Persistent fibroblast growth factor 23 signalling in the parathyroid glands for secondary hyperparathyroidism in mice with chronic kidney disease

Secondary hyperparathyroidism, in which parathyroid hormone (PTH) is excessively secreted in response to factors such as hyperphosphataemia, hypocalcaemia, and low 1,25-dihydroxyvitamin D (1,25(OH)2D) levels, is commonly observed in patients with chronic kidney disease (CKD), and is accompanied by high levels of fibroblast growth factor 23 (FGF23). However, the effect of FGF23 on the parathyroid glands (PG) remains controversial. To bind to FGF receptors, FGF23 requires αKlotho, which is highly expressed in the PG. Here, we examined the effects of Fgfr1–3, αKlotho, or Fgfr1–4 ablation specifically in the PG (conditional knockout, cKO). When mice with early to mid-stage CKD with and without cKO were compared, plasma concentrations of calcium, phosphate, FGF23, and 1,25(OH)2D did not change significantly. In contrast, plasma PTH levels, which were elevated in CKD mice, were significantly decreased in cKO mice. PG from CKD mice showed augmentation of cell proliferation, which was significantly suppressed by cKO. Parathyroid tissue cultured for 4 days showed upregulation of PTH secretion and cell proliferation in response to FGF23. Both these effects were inhibited by cKO. These findings suggest that FGF23 is a long-term inducer of parathyroid cell proliferation and PTH secretion, and is one cause of secondary hyperparathyroidism in CKD.

The PCR primers used with the BAC subcloning kit for subcloning the 5′ arm, floxed-out body, and 3′ arm were as follows. A PCR kit (GoTaq® Green Master Mix, M7123; Promega) was used for this purpose. The 5′ arm was cloned using the forward Targeted ES clones were selected by Sothern blotting and PCR using the probes and primers described below, and microinjected into 8-cell stage ICR embryos.
These injected embryos were transferred into pseudopregnant ICR females. The resulting chimaeras were bred with C57BL/6 mice, and heterozygous offspring were identified by Southern blotting (Supplementary Fig. S1b) and PCR ( Supplementary Fig.   S2) using the same probes and primers used for selecting targeted ES clones.
The probes used in the Southern blotting were as follows: the 5′ probe (1002 bases) was generated by PCR using the forward primer 5′-AGGTATCCTCCTTGAGACAGTGG-3′ and reverse primer 5′-GTTAGATAGGAAGTACCCATCGT-3′ with mouse genomic DNA as a template; the 3′ probe (1122 bases) was generated by PCR using the forward primer 5′-TCACCATGAACGAGCCAAACACA-3′ and reverse primer 5′-GGGAGGCCTATCAAGAGATAGAG-3′ with mouse genomic DNA as a template; and the Neo probe (877 bases) was generated by PCR using the forward primer 5′-CATTCTGCACGCTTCAAAAGCGCACG-3′ and reverse primer 5′-ACTCGTCAAGAAGGCGATAGAAGGCG-3′ with the targeting vector plasmid DT-ApA/conditional KO FW as a template. We screened the ES cells electroporated with the target vector for recombinant cells. More than ten recombinant ES clones were selected by performing Southern blotting with the 5′ and 3′ probes of the region that are described below, and PCR using several pairs of primers described below (primers FR4-F1, FR4-R1, FR4-F2, FR4-R2, and Neo-F) encompassing the targeted region.
The probes used in Southern blotting were as follows: the 5′ probe (794 bases) was generated by PCR using the forward primer 5′-ATAGCTAGGAGCTCTGCAGATGGC-3′ and reverse primer 5′-CCTTCTTCTGCTGTGTCTGAGAGC-3′ with mouse genomic DNA as a template; the 3′ probe (693 bases) was generated by PCR using the forward primer 5′-CTACATCCCTAGCCCGAAAGGCTTC-3′ and reverse primer 5′-ACAGATGGGGATCTGCAGGGTTAGC-3′ with mouse genomic DNA as a template; and the Neo probe (877 bases) was generated by PCR using the forward primer 5′-CATTCTGCACGCTTCAAAAGCGCACG-3′ and reverse primer 5′-ACTCGTCAAGAAGGCGATAGAAGGCG-3′ with the targeting vector plasmid DT-ApA/conditional KO FW as a template.
Targeted ES clones were microinjected into 8-cell stage ICR embryos, and the injected embryos were transferred into pseudo-pregnant ICR females. The resulting chimeric mice were bred with C57BL/6 mice, and heterozygous offspring were     Genomic DNA was digested with NheI-XmaI, AvrII, and BglII, and hybridised with the 5′ probe, 3′ probe, and Neo probe, respectively. Genomic DNA from WT, flox, and KO mice was also digested with AvrII-HindIII, and hybridised with the 3′ probe.       Parathyroid glands were cultured as described in the main text, except that the calcium in the medium was changed to two different concentrations (0.95 and 1.5 mM) following preincubation at 1.0 mM calcium. The ratio of 24-h PTH secretion on the 4th day of culture versus basal 2-h PTH secretion in 1.0 mM calcium is shown. The ratio representing incubation at 0.95 mM calcium was 5.8 ± 3.7 (mean ± SD), n = 4, and the ratio representing incubation at 1.5 mM calcium was 2.5 ± 1.7, n = 5. *P < 0.05.