Comparative effects of calcitriol and calcimimetic on bone health in renal insufficiency

Calcitriol and calcimimetics are used to treat hyperparathyroidism secondary to chronic kidney disease (CKD). Calcitriol administration and the subsequent increase in serum calcium concentration decrease parathyroid hormone (PTH) levels, which should reduce bone remodeling. We have previously reported that, when maintaining a given concentration of PTH, the addition of calcimimetics is associated with an increased bone cell activity. Whether calcitriol administration affects bone cell activity while PTH is maintained constant should be evaluated in an animal model of renal osteodystrophy. The aim of the present study was to compare in CKD PTH‐clamped rats the bone effects of calcitriol and calcimimetic administration. The results show that the administration of calcitriol and calcimimetic at doses that induced a similar reduction in PTH secretion produced dissimilar effects on osteoblast activity in 5/6 nephrectomized (Nx) rats with secondary hyperparathyroidism and in Nx rats with clamped PTH. Remarkably, in both rat models, the administration of calcitriol decreased osteoblastic activity, whereas calcimimetic increased bone cell activity. In vitro, calcitriol supplementation inhibited nuclear translocation of β‐catenin and reduced proliferation, osteogenesis, and mineralization in mesenchymal stem cells differentiated into osteoblasts. In conclusion, besides the action of calcitriol and calcimimetics at parathyroid level, these treatments have specific effects on bone cells that are independent of the PTH level.


| INTRODUCTION
Calcitriol therapy is used in the treatment of secondary hyperparathyroidism in chronic kidney disease (CKD) patients.Calcitriol reduces PTH synthesis and secretion by acting on the parathyroid vitamin D receptor (VDR) and also through its calcemic effect. 1 The calcemic effect of calcitriol may not be explained solely by the increase in the intestinal absorption of calcium.3][4] These effects are partially due to increased production of the receptor activator of NF-κB ligand (RANKL) by osteoblasts. 5At bone level, calcitriol administration reduces osteitis fibrosa, which should be expected if there is a concomitant decline in PTH.However, in some patients, the decrease in bone formation is not explained by moderate changes in PTH. 6These observations raise the question whether PTH-independent effects of therapeutic doses of calcitriol may affect bone remodeling.
In patients with moderate renal failure, the administration of calcitriol (0.5 μg/24 h) caused a reduction in PTH levels together with a decrease in osteoid thickness and bone resorption. 7More recently, Bernardor et al. 8 found in pediatric CKD patients that calcitriol reduced osteoclast differentiation of human peripheral blood mononuclear cells and also observed resistance to the action of calcitriol due to downregulation of the VDR in renal failure.Vitamin D deficiency is a risk factor for fracture in the general population, 9 and it has been also associated with cardiovascular diseases, among others. 10Nevertheless, the beneficial bone effects of calcitriol have been demonstrated in the prevention of fractures in postmenopausal osteoporosis. 11,12In CKD patients on hemodialysis, low vitamin D levels have been also related to defective mineralization and bone formation. 13These bone effects seem to be mainly due to the action of vitamin D on plasma calcium and phosphate homeostasis. 14,15ith respect to the bone effects of the activation of the calcium sensing receptor (CaSR) by calcimimetics, we have previously shown that in uremic animals with a clamp of PTH the administration of calcimimetics augments bone cell activity. 16This finding has been supported by clinical studies. 17ccording to the abovementioned findings, we hypothesize that the administration of either calcimimetic or calcitriol may induce differential osseous effects in the context of CKD and that this effect may be independent of the prevailing PTH level.Given that both calcitriol and calcimimetics decrease PTH secretion, it is essential to maintained PTH levels clamped to assess the possible effect of these molecules in the bone.To achieve this, we performed a parathyroidectomy followed by infusion of PTH levels, which should reduce bone remodeling.We have previously reported that, when maintaining a given concentration of PTH, the addition of calcimimetics is associated with an increased bone cell activity.Whether calcitriol administration affects bone cell activity while PTH is maintained constant should be evaluated in an animal model of renal osteodystrophy.The aim of the present study was to compare in CKD PTH-clamped rats the bone effects of calcitriol and calcimimetic administration.The results show that the administration of calcitriol and calcimimetic at doses that induced a similar reduction in PTH secretion produced dissimilar effects on osteoblast activity in 5/6 nephrectomized (Nx) rats with secondary hyperparathyroidism and in Nx rats with clamped PTH.Remarkably, in both rat models, the administration of calcitriol decreased osteoblastic activity, whereas calcimimetic increased bone cell activity.In vitro, calcitriol supplementation inhibited nuclear translocation of βcatenin and reduced proliferation, osteogenesis, and mineralization in mesenchymal stem cells differentiated into osteoblasts.In conclusion, besides the action of calcitriol and calcimimetics at parathyroid level, these treatments have specific effects on bone cells that are independent of the PTH level.

