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Targeted ablation of Fgf23 demonstrates an essential physiological role of FGF23 in phosphate and vitamin D metabolism
Takashi Shimada,1 Makoto Kakitani,1 Yuji Yamazaki,1 Hisashi Hasegawa,1 Yasuhiro Takeuchi,2 Toshiro Fujita,2 Seiji Fukumoto,3 Kazuma Tomizuka,1 and Takeyoshi Yamashita1
1Pharmaceutical Research Laboratories, KIRIN Brewery Co., Takasaki, Gunma, Japan2Department of Medicine, University of Tokyo School of Medicine, Bunkyo-ku, Tokyo, Japan3Department of Laboratory Medicine, University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan
Address correspondence to: Takeyoshi Yamashita, Pharmaceutical Research Laboratories, KIRIN Brewery Co., 3 Miyahara, Takasaki, Gunma 370-1295, Japan. Phone: 81-27-346-9789; Fax: 81-27-346-1672; E-mail: tyamashita@kirin.co.jp.
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1Pharmaceutical Research Laboratories, KIRIN Brewery Co., Takasaki, Gunma, Japan2Department of Medicine, University of Tokyo School of Medicine, Bunkyo-ku, Tokyo, Japan3Department of Laboratory Medicine, University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan
Address correspondence to: Takeyoshi Yamashita, Pharmaceutical Research Laboratories, KIRIN Brewery Co., 3 Miyahara, Takasaki, Gunma 370-1295, Japan. Phone: 81-27-346-9789; Fax: 81-27-346-1672; E-mail: tyamashita@kirin.co.jp.
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1Pharmaceutical Research Laboratories, KIRIN Brewery Co., Takasaki, Gunma, Japan2Department of Medicine, University of Tokyo School of Medicine, Bunkyo-ku, Tokyo, Japan3Department of Laboratory Medicine, University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan
Address correspondence to: Takeyoshi Yamashita, Pharmaceutical Research Laboratories, KIRIN Brewery Co., 3 Miyahara, Takasaki, Gunma 370-1295, Japan. Phone: 81-27-346-9789; Fax: 81-27-346-1672; E-mail: tyamashita@kirin.co.jp.
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1Pharmaceutical Research Laboratories, KIRIN Brewery Co., Takasaki, Gunma, Japan2Department of Medicine, University of Tokyo School of Medicine, Bunkyo-ku, Tokyo, Japan3Department of Laboratory Medicine, University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan
Address correspondence to: Takeyoshi Yamashita, Pharmaceutical Research Laboratories, KIRIN Brewery Co., 3 Miyahara, Takasaki, Gunma 370-1295, Japan. Phone: 81-27-346-9789; Fax: 81-27-346-1672; E-mail: tyamashita@kirin.co.jp.
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1Pharmaceutical Research Laboratories, KIRIN Brewery Co., Takasaki, Gunma, Japan2Department of Medicine, University of Tokyo School of Medicine, Bunkyo-ku, Tokyo, Japan3Department of Laboratory Medicine, University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan
Address correspondence to: Takeyoshi Yamashita, Pharmaceutical Research Laboratories, KIRIN Brewery Co., 3 Miyahara, Takasaki, Gunma 370-1295, Japan. Phone: 81-27-346-9789; Fax: 81-27-346-1672; E-mail: tyamashita@kirin.co.jp.
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1Pharmaceutical Research Laboratories, KIRIN Brewery Co., Takasaki, Gunma, Japan2Department of Medicine, University of Tokyo School of Medicine, Bunkyo-ku, Tokyo, Japan3Department of Laboratory Medicine, University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan
Address correspondence to: Takeyoshi Yamashita, Pharmaceutical Research Laboratories, KIRIN Brewery Co., 3 Miyahara, Takasaki, Gunma 370-1295, Japan. Phone: 81-27-346-9789; Fax: 81-27-346-1672; E-mail: tyamashita@kirin.co.jp.
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1Pharmaceutical Research Laboratories, KIRIN Brewery Co., Takasaki, Gunma, Japan2Department of Medicine, University of Tokyo School of Medicine, Bunkyo-ku, Tokyo, Japan3Department of Laboratory Medicine, University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan
Address correspondence to: Takeyoshi Yamashita, Pharmaceutical Research Laboratories, KIRIN Brewery Co., 3 Miyahara, Takasaki, Gunma 370-1295, Japan. Phone: 81-27-346-9789; Fax: 81-27-346-1672; E-mail: tyamashita@kirin.co.jp.
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1Pharmaceutical Research Laboratories, KIRIN Brewery Co., Takasaki, Gunma, Japan2Department of Medicine, University of Tokyo School of Medicine, Bunkyo-ku, Tokyo, Japan3Department of Laboratory Medicine, University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan
Address correspondence to: Takeyoshi Yamashita, Pharmaceutical Research Laboratories, KIRIN Brewery Co., 3 Miyahara, Takasaki, Gunma 370-1295, Japan. Phone: 81-27-346-9789; Fax: 81-27-346-1672; E-mail: tyamashita@kirin.co.jp.
