In the present studies, we employed a uremic mouse model to determine the effects of CKD and phosphate loading on the dentoalveolar complex. Sera levels of BUN, PTH and alkaline phosphatase were all significantly increased in U/NP and U/HP vs. sham controls, while serum calcium was increased in the U/HP group. No differences were noted in serum phosphate levels between groups. Micro-CT analyses showed a significant reduction in cortical bone thickness and an increase in trabecular thickness and trabecular bone volume/tissue volume in U/NP and U/HP groups compared to Sham/NP, with the most dramatic changes occurring in the U/HP group. A significant reduction in cortical bone thickness was also found in the Sham/HP vs. Sham/NP group. Histological evaluation confirmed increased trabeculation in the U groups. These data indicate that uremia, especially in the context of phosphate loading, has dramatic effects on the bones of the oral cavity.
While high phosphate feeding appeared to exacerbate skeletal changes in the uremic mice, serum phosphate levels in the mice were not elevated above sham control levels. This is likely due to compensatory increases in phosphate regulating hormones. Indeed, PTH was elevated in the uremic mice, and previously we showed that high serum levels of FGF23 are induced by phosphate feeding in uremic mice.12
Nevertheless, even small elevations in serum phosphate have been correlated with increased risk of cardiovascular disease and mortality in CKD patients,23
and vascular calcification is observed only in uremic mice treated with a high phosphate diet and not a normal phosphate diet.12
Thus, it is important to determine whether or not phosphate load in the CKD model even in the absence of measurable hyperphosphatemia, has an effect on bone activity.
Although no changes were noted in serum phosphate between the groups we did observe marked increases in serum BUN, PTH and ALP in U/NP and U/HP mice vs. sham/NP or sham/HP mice, indicative of a uremic condition and of altered bone metabolism. Further, decreased body weight was noted in U/NP and U/HP vs. sham groups. Not surprisingly, increased serum levels of PTH and ALP correlated with altered alveolar bone to include decreased cortical thickness and BV/TV and increased trabecular thickness and BV/TV. At the gross histological level this paralleled the disorientation of the PDL and increased trabeculation of alveolar bone, most apparent in the U/HP group. The bone changes occurred rather rapidly, i.e. 3 months following kidney ablation surgery and most likely sooner, but early samples were not taken for the studies here.
The decrease in cortical thickness and increase in trabeculation observed supports previous clinical reports of CKD patients that demonstrated thinning of cortical bone 15, 24
and increased trabeculation as determined by biopsies of the ilium.24
These data also support observations by Antonelli and Hottel, who noted a decrease in mineralization and alterations in trabeculae, producing a finely meshed pattern, in the mandible of a patient with CKD.25
A review by Jover-Cervero et al. suggested that patients had decreased trabeculation as determined by examining case reports of mandibles of CKD patients using dental panoramic radiographs.15
These differences may be attributed to several factors including stage and duration of disease,25
levels of serum Ca/P, Fgf23, ALKP, and BUN, diet (e.g. high protein/thus high protein diet), type of bone evaluated (long bones, iliac crest, mandibles) and mechanism of evaluation (bone biopsy vs. radiographs) and gender/species differences.
Surprisingly, while the effect on bone metabolism was modest, we noted a significant decrease in cortical thickness in samples from SHAM/HP mice vs. SHAM/NP mice. This finding coupled with a dramatic effect on alveolar bone in both U/NP and U/HP, with the greatest effect in U/HP, highlight the impact of CKD on bone metabolism, as well as potential effects of a high phosphate diet on bone homeostasis.
Previous studies from our group and others over the last decade have brought attention to phosphate as being more than a passive molecule controlled by calcium/calcium signaling.26
In fact, there is mounting evidence that inorganic phosphate acts as a signaling molecule, able to regulate genes associated with osteoblast differentiation, e.g. osteopontin, dentin matrix protein 1, bone sialoprotein and osteocalcin.27–31
Furthermore, data from humans, as well as rodent models, have shown that alterations in local as well as systemic levels of phosphate often result in profound and unique effects on mineralized tissues of the oral cavity vs. other mineralized tissues. These include inadequate cementum formation resulting in lack of PDL attachment, and subsequently severe periodontal disease, in humans and rodents with mutations in alkaline phosphatase, an enzyme that breaks down pyrophosphate (PPi) to inorganic phosphate (Pi) at local sites.32–34
In contrast, the reverse situation (i.e. excessive cementum formation) occurs in rodents exhibiting high levels of Pi relative to PPi locally, due to a mutation/knock out (KO) of either PC-1 (plasma cell glycoprotein 1, also known as ectonucleotide pyrophosphatase/phosphodiesterase 1, ENPP1) or ANK (progressive ankylosis protein).22
PC-1 is a membrane bound enzyme that can generate PPi for extracellular release35
while ANK encodes a transmembrane protein that regulates transport of PPi ions across the plasma membrane to the extracellular environment.36
At the systemic level, insufficient levels of Fgf23 in humans37
and in mice38
result in hyperphosphatemia, abnormally elevated levels of 1, 25 (OH)2
and increased PTH. We have reported (via abstract) a tooth phenotype in Fgf23−/−
mice including ectopic “mineral-like” tissue within the pulp chamber.39
In contrast, in murine models with mutation in PHEX, (phosphate regulating gene with homologies to endopeptidases on the X-chromosome), or DMP-1 (Dentin matrix protein 1), resulting in high levels of FGF23, hypophosphatemia and low levels of 1, 25 (OH)2
, there is evidence of deficient cementum formation40, 41
and previously reported defects in dentin formation with human counterparts.42, 43
Previous studies in humans and animal models focusing on non-oral bones report that CKD affects bone homeostasis.3, 9, 12, 21
The results of our studies, demonstrating that CKD in mice results in marked effects on alveolar bone homeostasis, seen by 3 months, and furthermore also noted in sham animals given HP diets for 3 months, suggest that bones of the oral cavity are highly sensitive to alterations related to uremic conditions, including dysregulation of phosphate metabolism.