. Russell J, Lettieri D, Sherwood LM: Suppression by 1,25(OH) D of overnight in 70% ethanol, and analyzed by microcomputed tomog- 2 3 transcription of the pre-proparathyroid hormone gene. Endocrinology raphy with a SkyScan 1072 scanner and associated analysis software 119: 2864–2866, 1986 (SkyScan, Antwerp, Belgium) as described.46 Scans were performed 5. Silver J, Naveh-Many T, Mayer H, Schmelzer JH, Popovtzer MM: on the distal part of the femur. Image acquisition was performed at Regulation by vitamin D metabolites of parathyroid hormone gene 100 kV and 98 ␮A with a 0.9° rotation between frames. Thresholding transcription in vivo in the rat. J Clin Invest 78: 1296–1301, 1986 was applied to the images to segment the bone from the background. 6. Yamamoto M, Igarashi T, Muramatsu M, Fukagawa M, Motokura T, Ogata E: Hypocalcemia increases and hypercalcemia decreases the Three-dimensional renderings of the distal 3.5 mm of the femora was steady-state level of parathyroid hormone messenger RNA in the rat. reconstructed using two-dimensional data from scanned slices with J Clin Invest 83: 1053–1056, 1989 the 3D Creator software supplied with the instrument. The trabecular 7. Moallem E, Silver J, Kilav R, Naveh-Many T: RNA protein binding and bone region of interest was drawn to include all cancellous bone in the post-transcriptional regulation of parathyroid hormone gene expres- metaphysis, and three-dimensional analysis was performed to calcu- sion by calcium and phosphate. J Biol Chem 273: 5253–5259, 1998 8. Wada M, Furuya Y, Sakiyama, J, Kobayashi N, Miyata S, Ishii H, late bone volume (BV)/tissue volume (TV). The resolution of the Nagano N: The calcimimetic compound NPS R-568 suppresses para- microcomputed tomography images was 18.2 ␮m. thyroid cell proliferation in rats with renal insufficiency. Control of parathyroid cell growth via a calcium receptor. J Clin Invest 100: 2977–2983, 1997 Computer-Assisted Image Analysis 9. Kremer R, Bolivar I, Goltzman D, Hendy GN: Influence of calcium and After H&E staining or histochemical staining of sections from repli- 1,25-dihydroxycholecalciferol on proliferation and proto-oncogene cate mice of each group, images of fields were photographed with a expression in primary cultures of bovine parathyroid cells. Endocrinol- Sony digital camera. Images of micrographs from single sections were ogy 125: 935–941, 1989 digitally recorded using a rectangular template, and recordings were 10. Cozzolino M, Lu Y, Finch J, Slatopolsky E, Dusso AS: p21WAF1 and TGF-alpha mediate parathyroid growth arrest by vitamin D and high processed and analyzed using Northern Eclipse image analysis soft- calcium. Kidney Int 60: 2109–2117, 2001 46 ware. To measure the size of the parathyroid glands, the border of 11. Slatopolsky E, Weerts C, Thielan J, Horst R, Harter H, Martin KJ: the glands were traced on micrographs of H&E-stained sections and Marked suppression of secondary hyperparathyroidism by intravenous

J Am Soc Nephrol 21: ●●●–●●●, 2010 Vitamin D and a Calcimimetic 9 BASIC RESEARCH www.jasn.org

