High-serum phosphate and parathyroid hormone distinctly regulate bone loss and vascular calcification in experimental chronic kidney disease

The regulation of the phosphaturic factor fibroblast growth factor 23 (FGF23) is not well understood. It was found that administration of 1,25-dihydroxyvitamin D(3) (1,25[OH](2)D(3)) to mice rapidly increased serum FGF23 concentrations from a basal level of 90.6 +/- 8.1 to 213.8 +/- 14.6 pg/ml at 8 h (mean +/- SEM; P < 0.01) and resulted in a four-fold increase in FGF23 transcripts in bone, the predominate site of FGF23 expression. In the Hyp-mouse homologue of X-linked hypophosphatemic rickets, administration of 1,25(OH)(2)D(3) further increased circulating FGF23 levels. In Gcm2 null mice, low 1,25(OH)(2)D(3) levels were associated with a three-fold reduction in FGF23 levels that were increased by administration of 1,25(OH)(2)D(3). In osteoblast cell cultures, 1,25(OH)(2)D(3) but not calcium, phosphate, or parathyroid hormone stimulated FGF23 mRNA levels and resulted in a dose-dependent increase in FGF23 promoter activity. Overexpression of a dominant negative vitamin D receptor inhibited 1,25(OH)(2)D(3) stimulation of FGF23 promoter activity, and mutagenesis of the FGF23 promoter identified a vitamin D-responsive element (-1180 GGAACTcagTAACCT -1156) that is responsible for the vitamin D effects. These data suggest that 1,25(OH)(2)D(3) is an important regulator of FGF23 production by osteoblasts in bone. The physiologic role of FGF23 may be to act as a counterregulatory phosphaturic hormone to maintain phosphate homeostasis in response to vitamin D.

Hyperphosphatemia, elevated calcium x phosphorus product (Ca x P), and calcium burden, major causes of vascular calcification, are correlated with increased cardiovascular morbidity and mortality in dialysis patients. To address the underlying mechanisms responsible for these findings, we have utilized an in vitro human smooth muscle cell (HSMC) model of vascular calcification. Previous studies using this system demonstrated enhanced calcification of HSMC cultures treated with phosphorus levels in the hyperphosphatemic range, and implicated a sodium-dependent phosphate cotransport-dependent mechanism in this effect. In the present study, we examine the effect of increasing calcium concentrations on HSMC calcification in vitro. Increasing calcium to levels observed in hypercalcemic individuals increased mineralization of HSMC cultures under normal phosphorus conditions. Importantly, at these total calcium concentrations, ionized calcium levels increased from 1.2 mmol/L to 1.7 mmol/L, consistent with levels observed physiologically in normocalcemic and hypercalcemic individuals, respectively. Furthermore, increasing both calcium and phosphorus levels led to accelerated and increased mineralization in the cultures. Calcium-induced mineralization was dependent on the function of a sodium-dependent phosphate cotransporter, since it was inhibited by phosphonoformic acid (PFA). While elevated calcium did not affect short-term phosphorus transport kinetics, long-term elevated calcium treatment of HSMCs induced expression of the sodium-dependent phosphate cotransporter, Pit-1. These studies suggest that elevated calcium may stimulate HSMC mineralization by elevating Ca x P product and enhancing the sodium-dependent phosphate cotransporter-dependent mineralization pathway previously observed in HSMCs.

Chronic kidney disease-mineral bone disorder (CKD-MBD) is defined by abnormalities in mineral and hormone metabolism, bone histomorphometric changes, and/or the presence of soft-tissue calcification. Emerging evidence suggests that features of CKD-MBD may occur early in disease progression and are associated with changes in osteocyte function. To identify early changes in bone, we utilized the jck mouse, a genetic model of polycystic kidney disease that exhibits progressive renal disease. At 6 weeks of age, jck mice have normal renal function and no evidence of bone disease but exhibit continual decline in renal function and death by 20 weeks of age, when approximately 40% to 60% of them have vascular calcification. Temporal changes in serum parameters were identified in jck relative to wild-type mice from 6 through 18 weeks of age and were subsequently shown to largely mirror serum changes commonly associated with clinical CKD-MBD. Bone histomorphometry revealed progressive changes associated with increased osteoclast activity and elevated bone formation relative to wild-type mice. To capture the early molecular and cellular events in the progression of CKD-MBD we examined cell-specific pathways associated with bone remodeling at the protein and/or gene expression level. Importantly, a steady increase in the number of cells expressing phosphor-Ser33/37-β-catenin was observed both in mouse and human bones. Overall repression of Wnt/β-catenin signaling within osteocytes occurred in conjunction with increased expression of Wnt antagonists (SOST and sFRP4) and genes associated with osteoclast activity, including receptor activator of NF-κB ligand (RANKL). The resulting increase in the RANKL/osteoprotegerin (OPG) ratio correlated with increased osteoclast activity. In late-stage disease, an apparent repression of genes associated with osteoblast function was observed. These data confirm that jck mice develop progressive biochemical changes in CKD-MBD and suggest that repression of the Wnt/β-catenin pathway is involved in the pathogenesis of renal osteodystrophy. Copyright © 2012 American Society for Bone and Mineral Research.