K E Y W O R D S
bone histomorphometry, calcimimetic, calcitriol, renal osteodystrophy, secondary hyperparathyroidism to maintain high and constant levels able to overcome the skeletal resistance to PTH, mimicking the conditions observed in 5/6 nephrectomized (Nx) rats.Thus, the aim of the present study was to compare the bone effects of calcitriol and a calcimimetic in a rat model of CKD with clamped PTH.The experiments were performed in matched groups of uremic rats with and without PTH clamp, thus excluding the bone effects triggered by treatment-induced PTH modulation.

| Study approval
Animal care and experimental procedures were approved by the Research and Ethics Committees of the Universidad de Córdoba (file number 14/03/2018/026) in accordance with Directive 2010/63/EU of the European Parliament and with institutional guidelines for the care and use of laboratory animals and the Declaration of Helsinki.

| Animal models and treatments
Three-to four-month-old male Wistar rats (Janvier Labs, Le Genest-Saint-Isle, France) were housed individually in cages in specific pathogen free (SPF) conditions, and each cage was tagged with a permanent numerical designation.Animals were anesthetized using sevoflurane (Sevorane, Abbvie, Madrid, Spain) to undergo a twostep 5/6 nephrectomy (Nx) as previously described. 18ll surgeries were done by the same surgeon.Animals were randomly distributed into four groups, housed individually, and standard diet was switched to a moderately high phosphate diet (0.9% phosphorus and 0.6% calcium, Altromin GmbH & Co. KG, Lage, Germany), with ab libitum access to water.Three groups were intraperitoneally treated with calcitriol (Kern Pharma, Barcelona, Spain) at 20, 40, or 60 ng/kg/48 h, respectively.A fourth group was treated with the calcimimetic AMG 641 (Amgen, Inc., CA, USA; 1.5 mg/kg/48 h; s.c) and the fifth group received vehicle (saline).Sham rats fed on a 0.6% phosphorus, and 0.6% calcium diet were also included as Sham group.
To decide the optimal dose of calcitriol that should be compared with calcimimetic (1.5 mg/kg), we measured plasma PTH for each dose of calcitriol (20, 40, and 60 ng/ kg).The dose of calcitriol that reduced the plasma concentration of PTH to a level similar to that of calcimimetic (1.5 mg/kg) was chosen for the comparative study.The expected reduction in PTH levels in treated animals could have effects on bone that must be separated from those directly induced by calcitriol and calcimimetic.Therefore, additional experiments were performed in Nx and parathyroidectomized (PTX) rats receiving constant infusion of rat recombinant PTH 1-34 (Sigma-Aldrich CO, St. Louis, MO, USA) at 0.132 μg/100 g/h using ALZET pumps (ALZET model 2ML4, Charles River Laboratories, Barcelona, Spain) as previously described. 19The dose of PTH chosen for the experiments was sufficient to overcome the skeletal resistance to PTH under uremic conditions, and the animals received constant PTH infusion for 28 days.The nephrectomy, the parathyroidectomy, and the pump insertion were performed consecutively in 20-25 min, and the administration of either calcimimetic or calcitriol began the following day.Those animals with evidence of pump malfunctioning were excluded from the study.
To evaluate bone mineralization, animals received calcein at 25 mg/kg/s.c(Sigma-Aldrich CO) at days 9 and 2 before sacrifice.After 28 days, rats were exsanguinated by aortic puncture under general anesthesia with sevoflurane and blood samples were collected to determine biochemical parameters.Bones were placed in 70% ethanol for subsequent histomorphometry analysis.A scheme of the in vivo experiment is represented in Figure 1.

| Blood biochemistry
Blood samples were centrifuged at 2000 × g for 10 min at 4°C for plasma separation.Serum ionized calcium level was measured using an ion selective electrode (RAPIDLab® 348EX, Siemens Healthcare Diagnostics, Frimley, Camberley, UK).Plasma intact PTH was assayed using enzyme-linked immunosorbent assay (ELISA) kit (Immutopic, San Clemente, CA, USA).Plasma concentration of the PTH 1-34 fragment was measured with a specific ELISA kit (Phoenix Pharmaceuticals, Burlingame, CA).Intact FGF23 level was also determined by ELISA (Kainos Laboratories, Tokyo, Japan), and the concentration of plasma calcitriol was measured using a radioimmunoassay kit (Immunodiagnostic Systems (IDS) Ltd, Boldon, UK).Total calcium, phosphate, and creatinine were determined by spectrophotometry using commercially available kits (BioSystems SA, Barcelona, Spain).

