Eight-week-old, male Wistar rats (n = 9 - 11) (Charles River Laboratories Inc., Tokyo, Japan) were housed in a controlled environment (temperature 23˚C ± 2˚C, humidity 40% ± 20%) with a 12-hour light-dark cycle with free access to water and rat food. The details were described in our previous study [14] . Rats were treated with a 0.75% adenine diet (Oriental Yeast Co., Ltd., Tokyo, Japan) for 4 weeks until 12 weeks of age and then fed a standard rodent chow (CE-7; Clea Japan, Tokyo, Japan) diet to generate CKD model rats (CKD group). The 4-week adenine diet treatment was selected based on previous studies [14] [15] , which reported that a 4-week adenine diet increased serum creatinine, phosphorus, and intact-PTH levels at 20 weeks of age and induced non-progressive, irreversible renal failure. In addition, Ferrari et al. demonstrated that the adenine-induced CKD model rats had a higher bone metabolism, as well as osteoclast area, than the 5/6 nephrectomized CKD model rats [16] . Based on these results, the adenine-induced CKD model rats at 20 weeks of age used in this study are considered to be appropriate for evaluating the effects of EC on cortical and trabecular bone microstructure in CKD conditions with SHPT.

At 20 weeks of age, the rats were divided into two groups: CKD group (vehicle administration) and EC group (0.6 mg/kg subcutaneous injection of EC daily). Vehicle or EC was injected into the CKD group or EC group, respectively, for 4 weeks starting at 20 weeks of age. EC was dissolved in vehicle (1 mass/volume percent (m/v%) glycine, 1 m/v% d (+)-trehalose dihydrate, 0.27 m/v% disodium succinate hexahydrate, 2 m/v% d-mannitol, 0.9 m/v% benzyl alcohol, pH 4.5), and administered subcutaneously daily for 4 weeks at a dose of 0.6 mg/kg body weight according to a previously reported protocol [17] . The non-CKD control (Cont group) was also included without the adenine diet and treated with vehicle for 4 weeks starting at 20 weeks of age. Body weight (BW) was measured at the beginning (20 weeks of age) and at the end of the experiment. The protocols for all animal experiments were approved in advance by the Animal Experimentation Committee of our institute (approval number a-1-0271), and all subsequent animal experiments adhered to the Guidelines for Animal Experimentation of our institution.

After sacrifice, the right femur was fixed in 10% neutral-buffered formalin (Wako Chemical Industries, Osaka, Japan) until preparation for measurement of BMD and micro-computed tomography (CT). The left femur was dissected free of soft tissue, wrapped in gauze moistened with saline, and frozen at −80˚C until biomechanical testing.

At 24 weeks of age, blood samples were collected from the vena cava of rats 24 - 36 hours after EC administration. Blood samples were centrifuged at 3000 rpm for 30 minutes at 4˚C to separate the serum, which was then stored in disposable aliquots at −80˚C until analysis. Blood urea nitrogen (BUN), creatinine (CRE), calcium (Ca), and inorganic phosphorus (IP) levels were measured using a Hitachi Automatic Biochemical Analyzer 7180 (Hitachi Ltd., Tokyo, Japan).

The excised right femur was secured in a specimen holder. Micro-CT was performed using the Cosmo Scan GX II (Rigaku Corporation, Tokyo, Japan) according to the manufacturer’s instructions with an isotropic voxel size of 36 μm, energy of 90 kVp, and current of 88 μA. The acquired images were rendered using TRI/3D BON software (Ratoc System Engineering Co., Ltd., Tokyo, Japan). Osteoporosis assessment was performed based on total area (Tt.Ar, mm²), cortical area (Co.Ar, mm²), cortical volume (Ct.V, mm³), cortical area/total area (Ct.Ar/Tt.Ar, %), cortical thickness (Ct.Th, mm), and cortical porosity (Co.Po, %) at the mid femur and bone volume/tissue volume (BV/TV, %), trabecular thickness (Tb.Th, mm), trabecular number (Tb.N, 1/mm), trabecular separation (Tb.Sp, mm), structure model index (SMI), and connectivity density (Conn.D) at the distal femur. The measurement area of the middle femur was a 1000-μm-high region centered on the midpoint of the entire femur. The measurement area of the distal femur was 2000 μm cranially from 500 μm from the reference line connecting the two ends of the growth plate of the sagittal image.

BMDs of the femur and lumbar vertebrae (L2-4) were measured using dual-energy X-ray absorptiometry (Horizon^® DXA System; Hologic, Bedford, MA, USA). Each region was scanned in “small animal” mode, with the “regional high resolution” scan option. Femoral BMD was measured at the proximal, middle, and distal thirds of the femur.

