Fibroblast growth factor 23 (FGF23) modulates mineral metabolism by promoting phosphaturia and decreasing the production of 1,25-dihydroxyvitamin D₃. FGF23 decreases parathyroid hormone (PTH) mRNA and secretion, but despite a marked elevation in FGF23 in uremia, PTH production increases. Here, we investigated the effect of FGF23 on parathyroid function in normal and uremic hyperplastic parathyroid glands in rats. In normal parathyroid glands, FGF23 decreased PTH production, increased expression of both the parathyroid calcium-sensing receptor and the vitamin D receptor, and reduced cell proliferation. Furthermore, FGF23 induced phosphorylation of extracellular signal–regulated kinase 1/2, which mediates the action of FGF23. In contrast, in hyperplastic parathyroid glands, FGF23 did not reduce PTH production, did not affect expression of the calcium-sensing receptor or vitamin D receptor, and did not affect cell proliferation. In addition, FGF23 failed to activate the extracellular signal–regulated kinase 1/2–mitogen-activated protein kinase pathway in hyperplastic parathyroid glands. We observed very low expression of the FGF23 receptor 1 and the co-receptor Klotho in uremic hyperplastic parathyroid glands, which may explain the lack of response to FGF23 in this tissue. In conclusion, in hyperparathyroidism secondary to renal failure, the parathyroid cells resist the inhibitory effects of FGF23, perhaps as a result of the low expression of FGF23 receptor 1 and Klotho in this condition.

Calcimimetics decrease parathyroid hormone (PTH) levels in uremic patients with secondary hyperparathyroidism without increasing serum calcium (Ca). The aim of this study was to evaluate the effect of calcimimetic R-568 alone or in combination with calcitriol on vascular and other soft tissue calcifications in uremic rats with secondary hyperparathyroidism. Sham-operated and 5/6 nephrectomized Wistar rats were studied. 5/6 Nephrectomized rats were treated with vehicle, calcitriol (80 ng/kg every other day), R-568 (1.5 and 3 mg/kg per d), and both calcitriol and R-568 1.5 mg/kg, as above. Rats were killed after 14 or 56 d of treatment. Blood was drawn for biochemical measurements. Aortic, heart, kidney, lung, and stomach tissue samples were processed for histopathology and measurement of tissue Ca and phosphorus content. PTH concentrations were significantly reduced by all treatments. Treatment with calcitriol induced significant vascular calcification (aortic Ca increased to 4.2 ± 1.2 mg/g at day 14 and to 11.4 ± 0.7 mg/g at day 56; P < 0.05 versus vehicle). Treatment with R-568 did not induce vascular calcification. Concurrent administration of R-568 with calcitriol reduced the aortic Ca (1.9 ± 0.2 mg/g at day 14 and 7.5 ± 1.4 mg/g at day 56) in relation to calcitriol alone. Soft tissue calcifications mirrored aortic mineralizations. Survival was significantly (P < 0.001) reduced in calcitriol-treated rats, and mortality was attenuated (P = 0.01) by concurrent treatment with R-568. In uremic rats, R-568 reduces elevated PTH levels without inducing vascular calcification, prevents calcitriol-induced vascular calcification, and decreases mortality.

Hyperphosphatemia is closely related to vascular calcification in patients with chronic kidney disease. Vascular smooth muscle cells (VSMCs) exposed to high phosphate concentrations in vitro undergo phenotypic transition to osteoblast‐like cells. Mechanisms underlying this transdifferentiation are not clear. In this study we used two in vitro models, human aortic smooth muscle cells and rat aortic rings, to investigate the phenotypic transition of VSMCs induced by high phosphate. We found that high phosphate concentration (3.3 mmol/L) in the medium was associated with increased DNA methyltransferase activity and methylation of the promoter region of SM22α. This was accompanied by loss of the smooth muscle cell–specific protein SM22α, gain of the osteoblast transcription factor Cbfa1, and increased alkaline phosphatase activity with the subsequent in vitro calcification. The addition of a demethylating agent (procaine) to the high‐phosphate medium reduced DNA methyltransferase activity and prevented methylation of the SM22α promoter, which was accompanied by an increase in SM22α expression and less calcification. Additionally, downregulation of SM22α, either by siRNA or by a methyl group donor (S‐adenosyl methionine), resulted in overexpression of Cbfa1. In conclusion, we demonstrate that methylation of SM22α promoter is an important event in vascular smooth muscle cell calcification and that high phosphate induces this epigenetic modification. These findings uncover a new insight into mechanisms by which high phosphate concentration promotes vascular calcification. © 2010 American Society for Bone and Mineral Research

To investigate whether the effect of the calcimimetic AMG 641 and calcitriol on CaSR and VDR expression could be separated from their ability to reduce parathyroid cell proliferation, five-sixth nephrectomized (5/6 Nx) rats received vehicle, AMG 641, calcitriol, or AMG 641+calcitriol either daily for 13 days (long-term protocol) or in a single dose (short-term protocol). In the long-term protocol, AMG 641, calcitriol, and their combination significantly reduced the percentage of proliferating parathyroid cells. Proliferation was uncontrolled in the short-term protocol. A significant increase in CaSR mRNA (% vs. β-actin) was detected in rats treated with both calcitriol (1.60 ± 0.30) and AMG 641 (1.66 ± 0.25) for 13 days ( P = 0.01 vs. 5/6 Nx+vehicle, 0.89 ± 0.09); and there was a further increase when both drugs were administered simultaneously (2.46 ± 0.33). In the short-term protocol, only rats receiving AMG 641 alone (2.01 ± 0.33, P < 0.001) showed increased expression of CaSR mRNA, whereas the combination (1.81 ± 0.20) produced no additional benefit. AMG 641 also increased CaSR mRNA expression in vitro. Changes in VDR mRNA paralleled those of CaSR mRNA. In the long-term treatment, both AMG 641 (0.87 ± 0.14) and calcitriol (0.99 ± 0.12) increased VDR mRNA ( P < 0.05 vs. 5/6 Nx+vehicle, 0.49 ± 0.10), and the increase was more accentuated when the drugs were combined (1.49 ± 0.45). In the short-term protocol, only treatment with AMG 641, alone (1.52 ± 0.41) or combined with calcitriol (1.86 ± 0.24), increased VDR mRNA. In conclusion, our results demonstrate an acute increase in CaSR mRNA and VDR mRNA in the parathyroid glands of uremic rats treated with AMG 641, in which cell proliferation was uncontrolled, thus supporting a direct effect of calcimimetics on CaSR and VDR expression by hyperplastic parathyroid cells.