| Bone histomorphometry
Right femurs were dissected at the time of euthanasia and placed in 70% ethanol for 3 days.Thereafter, bones were cut at the middle and distal femurs and were dehydrated in alcohol, cleared with xylene, and embedded in methyl methacrylate.Parameters of histomorphometry were quantified in undecalcified 5-μm sections stained with the Villanueva Goldner trichrome method. 20Green stained areas were considered as mineralized bone, and red stained areas measuring at least 1.5 μm were counted as osteoid.Kinetic parameters were calculated by analysis of the calcein labels lengths and distance in undecalcified 10-μm sections.
Bone histomorphometric parameters were assessed in cancellous bone within the secondary spongiosa (0.25 mm from endocortical bone and growth plate) under 200× magnification as previously described, 21 and derived indices were determined by standardized calculations and the nomenclature described by Dempster et al., 22 using the Osteometrics system (OsteoMetrics, Decatur, IL, USA) with a Leica DM4000D microscope (Leica Biosystems, Newcastle, UK).The parameters evaluated were bone volume per tissue volume (BV/TV), osteoid volume per bone volume (OV/BV), osteoid surface per bone surface (OS/BS), osteoblast surface per bone surface (Ob.S/BS), eroded surface per bone surface (ES/BS), osteoclast surface per bone surface (Oc.S/BS), osteoid thickness (O.Th), trabecular separation (Tb.Sp), trabecular number (Tb.N), mineralizing surface per bone surface (MS/BS), mineral apposition rate (MAR), adjusted apposition rate (Aj.AR), bone formation rate per bone surface (BFR/BS), mineralization lag time (Mlt), and osteoid maturation time (Omt).Bone histomorphometry parameters were measured and quantified by a single researcher.
Representative bright-field microphotographs were taken with a Leica DM2000 LED microscope and with a Leica MC190 HD camera using Leica Application Suite 4.8.0 software (Leica Biosystems).Fluorescence microphotographs were obtained with a Leica DMi8 digital using Leica Application Suite X Software (Leica Biosystems).

| Mesenchymal stem cell isolation and osteogenic differentiation
To evaluate the in vitro effects of calcitriol on osteogenic differentiation of bone marrow mesenchymal stem cells (MSC), male Wistar rats were euthanized as described above and tibias and femurs were collected, cut F I G U R E 1 Scheme of the procedure of in vivo experiments.Male Wistar rats underwent 5/6 nephrectomy (5/6Nx) and received either calcitriol, calcimimetic, or vehicle every other day (red arrows).The same treatments were administered to another set of with 5/6 nephrectomy rats with total parathyroidectomy and PTH replacement via ALZET Pump.Calcein was administered 9 and 2 days before sacrifice (green arrows).After 28 days, animals were sacrificed to obtain femurs and blood samples for bone histology and biochemical analyses.Sham rats were included as an additional control group (sham).

| Osteoclast differentiation of bone marrow cells
Male Wistar rats weighing approximately 250 g were anesthetized with sodium thiopental (50 mg/kg i.p) and sacrificed by aortic puncture.Rat femurs and tibias were obtained, and bone marrow was perfused as described above.Then, cells were centrifuged and suspended in ACK lysing buffer.After 2 min of incubation, cells were subsequently centrifuged and cultured with α-MEM with 10% FBS plus rat recombinant 10 ng/mL M-CSF (PeproTech) and incubated at 37°C, 5% CO 2 with saturated humidity.After 24 h, non-adherent cells were collected and cultured in 6-well plates with α-MEM plus 10% FBS containing rat recombinant 30 ng/mL M-CSF and maintained in the incubator.After 3 days, adherent cells were collected carefully using a scrapper, counted in a Neubauer chamber, and cultured in 24-well plates with α-MEM with FBS 10% containing rat recombinant 30 ng/mL M-CSF and rat recombinant 100 ng/mL RANKL.Additionally, calcitriol at 10 −10 M or AMG641 at 100 μM was added.Medium was replaced every 2 days, and after 5 days, cells were processed for quantification of Cathepsin K (CTSK) mRNA expression and tartrate-resistant acid phosphatase (TRAP) staining, both commonly used as osteoclastic markers.

| Identification of TRAP-positive cells
TRAP staining was performed using a commercially available kit (Sigma-Aldrich) according to manufacturer instructions.Briefly, cells were fixed with a citrateparaformaldehyde-acetone solution for 30 s at room temperature, rinsed three times with 37°C pre-warmed deionized distillated water (ddH2O), and incubated within TRAP-staining solution (0.25 M Acetate, 0.7 mg/mL Diazotized Fast Garnet GBC, 1.25 mg/mL Naphtol AS-BI phosphate, and 0.67 M tartrate) at 37°C for 1 h.Then, cells were rinsed twice with ddH2O, counterstained with hematoxylin for 2 min, rinsed with tap water, and air-dried.Microphotographs were taken with a Leica DM2000 LED microscope.

