The present study was approved by the Ethics Committee of Shandong Wendeng Orthopedic Hospital (Wendeng, China) and was performed in accordance with institutional guidelines. A total of 36 8-week-old male C57BL/6J mice (body weight, 20±2 g) were obtained from Shanghai Laboratory Animal Center (Shanghai, China) and acclimated to the environment for 1 week. Normal dietary Ca (TD.04200, containing 0.6% calcium carbonate and 0.4% phosphate by weight) and high dietary Ca (TD.96348, containing 2% calcium carbonate and 1.25% phosphate by weight) were purchased from Harlan Teklad (Madison, WI, USA). The mice individually caged in a temperature-controlled environment (23±2°C; humidity, 60±5%) with an artificial 12-h light/dark cycle and were provided with free access to food and tap water. The 5/6 Nx mouse model of CKD was established without angiotensin II infusion according to the method published by Souza et al (12) and upregulation of blood urea nitrogen and serum creatinine verified successful establishment of the animal model (12). The mice were randomly divided into three groups (n=12 in each group): Sham group, sham-operated mice (without removal of kidneys) treated with normal dietary Ca following sham surgery for 12 weeks; 5/6 Nx group, 5/6 Nx mice treated with normal dietary Ca following Nx surgery for 12 weeks; and 5/6 Nx+HCD group, 5/6 Nx mice treated with HCD following Nx surgery for 12 weeks. All mice were sacrificed at week 12 via an intraperitoneal injection of sodium pentobarbital (200 mg/kg; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Blood was harvested from the heart and centrifuged at 1,500 × g for 15 min at 4°C to obtain serum. Serum (~800 µl), urine (~2 ml) and lumbar vertebrae (L3-L5) tissues were immediately collected and maintained at −80°C for further analysis.

The concentrations of Ca (cat. no. C004-3; Nanjing Jiancheng Biology Engineering Institute, Nanjing, China) and creatinine (Cr; cat. no. C011-1; Nanjing Jiancheng Biology Engineering Institute) from the serum and urine were measured using standard colorimetric methods (13) and picric acid methods (14), respectively. The level of urinary Ca (UCa) was corrected according to the concentration of urinary Cr (UCr). Serum Cr (SCr; cat. no. C011-1; Nanjing Jiancheng Biology Engineering Institute) and blood urea nitrogen (BUN; cat. no. C013-2; Nanjing Jiancheng Biology Engineering Institute) levels were measured using an autoanalyzer and an enzymatic kinetic method with commercial kits following the manufacturer's protocol. Serum levels of FGF-23 (cat. no. 60-6800; Immutopics, Inc., San Clemente, CA, USA), PTH (cat. no. E-EL-M0709c; Elabscience Biotechnology Co., Ltd., Wuhan, China), tartrate resistant acid phosphatase-5b (TRAP-5b; cat. no. SB-TR201A; Immunodiagnostic Systems, Scottsdale, AZ, USA) and alkaline phosphatase (ALP; cat. no. E-EL-M0200c; Elabscience Biotechnology Co., Ltd.) were detected using murine ELISA assays with a SpectraMax M5 ELISA plate reader (Molecular Devices, LLC, Sunnyvale, CA, USA).

The lumbar vertebrae were incinerated using a muffle furnace (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 800°C for 12 h and 10 mg of bone ash was dissolved in 1 ml of 37% HCl diluted with Milli-Q^® water. The calcium content was determined using a kit (cat. no. C004-3; Nanjing Jiancheng Biology Engineering Institute).

Kidney tissues were collected immediately following sacrifice and fixed with 4% formalin at room temperature for 24 h and paraffin-embedded. Tissues were cut into ~5 µm-thick sections, which were stained with Masson's trichrome (cat. no. SBJ-0290; Nanjing SenBeiJia Biological Technology Co., Ltd., Nanjing, China) as previously described (15) and visualized under a microscope (magnification, ×200; Leica DM 2500; Leica Microsystems GmbH, Wetzlar, Germany). Renal injury was assessed using a previously described 0–4 scale (16) as follows: 0, none; 1, <10; 2, 10–25; 3, 25–75; or 4, >75%.

The lumbar vertebrae were collected immediately following sacrifice and fixed with 4% formalin at room temperature for 24 h, and then decalcified in 0.5 M EDTA (pH=8.0) and embedded in paraffin according to standard histological procedures. Sections of ~5 µm were cut and stained with hematoxylin and eosin (H&E; cat. no. C0105; Beyotime Institute of Biotechnology,) prior to being visualized under a microscope (magnification, ×200; Leica DM 2500).

Total RNA from lumbar vertebrae was extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. cDNA was synthesized by RT reactions with 2 µg of total RNA using moloney murine leukemia virus reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) following 37°C for 50 min and 70°C for 15 min. A total of 1 µl cDNA was used for PCR using a DNA Engine (ABI) with SYBR Green PCR Master Mix (Invitrogen; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were applied: 95°C for 10 min followed by 40 cycles of 95°C for 15 sec, 58°C for 30 sec and 72°C for 30 sec. The primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) as follows: osteoprotegerin (OPG), forward 5′-GCATTATGACCCAGAAACT-3′ and reverse 5′-ACCTGAGAAGAACCCATC-3′; receptor activator of nuclear factor κB ligand (RANKL), forward 5′-AACCAAGATGGCTTCTATTACC-3′ and reverse 5′-AAGGGTTGGACACCTGAATG-3′; and GAPDH, forward 5′-TCACTGCCACCCAGAAGA-3′ and reverse 5′-AAGTCGCAGGAGACAACC-3′. Signals were assessed using Quantity One^® software version 4.5 (Bio Rad Laboratories, Inc., Hercules, CA, USA) and normalized to the GAPDH. Analysis of relative gene expression data used real-time quantitative PCR and the quantitation cycle (Cq) method (17).