Mechanical testing of the left femoral shaft was performed at room temperature using a material testing machine (MZ500S; Maruto, Tokyo, Japan). The mid-diaphysis of the femur was stabilized by placing it on two supports of the test apparatus placed 18-mm apart. The load of a three-point bending test was applied in the anteroposterior direction midway between the two supports. Load-displacement curves were recorded at a crosshead speed of 5 mm/min. The maximum load (N), stiffness (N/mm), and breaking energy (N mm) were calculated using software for measuring bone strength (CTR win. Version 1.05; System Supply, Nagano, Japan), as described previously [18] . Following the three-point bending test, the distal part of the femur was evaluated using a compression test, as previously described [19] . Load-displacement curves were recorded, and the maximum load (N), stiffness (N/mm), and breaking energy (N mm) were calculated using the same software.

All data are expressed as mean ± standard deviation (SD) values. A Kolmogorov-Smirnov test showed that all data were normally distributed. Differences among the Cont, CKD, and EC groups were evaluated by one-way analysis of variance (ANOVA) and analyzed by Tukey’s multiple comparison test as a post hoc test. All statistical analyses were performed using EZR [20] , which is a modified version of R commander designed to add statistical functions frequently used in biostatistics. Values of p < 0.05 were considered significant.

To evaluate the health status of the animals, body weight and its changes during the experiment were measured. Although rats in the CKD and EC groups had significantly lower body weights than rats in the Cont group at the baseline and endpoints (p < 0.01) (Figure 1(a)), there were no significant differences between the CKD and EC groups (Figure 1(a)). The body weight change (%) from baseline to endpoint was significantly higher in the CKD group than in the Cont (p < 0.05) and EC (p < 0.01) groups (Figure 1(b)).

In addition, blood biochemical tests were performed to evaluate the effects of 4 weeks of treatment with EC. Serum BUN (p < 0.01) (Figure 2(a)), CRE (p < 0.01) (Figure 2(b)), and IP (p < 0.05) (Figure 2(d)) were significantly higher in

the CKD group than in the Cont group. Although the serum BUN level was significantly decreased (p < 0.01) in the CKD rats after 4 weeks of EC treatment (Figure 2(a)), it was still significantly higher than that in the Cont group (p < 0.01). The serum CRE level did not show a significant change with EC treatment compared with the CKD group, and it was significantly higher than that in the Cont group (p < 0.01) (Figure 2(b)). EC treatment significantly decreased serum Ca levels compared with the CKD group (p < 0.05) (Figure 2(c)), and serum IP decreased significantly compared with the CKD (p < 0.01) and Cont (p < 0.05) groups (Figure 2(d)).

Axial micro-CT images of the femoral diaphysis showed that rats in the CKD group had higher cortical porosity than rats in the Cont group (Figure 3(a)). In addition, treatment with EC decreased cortical porosity with an increase in cortical bone thickness in the EC rats (Figure 3(a)).

Analysis of cortical bone at the femoral diaphysis by micro-CT showed a significant increase in Ct.Po in the CKD group compared with normal rats (Cont group) (p < 0.01) (Figure 3(b)). There were no significant differences in other

cortical bone parameters between the Cont and CKD groups (Figures 3(c)-(g)). EC treatment significantly increased Ct.Ar (p < 0.01) (Figure 3(d)), Ct.V (p < 0.05) (Figure 3(e)), Ct.Ar/Tt.Ar (p < 0.05) (Figure 3(f)), and Ct.Th (p < 0.01)

(Figure 3(g)), and it significantly decreased Ct.Po (p < 0.01) (Figure 3(b)) compared with those in the CKD group.

Axial micro-CT images of the distal femoral metaphysis showed that the trabecular bone microstructure was degraded in the CKD group compared with the Cont group (Figure 4(a)).

Micro-CT analysis of the trabecular bone of the distal femur showed significantly reduced BV/TV (p < 0.05) (Figure 4(b)) and Tb.Th (p < 0.05) (Figure 4(c)) and significantly increased Tb.Sp (p < 0.05) (Figure 4(e)) in the CKD group compared with the Cont group. Although EC treatment for 4 weeks did not show a significant change in these parameters compared to the CKD group, there was no significant difference between the EC group and the Cont group. SMI and Conn D, which are parameters related to bone strength of trabecular bone, did not differ significantly among the three groups (Figure 4(f), Figure 4(g)).

The results for femur and lumbar BMDs are shown in Table 1. Compared with normal rats (Cont group), the BMDs of the femur at the total, proximal, middle, and distal and lumbar vertebrae were significantly lower in the CKD group and the EC group (p < 0.05). Compared with the CKD group, the EC group showed no significant changes in these BMDs.

Representative stress-strain curves from the three-point bending test are shown in Figure 5(a). In the three-point bending test, the CKD group had significantly lower maximum load (Figure 5(b)), stiffness (Figure 5(c)), and breaking energy (Figure 5(d)) compared to the Cont group (p < 0.05). Although EC treatment for 4 weeks did not show significant changes in these parameters compared to the CKD group, there was no significant difference between the EC group and the Cont group (Figures 5(b)-(d)).

In the compression test at the distal metaphysis of the femur, the parameters related to trabecular bone strength, which are maximum load (Figure 5(e)), stiffness (Figure 5(f)), and breaking energy (Figure 5(g)), were not significantly different among the three groups.