| Alkaline phosphatase activity and matrix mineralization analyses
Alkaline phosphatase activity (ALP) was measured using 20 μg of protein from the cytosolic enriched fraction incubated of lysate from culture cells in 2 mM p-nitrophenol phosphate (Sigma-Aldrich) for 30 min at 37°C.The reaction was stopped by adding 3 M NaOH, and the amount of hydrolyzed p-nitrophenol phosphate per minute (ALP activity) was measured by quantifying of absorbance at 405 nm.ALP activity was related to protein content measured by Bradford method and expressed as a fold change of the undifferentiated MSC.
Matrix mineralization was detected by alizarin red S staining.Cells were washed twice with PBS, fixed with 2% paraformaldehyde 1% sucrose for 15 min, and subsequently washed with PBS three times.Then, cells were stained with alizarin red S 40 mM pH 4.1 (Sigma-Aldrich) for 20 min and washed 4 times for 5 min with water at pH 7. Thereafter, water was removed, and samples were dried at room temperature.Plates were scanned in a WIFI OKI Scanner (OKI, Madrid, Spain).Microphotographs were obtained at 100x using a Leica DM2000 LED microscope.

| Statistical analysis
Statistics and plots were performed using Graphpad Prism 8 (GraphPad Software, San Diego, CA, USA).Differences between two groups were analyzed by two-tailed T-test.One-way ANOVA was used to analyze differences among 3 or more groups.P-values lower than .05were considered significant.In tables, data are presented as mean ± SD.In figures, data are presented as mean ± SD shown all data points.

| Calcitriol dose titration
The dose of calcitriol was titrated to reduce plasma PTH concentration to levels similar to those observed in the calcimimetic group.Doses of 40 and 60 ng/kg of calcitriol were necessary to reduce the PTH concentration to target levels (Supplemental Figure S1).The highest dose (60 ng/ kg) produced hyperphosphatemia and hypercalcemia.Animals treated with calcitriol at 40 ng/kg did not significantly increase plasma ionized calcium and phosphate levels.These rats presented a plasma PTH concentration similar to that of the Nx-CM group.Thus, Nx rats treated with calcitriol at 40 ng/kg (from here on Nx-CTR) were appropriate for comparison with Nx animals treated with calcimimetic.

|
The effects of calcitriol and calcimimetic in 5/6Nx rats Biochemical parameters are presented in Table 1.All Nx rats had higher plasma creatinine and phosphate levels and lower ionized calcium levels than the sham group.The Nx group developed secondary hyperparathyroidism and had increased plasma concentration of intact PTH as compared with the sham group.As expected, plasma intact FGF23 levels were also increased in Nx rats as compared with the sham group and the administration of calcitriol further increased plasma levels of intact FGF23 while calcimimetic did not.Calcitriol administration also produced an increase in plasma creatinine concentration as compared with Nx rats receiving vehicle; this effect was not observed in animals treated with calcimimetic.Calcimimetic administration reduced plasma ionized calcium as compared with the Nx group treated with vehicle, while calcitriol prevented the decrease of plasma calcium.
With respect to bone histology, Nx rats had lower bone volume (Figure 2A) and higher osteoid volume and surface than sham rats (Figure 2B,C, respectively); this was accompanied by an increase in the bone surface covered by osteoblasts (Figure 2D).As compared with sham animals, Nx rats showed an increase in the eroded surface (Figure 2F) and in the bone surface covered by osteoclasts (Figure 2G), also had higher trabecular separation (Figure 2H) and a decrease in trabecular number (Figure 2I), all of these findings consistent with the lower bone volume.Treatment with calcitriol, which reduced the concentration of PTH, resulted in higher bone volume, lower trabecular separation, and higher trabecular number as compared with the Nx group treated with vehicle, indicating a protective effect of calcitriol on bone loss in renal insufficiency.As compared with the Nx-CTR group, the treatment with calcimimetic resulted in higher osteoblast activity, as indicated by the bone surface covered by osteoid and osteoblasts, although the PTH levels were similar in both groups.The Nx-CM group showed similar bone volume than the Nx group, whereas the addition of calcitriol resulted in higher bone volume.
Kinetic parameters of bone formation rate and mineralizing surface were increased in Nx rats as compared with the Sham group and remained similar among Nx groups (Figure 3A,B, respectively), whereas mineral apposition rate did not reach significant differences (Figure 3C).Mineralization lag time was significantly increased in the calcimimetic group as compared with the group treated with calcitriol (Figure 3D).The adjusted apposition rate was greater in Sham rats as compared with the Nx group, and it was similar among all groups of uremic animals (Figure 3E).Osteoid maturation time was comparable in all groups (Figure 3F).Representative microphotographs are shown in Figure 3G-J.