administration of 1,25-dihydroxycholecalciferol in uremic patients. Riccardi D: Physiological changes in extracellular calcium concentra- J Clin Invest 74: 2136–2143, 1984 tion directly control osteoblast function in the absence of calciotropic 12. Coburn JW, Maung HM, Elangovan L, Germain MJ, Lindberg JS, hormones. Proc Natl Acad SciUSA101: 5140–5145, 2004 Sprague SM, Williams ME, Bishop CW: Doxercalciferol safely sup- 28. Chang W, Tu C, Chen TH, Komuves L, Oda Y, Pratt SA, Miller S, presses PTH levels in patients with secondary hyperparathyroidism Shoback D: Expression and signal transduction of calcium-sensing associated with chronic kidney disease stages 3 and 4. Am J Kidney receptors in cartilage and bone. Endocrinology 140: 5883–5893, 1999 Dis 43: 877–890, 2004 29. Pi M, Faber P, Ekema G, Jackson PD, Ting A, Wang N, Fontilla-Poole 13. Nemeth EF, Steffey ME, Hammerland LG, Hung BCP, Van Wagenen M, Mays RW, Brunden KR, Harrington JJ, Quarles LD: Identification of BC, Delmar EG, Balandrin MF: Calcimimetics with potent and selec- a novel extracellular cation-sensing G-protein-coupled receptor. J Biol tive activity on the parathyroid calcium receptor. Proc Natl Acad Sci U Chem 280: 40201–40209, 2005 SA95: 4040–4045, 1998 30. House MG, Kohlmeier L, Chattopadhyay N, Kifor O, Yamaguchi T, 14. Block GA, Martin KJ, de Francisco ALM, Turner SA, Avram MM, Leboff MS, Glowacki J, Brown EM: Expression of an extracellular Suranyi MG, Hercz G, Cunningham J, Abu-Alfa AK, Messa P, Coyne calcium-sensing receptor in human and mouse bone marrow cells. DW, Locatelli F, Cohen RM, Evenepoel P, Moe SM, Fournier A, Braun J Bone Miner Res 12: 1959–1970, 1997 J, McCary LC, Zani VJ, Olson KA, Dru¨eke TB, GoodmanWG: Cinacal- 31. Kanatani M, Sugimoto T, Kanzawa M, Yano S, Chihara K: High extra- cet for secondary hyperparathyroidism in patients receiving hemodi- cellular calcium inhibits osteoclast-like cell formation by directly acting alysis. N Engl J Med 350: 1516–1525, 2004 on the calcium-sensing receptor existing in osteoclast precursor cells. 15. Silverberg SJ, Rubin MR, Faiman C, Peacock M, Shoback DM, Small- Biochem Biophys Res Commun 261: 144–148, 1999 ridge RC, Schwanauer LE, Olson KA, Klassen P, Bilezikian JP: Cinacal- 32. Kameda T, Mano H, Yamada Y, Takai H, Amizuka N, Kobori M, Izumi cet hydrochloride reduces the serum calcium concentration in inop- N, Kawashima H, Ozawa H, Ikeda K, Kameda A, Hakeda Y, Kumegawa erable parathyroid carcinoma. J Clin Endocrinol Metab 92: 3803– M: Calcium-sensing receptor in mature osteoclasts, which are bone 3808, 2007 resorbing cells. Biochem Biophys Res Commun 245: 419–422, 1998 16. Alon US, Levy-Olomucki R, Moore WV, Stubbs J, Liu S, Quarles LD: 33. Dvorak MM, Chen TH, Orwoll B, Garvey C, Chang W, Bikle DD, Calcimimetics as an adjuvant treatment for familial hypophosphatemic Shoback DM: Constitutive activity of the osteoblast Ca2ϩ-sensing rickets. Clin J Am Soc Nephrol 3: 658–664, 2008 receptor promotes loss of cancellous bone. Endocrinology 148: 3156– 17. Panda DK, Miao D, Tremblay ML, Sirois J, Farookhi R, Hendy GN, 3163, 2007 Goltzman D: Targeted ablation of the 25-hydroxyvitamin D 1alpha- 34. Richard C, Huo R, Samadfam R, Bolivar I, Miao D, Brown EM, Hendy hydroxylase enzyme: Evidence for skeletal, reproductive, and immune GN, Goltzman D: The calcium sensing receptor and 25-hydroxyvita- dysfunction. Proc Natl Acad SciUSA98: 7498–7503, 2001 min D-1alpha-hydroxylase interact to modulate skeletal growth and 18. Panda DK, Miao D, Bolivar I, Li J, Huo R, Hendy GN, Goltzman D: bone turnover. J Bone Miner Res 25: 1627–1636, 2010 Inactivation of the 25-hydroxyvitamin D 1alpha-hydroxylase and vita- 35. Malluche HH, Monier-Faugere MC, Wang G, Fraza˜O JM, Charytan C, min D receptor demonstrates independent and interdependent ef- Coburn JW, Coyne DW, Kaplan MR, Baker N, McCary LC, Turner SA, fects of calcium and vitamin D on skeletal and mineral homeostasis. Goodman WG: An assessment of cinacalcet HCl effects on bone J Biol Chem 279: 16754–16766, 2004 histology in dialysis patients with secondary hyperparathyroidism. Clin 19. Xue Y, Karaplis AC, Hendy GN, Goltzman D, Miao D: Genetic models Nephrol 69: 269–278, 2008