Data are presented as the mean + or ± standard deviation as indicated for each group. All statistical analyses were performed using GraphPad Prism software version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Inter-group differences were analyzed using one-way analysis of variance with a post hoc Tukey test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the role of HCD supplementation in 5/6 Nx-induced renal dysfunction, serum levels of SCr and BUN were compared across groups. The levels of SCr and BUN were significantly increased in the 5/6 Nx group compared with the sham-operated group at week 6 and week 12 (P<0.05; Fig. 1A and B); however, HCD supplementation in 5/6 Nx mice did not significantly improve these parameters (Fig. 1A and B). Masson staining was performed to observe the extent of renal interstitial fibrosis. As indicated in Fig. 1C and D, increased matrix deposition, interstitial fibrosis and renal lesions were observed in the kidneys from 5/6 Nx mice compared with sham mice, and injury scores were significantly increased (P<0.05; Fig. 1D). Furthermore, HCD supplementation had no marked effect on renal pathological changes in 5/6 Nx mice. These data suggest that the CKD mice models were successfully established and that HCD did not improve renal function in 5/6 Nx mice.

Acute kidney injury (AKI) or CKD-induced GFR regression predisposes to bone metabolism disturbances via upregulating serum P (18), which accelerates FGF-23 and PTH secretion. Previous studies have demonstrated that increased FGF-23 is associated with CKD or AKI severity (19,20). FGF-23 is a peptide released from osteocytes and osteoblasts, whilst PTH is associated with Ca, P and FGF-23 levels in CKD (21). To evaluate the effect of an HCD in CKD-MBD, the levels of FGF-23 and PTH in the serum of 5/6 Nx mice were measured. Results indicated that serum FGF-23 and PTH levels were significantly increased in the 5/6 Nx group compared with the sham group (P<0.05; Fig. 2A and B). Notably, HCD significantly reversed CKD-induced upregulation of serum FGF-23 and PTH levels in 5/6 Nx mice (P<0.05; Fig. 2A and B). In addition, the effect of 5/6 Nx on markers of bone resorption and formation, ALP and TRAP-5b (22,23), in the serum of 5/6 Nx mice was assessed (Fig. 2C and D). 5/6 Nx induced a significant increase in serum TRAP-5b concentration compared with the sham group; however, HCD supplementation significantly decreased TRAP-5b levels in CKD mice (P<0.05). No significant difference in ALP concentration was observed in any of the experimental groups.

HCD has an important role in the bone-kidney axis and the regulation of calcium homeostasis in CKD (24,25). The Ca content in serum, urine and bone was measured in CKD mice. Results indicated that 5/6 Nx mice exhibited decreased SCa levels and an ~3-fold increase in UCa excretion compared with the sham group, whereas HCD supplementation significantly increased SCa content and decreased UCa excretion in CKD mice (P<0.05; Fig. 3A and B). At week 12, comparison of the results of bone Ca content between sham and 5/6 Nx groups indicated 5/6 Nx significantly decreased bone Ca content; however, the bone calcium content was significantly increased in 5/6 Nx mice with HCD supplementation (both P<0.05; Fig. 3C). A similar result was obtained for Ca content in the ash of lumbar vertebrae (Fig. 3D). Furthermore, H&E staining was conducted to observe the trabecular bone microstructure of lumbar vertebrae. As indicated in Fig. 3E, a loss of network connection in the trabecular bone was observed in the 5/6 Nx group compared with the sham group; however, the increased disconnections and separation among trabecular bone network were improved by HCD supplementation in CKD mice.

The maturation and formation of osteoclasts is primarily regulated by the balance of extracellular OPG and RANKL (26). OPG and RANKL cytokines affect the activity of osteoblast cells and osteoclastogenesis (26). OPG cytokine binds to RANKL and prevents RANKL from binding to the RANK receptor on osteoclast cells, subsequently inhibiting bone resorption and osteoclastogenesis (27). Thus, the ratio of OPG/RANKL expression in lumbar vertebrae was determined in the present study (Fig. 4). RT-qPCR results revealed a significant decrease in the OPG mRNA expression levels and ratio of OPG/RANKL compared with the sham group (P<0.05; Fig. 4A, B and D). Furthermore, RANKL mRNA expression levels were significantly increased in mice treated with 5/6 Nx compared with the sham group (P<0.05; Fig. 4A and C). HCD supplementation significantly reversed the effects of 5/6 Nx on OPG/RANKL and RANKL mRNA expression (P<0.05; Fig. 4C and D). However, no significant difference was observed between the mRNA expression levels of OPG in the 5/6 Nx and 5/6 Nx + HCD groups.