| The effects of calcitriol and calcimimetic in 5/6Nx rats with PTH clamp
The decreased bone cell activity observed in the calcitriol group could have been caused in part by the reduction of PTH.Therefore, we performed additional experiments in Nx animals undergoing PTX and receiving a constant infusion of PTH sufficient to achieve plasma concentrations  1 and 2).The biochemical data of Nx-PTX rats with a clamp of PTH (Nx-PTX-PTH) are shown in Table 2. Nx-PTX-PTH rats treated with calcitriol presented slightly but significant higher plasma concentration of creatinine and phosphate as compared with the Nx-PTX-PTH group treated with calcimimetic.As expected, treatment with calcitriol increased plasma intact FGF23 levels, whereas administration of calcimimetic did not change plasma intact FGF23.Concentrations of the infused 1-34 PTH were similar in all groups.
Regarding bone histomorphometry, treatment with calcitriol resulted in higher bone volume than the vehicle and calcimimetic groups (Figure 4A).In addition, calcimimetic treatment produced an increase in osteoid volume and surface as compared with the animals treated with calcitriol (Figure 4B,C, respectively), which was associated with a higher surface of bone covered by osteoblasts (Figure 4D).Osteoid thickness remained similar among groups (Figure 4E).In addition, the bone surface covered by osteoclasts (Figure 4G) was also increased in the Nx-PTX-PTH group treated with calcimimetic as compared with those that received vehicle or calcitriol.Overall, as compared with the Nx-PTX-PTH-CTR group, the calcimimetic group had a greater osteoid volume, osteoid surface, osteoblast surface, osteoclast surface, and trabecular separation, while trabecular number was reduced.
With respect to kinetic parameters, the Nx-PTX-PTH-CM group had an increased bone formation rate as compared to the Nx-PTX-PTH-CTR group (Figure 5A).The mineralizing surface in the Nx-PTX-PTH-CM group did not reach significance as compared with the Nx-PTX-PTH-CTR group (Figure 5B, p = .054).Representative microphotographs are shown in Figure 5G-I.

| In vitro effects of increasing concentration of calcitriol on mesenchymal stem cell osteogenic differentiation
The effect of different concentrations of calcitriol (10 −11 to 10 −9 M (4.17 to 417 pg/mL)) on rat bone marrow mesenchymal stem cell (MSC) osteogenesis was evaluated.The MSC osteogenic differentiation was demonstrated   by the presence of matrix calcification, assessed by alizarin red staining (Figure 6B), and increased calcium content in the mineralized matrix (Figure 6F) and alkaline phosphatase activity (Figure 6G) after a 21-day period of osteogenic stimuli.Moreover, the expressions of the osteogenic genes Runx2 and Osterix were increased in osteoblast-like MSC as compared with undifferentiated control (Figure 6H,I, respectively).In these osteoblastlike cells, the addition of calcitriol dose-dependently decreased alizarin red staining (Figure 6C-E), amount of matrix calcium, alkaline phosphatase activity, and mRNA expression of osteogenic genes Runx2 and Osterix, suggesting an inhibitory effect of calcitriol on the osteogenic differentiation of MSC.

| Inhibition of β-catenin nuclear translocation by calcitriol
Next, we explored the possibility that the lower osteoblastic differentiation induced by calcitriol could be mediated by an inhibition of the canonical Wnt/βcatenin pathway, which is known to be involved in osteoblast differentiation.Confocal microscopy analysis showed an increased nuclear translocation of βcatenin in osteoblast-like MSC (Figure 7B) as compared with undifferentiated controls (Figure 7A).Of note, the presence of calcitriol dose-dependently decreased the expression of βcatenin in the nuclei (Figure 7C-E).Moreover, a decrease in proliferation and a decrease in the number of cells were also observed after calcitriol treatment.Additionally, western blot analysis revealed a reduction of the amount of PCNA in enriched nuclear protein extracts (Figure 7F) indicating an inhibitory effect of calcitriol on cellular proliferation, a target of the canonical Wnt/β-catenin pathway.

| Effects of calcitriol and calcimimetic in osteoblastic-like UMR-106 cells
The addition of calcitriol did not influence Runx2 and Osterix gene expressions (Supplemental Figure S2A,B, respectively); however, Osteocalcin gene expression was upregulated (Supplemental Figure S2C).The addition of calcimimetic (100 μM) resulted in increased both Runx2 and Osterix mRNA expression, and only Osterix mRNA was significantly increased with respect to calcitriol.In comparison with calcitriol, calcimimetic did not induce the upregulation of Osteocalcin gene expression.

| In vitro effects of calcitriol and calcimimetic on osteoclastic differentiation
Osteoclastic stimuli resulted in TRAP-positive multinuclear cells formation (Supplemental Figure S3A,B) and increased Cathepsin K (CTSK) mRNA expression (Supplemental Figure S3E).Administration of both treatments, calcitriol 10 −10 M and calcimimetic 100 μM, throughout osteoclastogenesis resulted in increased osteoclastic differentiation at a similar level as assessed by TRAP staining (Supplemental Figure S3C,D) and higher CTSK mRNA expression (Supplemental Figure S3E), indicating a direct pro-osteoclastogenic effect of both treatments.

