High calcium diet alleviates 5/6 nephrectomy-induced bone deteriorations of lumbar vertebrae in mice

Abstract

Dietary calcium (Ca) supplementation has beneficial effects on bone health. However, it is not clear whether a high calcium diet (HCD) following 5/6 nephrectomy (5/6 Nx) is beneficial to bone health. The aim of the present study was to examine the effects of an HCD on bone metabolism using a chronic kidney disease (CKD) mouse model. Male C57BL/6J mice were divided into three groups: Sham group, 5/6 Nx group and 5/6 Nx + HCD group. Mice were sacrificed 12 weeks post-surgery. Calcium (Ca) and creatinine (Cr) were measured using standard colorimetric methods and picric acid methods, respectively. Bone metabolism-associated markers, FGF-23, PTH, ALP-b and TRAP-5b were measured using ELISA kits. Lumbar vertebrae histomorphological analysis was performed using hematoxylin and eosin staining. The expression of osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) mRNA was detected using reverse transcription-quantitative polymerase chain reaction. Impaired renal function and histopathological damage was indicated in 5/6 Nx mice. However, HCD had no significant effects on these changes in 5/6 Nx mice. Notably, mineral metabolism disorder and histopathological damage to lumbar vertebrae were markedly improved in HCD-treated 5/6 Nx mice. Compared with 5/6 Nx mice, HCD supplementation significantly elevated the ratio of OPG/RANKL and inhibited RANKL mRNA expression in lumbar vertebrae. To conclude, the present findings indicated that increased Ca intake is effective in increasing bone mineral content of the lumbar vertebrae in 5/6 Nx mice. These results may provide a basis for the clinical use of dietary Ca supplementation as a therapeutic approach to treat CKD-induced disturbance of mineral metabolism and bone loss.

Keywords: bone injury; chronic kidney disease; high calcium diet.

Figure 1.

Changes in renal function in 5/6 Nx mice. (A) SCr and (B) BUN levels in 5/6 Nx mice with or without HCD supplementation were measured. Data are presented as the mean ± standard deviation. (C) Masson's trichrome staining was performed in 5/6 Nx mice with or without HCD supplementation to evaluate renal injury (magnification, ×200). (D) Kidney injury score for each group. Data are presented as the mean ± standard deviation. *P<0.05 vs. Sham group. 5/6 Nx, 5/6 nephrectomy; SCr, serum creatinine; BUN, blood urea nitrogen; HCD, high calcium diet; ND, not detected.

Figure 2.

HCD supplementation regulates bone metabolism-associated biomarkers. Serum levels of (A) FGF-23, (B) PTH, (C) ALP and (D) TRAP-5b mice were measured in 5/6 Nx with or without HCD supplementation. Data are presented as the mean ± standard deviation. *P<0.05 vs. Sham group; ^#P<0.05 vs. 5/6 Nx group. 5/6 Nx, 5/6 nephrectomy; HCD, high calcium diet; FGF-23, fibroblast growth factor-23; PTH, parathyroid hormone; ALP, alkaline phosphatase; TRAP-5b, tartrate resistant acid phosphatase-5b.

Figure 3.

HCD supplementation regulates Ca metabolism and bone remodeling. Concentrations of (A) SCa (B) UCa, (C) bone Ca and (D) Ca/Ash were measured in 5/6 Nx mice with or without HCD supplementation. (E) H&E staining (magnification, ×200) was performed in 5/6 Nx mice with or without HCD supplementation to evaluate trabecular bone injury. Data are presented as the mean ± standard deviation*P<0.05 vs. Sham group; ^#P<0.05 vs. 5/6 Nx group. 5/6 Nx, 5/6 nephrectomy; Ca, calcium; SCa, serum calcium; UCa, urinary Ca; HCD, high calcium diet; H&E, hematoxylin & eosin.

Figure 4.

HCD supplementation regulated OPG/RANKL ratio. (A) mRNA expression levels of OPG and RANKL were determined. Quantified results of the mRNA expression levels of (B) OPG and (C) RANKL and (D) the ratio of OPG/RANKL were demonstrated in 5/6 Nx mice with or without HCD supplementation. Data are presented as the mean ± standard deviation. *P<0.05 vs. Sham group; ^#P<0.05 vs. 5/6 Nx group. OPG/RANKL; osteoprotegerin/receptor activator of nuclear factor kappa B ligand; 5/6 Nx, 5/6 nephrectomy; HCD, high calcium diet.