FGF23 fails to inhibit uremic parathyroid glands

Abstract

Fibroblast growth factor 23 (FGF23) modulates mineral metabolism by promoting phosphaturia and decreasing the production of 1,25-dihydroxyvitamin D(3). 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.

Figure 1.

FGF23 reduces PTH secretion in normal rat parathyroid glands. (A) PTH secretion. Parathyroid glands are incubated in 1.25 mM Ca and then shifted to 0.8 mM to show a functional response. Next, the effect of the addition of 200 nM rat FGF23 (5 × 10³ ng/ml) is evaluated in low-Ca (0.8 mM) and high-Ca (1.5 mM) concentrations. Data are means ± SEM (n = 4 in each group; the experiment was repeated three times). ^#P < 0.01 versus vehicle-treated glands. (B) Regulation of PTH, VDR, and CaR mRNA levels by FGF23 in vitro. Intact rat parathyroid glands are incubated for 6 hours in Ca concentrations of 0.8 and 1.5 mM with or without FGF23 (200 nM). Data are means ± SEM (n = 4 in each group; the experiments were repeated three times). *P < 0.05 versus 0.8 mM Ca with no added FGF23.

Figure 2.

FGF23 increases VDR and CaR expression and ERK1/2 phosphorylation in normal rat parathyroid glands. Intact normal rat parathyroid glands are incubated for 6 hours with low Ca (0.8 mM), with or without FGF23 (200 nM; 5 × 10³ ng/ml), or high Ca (1.5 mM) for immunohistochemical evaluation of VDR, CaR, and phospho-ERK1/2. Sections were counterstained with hematoxylin. Magnification, ×200.

Figure 3.

FGF23 increases ERK1/2 phosphorylation in normal rat parathyroid glands. Western blot of phospho-ERK1/2 in bovine parathyroid glands incubated for 6 hours with low-Ca (0.8 mM) and high-Ca (1.5 mM) concentration with and without FGF23 (200 nM) added. PD98059 (10 μM), an inhibitor of ERK1/2 phosphorylation, is added as control. Quantification is performed by measurement of the integrated OD. Image is representative of three different experiments. The values presented in the figure are related to that specific experiment; the statistical data of all of the experiments (means ± SEM) are 100 ± 9 in 0.8 mM Ca, 185 ± 11* in 0.8 mM Ca + FGF23, 60 ± 7*# in 0.8 mM Ca + FGF23 + PD, 182 ± 8* in 1.5 mM Ca, 200 ± 12* in 1.5 mM Ca + FGF23, and 66 ± 5*# in 1.5 mM Ca + FGF23 + PD; *P < 0.01 versus 0.8 mM Ca; #P < 0.01 versus 0.8 or 1.5 mM Ca with added FGF23.

Figure 4.

FGF23 decreases parathyroid cell proliferation in normal rat parathyroid glands in low-Ca medium. Intact normal rat parathyroid glands are incubated for 24 h with low Ca (0.8 mM) with or without FGF23 (200 nM; 5 × 10³ ng/ml), and an additional group is treated with calcitriol 10⁻⁷ M as a positive control of parathyroid cell proliferation inhibition. (A and B) Proliferation is assessed by the number of cells entering the S phase of the cell cycle by flow cytometry (data are means ± SEM; n = 4 in each group; the experiment is repeated three times; ^#P < 0.01 versus 0.8 Ca mM; A) and by the immunohistochemical determination of p21 (an inhibitory protein of the cell cycle; B). Images are representative of four glands per three different experiments. (C) Intact normal rat parathyroid glands are treated as in A but with high Ca (1.5 mM) with or without the specific ERK1/2 inhibitor PD98059 (10 μM). Data are means ± SEM (n = 4 in each group; the experiment is repeated three times). ^#P < 0.01 versus 1.5 mM Ca. Magnification, ×200.

Figure 5.

FGF23 reduces PTH secretion and synthesis in normal but not in uremic parathyroid glands. In vivo studies: Renal failure in rats is induced by two-step 5/6 Nx. (A and B) Sham-operated and 5/6 Nx rats are administered FGF23 (15 μg/d per rat) or vehicle for 2 consecutive days, and PTH secretion (A) and PTH mRNA (B) are evaluated. Data are means ± SEM (n = 4 in each group; the experiments are repeated three times). *P < 0.05 versus sham with vehicle. (C) In vitro studies: After a stabilization period of 6 hours, parathyroid glands from normal and uremic rats are incubated with increasing concentrations of FGF23. The incubation medium is replaced hourly, and PTH secretion is evaluated. Data are means ± SEM (n = 4 in each group; the experiments are repeated three times). *P < 0.05 versus 0.8 mM Ca with added FGF23.