| DISCUSSION
The present study compares the effects of calcitriol and a calcimimetic on bone histomorphometry in an experimental model of CKD.Both molecules exhibited specific effects on bone, which are independent of PTH.To accomplish this goal, it was necessary to maintain serum levels of PTH unchanged.Thus, the experiments were performed in a PTX rat model of CKD with clamped PTH.Aadditional in vitro experiments were also required.The information provided here is relevant considering that both calcitriol and calcimimetics are used to treat secondary hyperparathyroidism in chronic renal failure.
In the setting of CKD, calcimimetics and calcitriol are used to control PTH production.PTH is a potent regulator of bone metabolism.This regulation may be dual according to the duration of the exposure to PTH. 23 Parathyroid hormone modulates bone synthesis and resorption by acting through different cell types.Thus, the use of molecules that lead to changes in PTH may have relevant effects on bone remodeling.To avoid this confounding effect, as abovementioned, in our study 5/6Nx rats underwent parathyroidectomy and had clamped levels of PTH.In these animals, calcitriol decreased osteoblast activity and reduced the bone surface covered by osteoblasts and the amount of osteoid, whereas bone resorption was not changed significantly, which helps to maintain trabecular bone volume.Clinical studies by Costa et al. 24 show results supporting our experimental data, indicating that calcitriol may reduce osteoblast activity independently of its action on PTH; they described that calcitriol treatment may reduce PTH secretion in some hemodialysis patients with severe secondary hyperparathyroidism (SHPT), classified as responders.Nevertheless, both responding and non-responding patients showed a 50% reduction in osteoblast surface and decreased osteoid surface after 6 months of calcitriol treatment.Non-responding patients showed increased eroded surface that may be due to the higher PTH levels.In a cohort study of patients undergoing continuous cycling peritoneal dialysis, Salusky et al. 25 investigated the differential effects of intermittent calcitriol administration given orally or intraperitoneally.
They found that patients treated with calcitriol intraperitoneally showed a significant reduction of serum PTH concentration after 9 months, while oral calcitriol did not produce significant changes, which could be due to different bioavailability.Of note, both calcitriol treatments decreased bone turnover and 33% of patients developed adynamic bone disease.These results indicate an inhibitory effect of calcitriol on bone cell activity that is not explained by the reduction in circulating PTH concentration.A case report with similar findings was published by Pahl et al., 26 showing that calcitriol treatment in a hemodialysis patient decreased calcium efflux from bone despite no difference in the PTH response to dialysate calcium concentration.The findings suggest that calcitriol may have a direct suppressive effect on bone.Similarly, Ureña et al. 27 reported the case of a hemodialysis patient with decreased plasma bone specific alkaline phosphatase after treatment with calcitriol that was not accompanied by a reduction in plasma PTH.These results agree with our observations in animal models.
Overall, the effect of active vitamin D treatment on bone health has yielded variable results.Thus, the effect of vitamin D on fracture prevention is controversial.Laird et al. have reviewed the results reported by a number of studies in which vitamin D has been beneficial or had no effect on bone. 28More recently, two studies have reported reductions in the rate of vertebral fractures following administration of oral calcitriol. 29,30When it comes to bone mineral density, the results offered by several studies suggest that vitamin D therapy may, at least, reduce the loss of bone mineral density in CKD. 31,32Nevertheless, the effect of vitamin D on bone status seems to be influenced by the administration regime, continuous versus intermittent, and the dosage, with no effect observed with very low doses of vitamin D. 33 As compared with calcitriol, calcimimetic increased bone remodeling, assessed by the greater amount of osteoid and the more bone surface covered by osteoblasts and osteoclasts.Regarding the bone effects of calcimimetics in hemodialysis patients with established SHPT, Malluche et al. showed that the reduction of PTH concentration with calcimimetics is accompanied by a decrease in bone remodeling; however, simultaneous uses of phosphate binders and calcitriol were allowed in this study cohort, which could modify the effects of calcimimetic. 34imilarly, Behets et al. reported that bone formation and circulating markers of bone turnover were reduced in dialysis patients after 12 months of treatment with cinacalcet.In these patients, vitamin D sterols were allowed throughout the study, which may have reduced bone turnover. 35e have previously demonstrated the direct osteoanabolic effect of calcimimetics. 16These observations are in line with findings from clinical studies.Tsuruta et al. reported an improved bone mineral in patients treated with cinacalcet for one year. 36Very recently, it has been demonstrated a similar effect induced by the novel molecule etelcalcetide, without affecting bone material properties. 37The beneficial effect of calcimimetics in bone is associated with increases in bone formation markers and a decrease in molecules that are markers of bone resorption. 38,39Accordingly, the use of calcimimetics has been shown to reduce the risk of fractures associated to renal disease.This effect has been confirmed in studies using both oral 40,41 and intravenous calcimimetics. 42lthough previous studies have reported that calcitriol increases osteoclastogenesis and bone resorption in vitro, [2][3][4] we did not observe higher osteoclast activity in the group of rats with 5/6Nx and clamped PTH, suggesting additional mechanisms or impaired osteoclastic response to calcitriol in renal insufficiency.Nevertheless, our in vitro results showed that, at the concentrations studied, both calcitriol and calcimimetic had pro-osteoclastogenic effects.