show that parathyroid hormone and 1,25-dihydroxyvitamin D3 play 36. Dardenne O, Prud’homme J, Hacking SA, Glorieux FH, St-Arnaud R: distinct and synergistic roles in postnatal mineral ion homeostasis and Correction of the abnormal mineral ion homeostasis with a high- skeletal development. Hum Mol Genet 14: 1515–1528, 2005 calcium, high-phosphorus, high-lactose diet rescues the PDDR phe- 20. Nemeth EF, Heaton WH, Miller M, Fox J, Balandrin MF, Van Wagenen notype of mice deficientfor the 25-hydroxyvitamin D-1alpha-hydroxy- BC, Colloton M, Karbon W, Scherrer J, Shatzen E, Rishton G, Scully S, lase (CYP27B1). Bone 32: 332–340, 2003 Qi M, Harris R, Lacey D, Martin D: Pharmacodynamics of the type II 37. Amling M, Priemel M, Holzmann T, Chapin K, Rueger JM, Baron R, calcimimetic compound cinacalcet HCl. J Pharmacol Exp Ther 308: Demay MB: Rescue of the skeletal phenotype of vitamin D receptor- 627–635, 2004 ablated mice in the setting of normal mineral ion homeostasis: formal 21. Finch JL, Tokumoto M, Nakamura H, Yao W Dr, Shahnazari M, Lane N histomorphometric and biomechanical analyses. Endocrinology 140: Md, Slatopolsky E: Effect of paricalcitol and cinacalcet on serum 4982–4987, 1999 phosphate, FGF-23, and bone in rats with chronic kidney disease. 38. Chang W, Tu C, Chen TH, Bikle D, Shoback D: The extracellular Am J Physiol Renal Physiol 298: F1315–F1322, 2010 calcium-sensing receptor (CaSR) is a critical modulator of skeletal 22. Colloton M, Shatzen E, Miller G, Stehman-Breen C, Wada M, Lacey D, development. Sci Signal 1: ra1, 2008 Martin D: Cinacalcet HCl attenuates parathyroid hyperplasia in a rat 39. Kawata T, Imanishi Y, Kobayashi K, Kenko T, Wada M, Ishimura E, Miki model of secondary hyperparathyroidism. Kidney Int 67: 467–476, 2005 T, Nagano N, Inaba M, Arnold A, Nishizawa Y: Relationship between 23. Herbert SC, Cheng S, Geibel J: Functions and roles of the extracellular parathyroid calcium-sensing receptor expression and potency of the Ca2ϩ-sensing receptor in the gastrointestinal tract. Cell Calcium 35: calcimimetic, cinacalcet, in suppressing parathyroid hormone secre- 239–247, 2004 tion in an in vivo murine model of primary hyperparathyroidism. Eur J 24. Samadfam R, Richard C, Nguyen-Yamamoto L, Bolivar I, Goltzman D: Endocrinol 153: 587–594, 2005 Bone formation regulates circulating concentrations of fibroblast 40. Miao D, Bai X, Panda D, McKee MD, Karaplis AC, Goltzman D: growth factor 23. Endocrinology 150: 4835–4845, 2009 Osteomalacia in Hyp mice is associated with abnormal Phex expres- 25. Xue Y, Karaplis AC, Hendy GN, Goltzman D, Miao D: Exogenous sion and with altered bone matrix protein expression and deposition.

1,25-dihydroxyvitamin D3 exerts a skeletal anabolic effect and im- Endocrinology 142: 926–939, 2001 proves mineral ion homeostasis in mice that are homozygous for both 41. Miao D, Scutt A: Recruitment, augmentation and apoptosis of rat

the 1alpha-hydroxylase and parathyroid hormone null alleles. Endo- osteoclasts in 1,25-(OH)2D3 response to short-term treatment with crinology 147: 4801–4810, 2006 1,25-dihydroxyvitamin D3 in vivo. BMC Musculoskelet Disord 3: 16– 26. Chattopadhyay N, Yano S, Tfelt-Hansen J, Rooney P, Kanuparthi D, 26, 2002 Bandyopadhyay S, Ren X, Terwilliger E, Brown EM: Mitogenic action 42. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, of calcium-sensing receptor on rat calvarial osteoblasts. Endocrinol- Ott SM, Recker RR: Bone histomorphometry: Standardization of nomen- ogy 145: 3451–3462, 2004 clature, symbols, and units. Report of the ASBMR Histomorphometry 27. Dvorak MM, Siddiqua A, Ward DT, Carter DH, Dallas SL, Nemeth EF, Nomenclature Committee. J Bone Miner Res 2: 595–610, 1987

10 Journal of the American Society of Nephrology J Am Soc Nephrol 21: ●●●–●●●, 2010 www.jasn.org BASIC RESEARCH

43. Samadfam R, Xia Q, Goltzman D: Co-treatment of PTH with osteo- 45. Miao D, Li J, Xue Y, Su H, Karaplis AC, Goltzman D: Parathyroid protegerin or alendronate increases its anabolic effect on the skeleton hormone-related peptide is required for increased trabecular bone of oophorectomized mice. J Bone Miner Res 22: 55–63, 2007 volume in parathyroid hormone-null mice. Endocrinology 145: 3554– 44. Miao D, He B, Jiang Y, Kobayashi T, Soroceanu MA, Zhao J, Su H, 3562, 2004 Tong X, Amizuka N, Gupta A, Genant HK, Kronenberg HM, Goltz- 46. Samadfam R, Xia Q, Goltzman D: Pretreatment with anticatabolic man D, Karaplis AC: Osteoblast-derived PTHrP is a potent endog- agents blunts but does not eliminate the skeletal anabolic response to enous bone anabolic agent that modifies the therapeutic efficacy of parathyroid hormone in oophorectomized mice. Endocrinology 148: administered PTH 1–34. J Clin Invest 115: 2402–2411, 2005 2778–2787, 2007

J Am Soc Nephrol 21: ●●●–●●●, 2010 Vitamin D and a Calcimimetic 11