Figure 6.

FGF23 has no effect on parathyroid cell proliferation in uremic rats. (A through C) Parathyroid cell proliferation is studied both in vivo (A and B) and in vitro (C). (A) In the in vivo studies, the uremic rats receive FGF23 (15 μg/d for 2 consecutive days). Rats are killed and parathyroid glands are removed for analysis of cell proliferation (number of cells entering the S phase of the cell cycle). Data are means ± SEM (n = 8 rats in each group; the experiment is repeated three times). *P < 0.05 versus 5/6 Nx rats. (B) Immunohistochemical determination of p21. (C) Intact parathyroid glands from uremic animals are incubated for 24 hours with low Ca (0.8 mM) with and without FGF23 (200 nM; 5 × 10³ ng/ml). The proliferation is assessed by number of cells entering the S phase. Data are means ± SEM (n = 4 in each group; experiments are repeated at least three times). (D) Immunohistochemical determination of VDR, CaR, and phospho-ERK1/2 (activation of ERK1/2). Images are representative of three rats per three different experiments. Sections are counterstained with hematoxylin. Magnification, ×200.

Figure 7.

FGF23 does not increase VDR expression or activate ERK1/2 in parathyroid glands from uremic rats. Nx rats receive FGF23 (15 μg/d for 2 consecutive days). (A and B) Parathyroid glands are removed for immunohistochemical analysis of VDR expression and phospho-ERK1/2 (ERK1/2 activation; images are representative of four rats from three different experiments; A) and Western blot evaluation of ERK1/2 phosphorylation (B). Quantification is performed by measuring the integrated OD. Image is representative of three different experiments. The values presented in the figure are related to that specific experiment; the statistical data of all of the experiments (means ± SEM) are 100 ± 9 in Nx, 94 ± 4 in Nx + FGF23, and 144 ± 21* in Sham. *P < 0.01 versus Nx. Magnification, ×200.

Figure 8.

FGF23 increases VDR expression and activates ERK1/2 in normal rats in vivo. Normal rats receive FGF23 (15 μg/d for 2 consecutive days). The glands are processed for immunohistochemical determination of VDR and ERK1/2 phosphorylation (activation). Images are representative of four glands per three different experiments. Magnification, ×200.

Figure 9.

FGFR1 and Klotho expression are reduced in parathyroid glands from uremic rats in vivo. (A) FGFR1 and Klotho mRNA levels are measured by quantitative real-time reverse transcriptase–PCR (versus β-actin mRNA). Data are means ± SEM (n = 4 in each group; the experiment is repeated three times). ^#P < 0.01 versus normal (sham) rats. (B) FGFR1 and Klotho protein expression in hyperplastic parathyroid glands from uremic rats. Parathyroid glands are processed for immunohistochemical evaluation of FGFR1 and Klotho protein. Images are representative of four glands from three different experiments. Positive reaction (brown deposits) is revealed by the diaminobenzidine-tetrachloride system. Sections are counterstained with hematoxylin. Magnification, ×200.

Figure 10.

Effect of extracellular Ca (A), phosphate (B), and FGF23 (C and D) on FGFR1 and Klotho expression in parathyroid glands from uremic rats in vitro. (A through C) Intact normal rat parathyroid glands are incubated for 6 hours with low-Ca (0.8 mM) or high-Ca (1.5 mM) concentration in the medium with normal (1.0 mM) phosphate (A), high (4.0 mM) or normal (1.0 mM) phosphate concentration in the medium with normal (1.25 mM) Ca (B), and increasing concentrations of FGF23 in the medium with normal Ca (1.25 mM) and phosphate (1.0 mM; C). FGFR1 and Klotho mRNA levels are measured by quantitative real-time reverse transcriptase–PCR (versus β-actin mRNA). Data are means ± SEM (n = 4 in each group; the experiment is repeated three times). ^#P < 0.01 versus 0.8 mM Ca.