Currently, the KDIGO guidelines recommend to use calcitriol, calcimimetic, or combination in patients with CKD G5D requiring PTH-lowering therapy, but evidence for benefit or harm of one or the other remains unclear. 43The present study suggests that the use of calcimimetic could be more appropriate to control PTH in those patients with low bone turnover, while calcitriol administration, which decreases bone turnover and contributes to the development of adynamic bone disease, is more appropriate in case of high bone turnover disease.However, a limitation of our study is the difficulty of comparing patients treated with calcitriol or calcimimetics with similar PTH levels, since more effective PTH modulation may be expected with the use of calcimimetic.
To further assess the bone effects of calcitriol, we examined its action on the osteogenic differentiation of MSC and found that calcitriol dose-dependently decreased osteogenic gene expressions and blocked mineralization.These effects seem to be mediated, at least partially, by inhibition of the canonical Wnt/β-catenin signaling, a key osteoanabolic pathway. 44The reduction of osteoblast differentiation induced by calcitriol administration supports our in vivo results, suggesting that the decrease in the bone surface covered by osteoblast and the amount of osteoid may be due to a direct inhibitory effect of the high concentrations of calcitriol on Wnt/β-catenin pathway and osteoblast differentiation.We also examined in UMR-106 cells whether osteogenic gene expression is affected by calcitriol or calcimimetic; it was observed that as compared with calcitriol, calcimimetic upregulated osteoblastic gene expression.We also evaluated the in vitro effect of calcitriol or calcimimetics on osteoclastic differentiation and we found that both, calcitriol and calcimimetic, produced an additional increase of cathepsin K expression, also pointing to a direct effect of CaSR modulation that has been reported previously. 45e have reported recently that calcimimetics maintain bone remodeling in a rat model of renal insufficiency by acting directly on osteoblasts. 16These results were subsequently supported by clinical studies. 17Therefore, in the current study, we attempt to compare the bone effects of calcitriol and calcimimetic using in vivo and in vitro models.These two treatments are primary choices for the management for SHPT.Our results indicate that the effects of these treatments on bone histomorphometry in rats with renal insufficiency can be opposed.Thus, for the same PTH, calcimimetic administration increased bone cells activity whereas calcitriol produced the opposite effect.Bone histology in renal diseases may not only be the result of the prevailing PTH levels and bone health in CKD patients may depend on the strategy used to control PTH level.
The results shown here may help clarify the therapeutic consequences of the use of calcimimetics and calcitriol in the context of renal dysfunction.However, the administration of both molecules in subjects free of renal disease may contribute to define their effects on bone metabolism.7][48][49] The phase I trials of calcimimetics, carried out in healthy subjects, were intended to assess pharmacokinetics, pharmacodynamics, safety, tolerability, and the efficacy of oral and intravenous molecules on the levels of PTH and other factors involved in the phosphate and calcium metabolism.1][52][53][54] With regard to vitamin D, and also in the context of primary hyperparathyroidism without CKD, it has been documented a decreased bone turnover due to vitamin D-mediated inhibition of PTH secretion. 55,56The administration of vitamin D has been also reported in osteoporosis.In this regard, decreased bone loss associated with vitamin D therapy has been observed repeatedly. 12et, the use of vitamin D under these circumstances is associated with a higher risk of developing hypercalcemia and hypercalciuria.
Apart from the bone effects of calcitriol vs calcimimetic, it is also interesting to note that, as compared with calcimimetic, calcitriol administration led to a significant increase of serum phosphate, creatinine, and FGF23 levels.These findings are probably due to the higher intestinal absorption of phosphate promoted by calcitriol and, consequently, the increased phosphatemia leads to further elevations in FGF23.As shown in the classical study carried out by Sprague et al., 57 the administration of calcitriol was associated with frequent episodes and/or elevations in the Ca x P product in CKD patients.Undoubtedly, the elevations in these parameters may promote faster progression of renal disease, as evidenced by the higher values of creatinine shown in the group of animals treated with calcitriol.Such undesirable consequence may be prevented by administrating vitamin D sterols with a less calcemic profile, such as paricalcitol. 58n conclusion, in our rat model of renal insufficiency with clamped PTH, the administration of calcitriol reduces osteoblast activity, whereas calcimimetic increases bone turnover.