Few reports have investigated the effects of EC on bone, including bone microstructure and bone strength in trabecular and cortical bone. To the best of our knowledge, the present study is the first to report them in a rat model of adenine-induced CKD. In the adenine-induced CKD rat model in the present study, bone microstructures were not altered in cortical bone at the relatively early stage of 12 weeks after CKD induction, but BMD and cortical bone strength were reduced by increased cortical porosity. On the other hand, in trabecular

Data are mean ± standard deviation values. Cont: non-CKD control rats, CKD: CKD rats administered vehicle, EC: CKD rats administered etelcalcetide. ^ap < 0.05 vs Cont group (Tukey’s multiple comparison test).

bone, a decrease in BMD and impairment of trabecular bone microstructure were observed, but no significant deterioration in bone strength was observed. In cortical bone, EC improved bone microstructure, reduced porosity, and inhibited the decrease in bone strength after 4 weeks of treatment. In trabecular bone, EC inhibited the progression of degradation of trabecular bone microstructure, but no significant improvement in bone strength was observed.

Administration of EC to SHPT has an effect on bone by reducing blood PTH levels and improving the overactive bone metabolic turnover. EC has also been reported to inhibit the development and progression of cortical bone porosity in a rat model of CKD [17] . Li et al. reported that 6 weeks of EC treatment improved failure energy of cortical bone in a rat model of CKD by subtotal nephrectomy [21] . Furthermore, Damrath et al. also demonstrated that calcimimetics may help prevent CKD-induced bone deterioration by improving bone quality in new periosteal bone and in bone tissue near osteocyte lacunae in the Cy/+ rats, which was a model of spontaneous and progressive CKD-mineral and bone disorder [22] . In the present study, EC also significantly reduced cortical bone porosity in the femoral diaphysis and significantly improved cortical bone area and width, but it did not significantly improve BMD in the femoral diaphysis by DXA or bone strength in the femoral diaphysis by the 3-point bending test compared to the CKD group. A slightly longer duration of EC administration may confirm its effect on cortical bone strength.

On the other hand, in the trabecular bone area, the 4 weeks of EC treatment had no effect on the parameters of BMD and microstructure in trabecular bone and bone strength. Li et al. reported that bone morphometry of trabecular bone in CKD model rats treated with EC for 6 weeks showed a decrease in osteoid volume, but no change in bone volume, trabecular bone microstructure, or osteoclast

surface [21] . A recent study showed that EC treatment improved BMD and trabecular bone quality and reduced bone metabolism without altering bone material properties [23] . Although a simple comparison between humans on hemodialysis and CKD model rats may not be possible, a clearer effect on trabecular bone may be confirmed in rats by extending the duration of etelcalcetide treatment.

In the present study, EC alone did not significantly improve BMD or bone strength in CKD model rats, and it was found that EC inhibited CKD-induced bone strength loss. Swallow et al. pointed out that EC mitigates, but does not reverse the progression of cortical bone deterioration in CKD, so combination therapy with osteoporosis drugs should be considered to more effectively improve bone loss in CKD [17] . However, there have been no randomized, controlled trials investigating the effects of osteoporosis medications in hemodialysis patients. In addition, there are no reports detailing the effects of the combination of calcimimetics and osteoporosis medications in CKD with SHPT. Osteoporosis medications include anti-resorptive agents and osteoanabolic agents, the most common of which are bisphosphonates and denosumab, and teriparatide [24] . However, in CKD or hemodialysis patients, bisphosphonates may cause over-suppression of bone turnover and development of adynamic bone disease, denosumab may cause severe hypocalcemia, and teriparatide may affect the pathophysiology of hyperparathyroidism [24] [25] . Furthermore, hypocalcemia is one of the side effects of etelcalcetide, so measures against hypocalcemia are necessary when etelcalcetide is used in combination with those bone resorption inhibitors. Iwasaki et al. reported that, in rats with SHPT induced by 5/6 nephrectomy, the elastic mechanical properties of cortical bone deteriorated regardless of bone metabolism or bone mass, and the factors determining bone elasticity were associated with the severity of uremia [26] . Therefore, normalization of PTH secretion alone is insufficient to improve bone fragility in CKD, and a more beneficial effect may be confirmed by concurrent treatment of uremic toxins. However, there is a report that the combination with the nicotinamide adenine dinucleotide phosphate oxidase (NOX1/4) inhibitor GKT-137831, which has a suppressive effect on oxidative stress, had no effect in combination with EC [27] . In the future, the combined effect of EC with a treatment that improves bone metabolism should be studied, rather than the effect of EC alone on bone.

This study has several limitations. First, the duration of EC treatment was only four weeks. The effect of EC on cortical and trabecular bone over a longer period of time needs to be investigated. Second, the effect of EC treatment on bone metabolism has not been investigated. We expect to elucidate these limitations in future studies. Finally, we did not evaluate the effect sizes of intergroup differences of all parameters. We should consider the effect sizes in the small number of each group in this study.