High phosphate feeding promotes mineral and bone abnormalities in mice with chronic kidney disease

Abstract

Background: Chronic kidney disease-mineral bone disorder (CKD-MBD) is a systemic syndrome characterized by imbalances in mineral homeostasis, renal osteodystrophy (ROD) and ectopic calcification. The mechanisms underlying this syndrome in individuals with chronic kidney disease (CKD) are not yet clear.

Methods: We examined the effect of normal phosphate (NP) or high phosphate (HP) feeding in the setting of CKD on bone pathology, serum biochemistry and vascular calcification in calcification-prone dilute brown non-agouti (DBA/2) mice.

Results: In both NP and HP-fed CKD mice, elevated serum parathyroid hormone and alkaline phosphatase (ALP) levels were observed, but serum phosphorus levels were equivalent compared with sham controls. CKD mice on NP diet showed trabecular alterations in the long bone consistent with high-turnover ROD, including increased trabecular number with abundant osteoblasts and osteoclasts. Despite trabecular bone and serum biochemical changes, CKD/NP mice did not develop vascular calcification. In contrast, CKD/HP mice developed arterial medial calcification (AMC), more severe trabecular bone alterations and cortical bone abnormalities that included decreased cortical thickness and density, and increased cortical porosity. Cortical bone porosity and trabecular number strongly correlated with the degree of aortic calcification.

Conclusions: HP feeding was required to induce the full spectrum of CKD-MBD symptoms in CKD mice.

Fig. 1.

(A) Aortic calcium content in CKD mice and sham controls under normal and HP feeding conditions. Bars represent mean ± SEM. Letters above the graph denote significance (P< 0.01), A compared with the sham/NP group; B compared with the sham/HP group; C compared with the CKD/NP group. Sham/NP n= 5; sham/HP n= 6; CKD/NP n= 6; CKD/HP n= 4. (B) Representative micrographs of Alizarin Red-stained sections of the abdominal aorta from each group (scale bar = 200 μm). (i) Sham/NP, (ii) sham/HP, (iii) CKD/NP, (iv) CKD/HP. Mineralization of the tunica media was observed only in the CKD/HP group (iv).

Fig. 2.

Representative femur micro-CT sections from each group. (A) Longitudinal sections of the femurs; (B) cross-sectional sections in the diaphysis of the femurs and (C) sagittal sections in the epiphysis of the femurs.

Fig. 3.

Femur morphometrics based on micro-CT analysis. (A) Trabecular number (per mm), (B) trabecular bone density (g/cm³), (C) trabecular separation (mm), (D) trabecular thickness (mm), (E) cortical porosity (%), and (F) cortical thickness (mm), (G) ortical bone density (g/cm³), (H) Cortical BV/TV, %. (I) Cortical cross-sectional area (mm²). Bars represent mean ± SEM. Letters above the graph denote significance (P< 0.05), A compared with the sham/NP group; B compared with the sham/HP group; C compared with the CKD/NP group. Sham/NP n= 5; sham/HP n= 5; CKD/NP n= 6; CKD/HP n= 4.

Fig. 4.

Representative micrographs of H&E stained sections of femurs from each group. (A–D) Longitudinal femur sections. (E–H) Diaphyseal cortical bone sections. Note the dramatic decrease in cortical bone width, altered lamellar organization and the presence of trabecular protrusions into the marrow cavity (blue arrows) in the CKD/HP group compared with the CKD/NP group. Also, note the dramatic increase in cortical porosity in the CKD/HP group (asterisk). (I–L) Trabecular bone and marrow sections. The CKD/NP group showed increased trabeculae and increased osteoblast numbers on trabecular surfaces, while the CKD/HP group exhibited more dramatic remodeling such as increased trabeculae, increased osteoblasts and mineralization on trabecular surfaces, and numerous osteoclasts associated with scalloped trabecular surfaces (black arrowheads). Note that bone labeling studies were not done, therefore we could not precisely quantify bone-turnover rates.