F I G U R E 2
Bone histomorphometry of Nx rats treated with either calcitriol or calcimimetic.Nx rats treated with calcitriol showed higher bone volume than those treated with vehicle or calcimimetic (A).Osteoid volume (B), osteoid surface (C), and the surface of bone covered by osteoblasts (D) were higher in the Nx rats treated with calcimimetic than in those receiving calcitriol.The osteoid thickness (E) remained similar in all Nx rats.The bone eroded surface (F) and the bone surface covered by osteoclasts (G) were similar in all Nx groups.Consistent with the data on bone volume, calcitriol treatment decreased trabecular separation (H) and increased trabecular number (I) as compared with the other Nx groups.Bars represent mean ± SD.One-way ANOVA with Tukey test.*p < .05;**p < .01;***p < .001;****p < .0001.CM, calcimimetic; CTR, calcitriol.

F I G U R E 3
Effects of the treatment with either calcitriol or calcimimetic on kinetic parameters of bone formation.Bone formation rate (A) and mineralizing surface (B) are greater in all Nx than in sham rats, while mineral apposition rate (C) was only increased in the Nx treated with calcimimetic as compared with sham animals.Mineralization lag time (D) was also increased in Nx rats treated with calcimimetic as compared with calcitriol and sham groups.The adjusted apposition rate (E) was reduced in Nx rats including those receiving calcimimetic as compared with sham, while the osteoid maturation time (F) was similar in all groups.Representative microphotograph of Goldner trichrome staining and calcein labeling (G-J).Bars represent mean ± SD.One-way ANOVA with Tukey test.*p < .05;**p < .01.Magnification: 200×.Scale bar: 100 μm.Arrows indicate distances between both calcein labels.Ob, Osteoblasts; Oc, Osteoclasts.One-way ANOVA with Tukey test.

Note:T A B L E 2 F I G U R E 4
Values shown are mean ± SD.One-way ANOVA with Tukey test.a p < .05versus Nx-PTX-PTH-Veh.b p < .05versus Nx-PTX-PTH-CTR.Plasma biochemistry of 5/6Nx rats with total PTX and PTH replacement treated with either calcitriol or calcimimetic during 28 days.Bone histomorphometry of Nx rats with total PTX and PTH replacement treated with calcitriol or calcimimetic.In Nx with total PTX and PTH replacement, treatment with calcitriol prevented the loss of bone volume (A).As compared with calcitriol, calcimimetic treatment increased the osteoid volume (B), osteoid surface (C), and osteoblast surface (D).Osteoid thickness remained similar in all groups (E).The eroded surface was similar in all groups (F), and the bone surface covered by osteoclasts (G) was increased in the calcimimetic group.Rats treated with calcimimetic also increased trabecular separation and decreased trabecular number (H and I, respectively).Bars represent mean ± SD.One-way ANOVA with Tukey test.*p < .05;**p < .01;****p < .0001.

F I G U R E 5
Effects of the administration of either calcitriol or calcimimetic on kinetic indices in Nx rats with total PTX and PTH replacement.Treatment with calcimimetic increased the bone formation rate as compared with rats on calcitriol (A).Mineralizing surface (B), mineral apposition rate (C), mineralization lag time (D), adjusted apposition rate (E), and osteoid maturation time remained similar in all groups.Representative microphotographs of Goldner trichrome staining and calcein labeling (G-I).Bars represent mean ± SD.One-way ANOVA with Tukey test.*p < .05.Augmentation: 200×.Scale bar: 100 μm.Arrows indicate distances between calcein labels.Ob, osteoblasts; Oc, osteoclasts.

F I G U R E 6
Effects of calcitriol treatment on MSC osteogenesis.Microphotographs for bright field and alizarin red staining showed a decreased mineralization in osteoblast-like MSC treated with calcitriol (A-E).Quantification of the calcium content in the matrix eluted with hydrochloric acid (F) and alkaline phosphatase activity assesses by p-nitrophenol hydrolysis (G) showed a consistent decrease in calcitriol-treated cells.Likewise, the mRNA expressions of the osteogenic master genes Runx2 (H) and Osterix (I) were also downregulated.Bars represent mean ± SD.One-way ANOVA with Tukey test.*p < .05;***p < .001;****p < .0001.ALP, alkaline phosphatase; MSC, mesenchymal stem cells; OB, osteoblast-like MSC cells.Bright-field microscopy magnification: 100×.Scale bar: 200 μm.

7
Effects of calcitriol treatment on nuclear translocation of βcatenin and cell proliferation.Representative microphotographs from confocal microscopy analysis shown the decrease in nuclear amount of βcatenin-488 in response to the increasing doses of calcitriol.Submask analysis is also showed for greater visual clarity (A-E).Western blot analysis shows that calcitriol induced dose-dependent reduction of PCNA (proliferating cell nuclear antigen) amount (F).TFIIB (transcription factor II B) was used as loading control.Augmentation: 630×.Scale bar: 25 μm.