Find articles by Tomizuka, K. in: JCI | PubMed | Google Scholar
1Pharmaceutical Research Laboratories, KIRIN Brewery Co., Takasaki, Gunma, Japan2Department of Medicine, University of Tokyo School of Medicine, Bunkyo-ku, Tokyo, Japan3Department of Laboratory Medicine, University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan
Address correspondence to: Takeyoshi Yamashita, Pharmaceutical Research Laboratories, KIRIN Brewery Co., 3 Miyahara, Takasaki, Gunma 370-1295, Japan. Phone: 81-27-346-9789; Fax: 81-27-346-1672; E-mail: tyamashita@kirin.co.jp.
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Published February 15, 2004 - More info
J Clin Invest. 2004;113(4):561–568. https://doi.org/10.1172/JCI19081.
© 2004 The American Society for Clinical Investigation
Received: May 30, 2003; Accepted: December 16, 2003
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Methods
Establishment of the FGF23-null mice. The genomic DNA corresponding to 5.2 kb of the 5′-flanking region, a part of exon 1, and the following 3′-intronic sequence in the Fgf23 gene were isolated from a C57BL/6 genomic bacterial artificial chromosome library. These DNA fragments were subcloned into 5′ and 3′ sites of a neomycin-resistance gene to construct the targeting vector (Figure 1a). This targeting vector was introduced into TT2 ES cells (13) by electroporation to replace the 0.2-kb fragment of DNA of exon 1. The transformed clones carrying a targeted allele were selected by Southern blot analysis and microinjected into the eight-cell embryo prepared from ICR mice. Male chimeric mice with a germline transmission of the targeted allele were mated with female C57BL/6 mice to generate heterozygous offspring. Homozygous founders were generated by mating these heterozygous mice. All mice were fed with a standard rodent chow containing 1.1% P and 1.0% Ca (CLEA Japan Inc., Tokyo, Japan) and tap water ad libitum. All studies using animals were reviewed and approved by the Institutional Animal Care and Use Committee at the Pharmaceutical Research Laboratories, KIRIN Brewery Co. (Takasaki, Japan).
Figure 1Establishment of FGF23-null mice. (a) Targeted ablation of Fgf23 gene. Construct of a targeting vector (top), wild-type allele (middle), and targeted mutant allele (bottom) are shown. DT-A, diphtheria toxin-A fragment; H, HindIII. (b) Genomic Southern blot analysis. Genomic DNAs (5 μg) isolated from transfected ES clones were digested with HindIII and hybridized to the indicated probe. (c) Gross phenotypes of heterozygous and homozygous founders at 6 weeks of age. (d) Growth curves and (e) survival ratios for male (filled) or female (open) WT (circles), heterozygotes (triangles), and homozygotes (squares). Data are represented as mean ± SEM. Statistical analysis in each sex was carried out by Dunnett’s method. Statistically significant results are marked by asterisk (male) or cross (female) (*P < 0.05, §P < 0.01, †P < 0.05, #P < 0.01). Numbers of animals used in this study: male WT (+/+), n = 9; female WT, n = 15; male heterozygotes (+/–), n = 20; female heterozygotes, n = 19; male homozygotes (–/–), n = 7; female homozygotes, n = 6.
Soft roentgenogram of femurs. The femurs were fixed in 4% paraformaldehyde and were exposed to X-rays at 0.1 mA and 25 kV for 5 seconds using μFX-1000 (Fujifilm Co., Tokyo, Japan). The image was developed by a BAS image analyzer (Fuji Photo Film Co.).
Bone histology. For double-labeling analysis, 30 mg/kg of tetracycline and then 30 mg/kg of calcein were injected intraperitoneally with a 3-day interval between injections. The isolated femurs were fixed and stained with Villanueva bone stain. Tissues were then embedded in a methyl methacrylate resin (Wako Pure Chemical Industries Ltd., Osaka, Japan), and undecalcified sections with 4 μm thickness were counterstained with Villanueva-Goldner counterstain. Histomorphometric analysis was carried out by choosing 24–26 fields in a femoral secondary cancellous bone.
Measurement of serum parameters. Blood samples were collected from the carotid artery (day 1 and 6) or heart (day 10 and thereafter) under anesthesia. Phosphate and calcium concentrations were determined using P-test WAKO and Ca-test WAKO, respectively (Wako Pure Chemical Industries Ltd.). Serum PTH and 1,25(OH)2D were determined by Rat Intact PTH RIA Kit (Immutopics, Inc., San Clemente, California, USA) and RIA Kit (TFB Co., Tokyo, Japan), respectively. Serum FGF23 was measured by a sandwich ELISA (12) established for a detection of full-length human FGF23. Immunoprecipitation followed by Western blotting demonstrated that the two mAbs used in this ELISA recognize mouse as well as human FGF23. In addition, a titration curve of serial dilutions of recombinant mouse FGF23 prepared by this ELISA was virtually identical to the standard curve produced using purified recombinant human FGF23 protein (data not shown), indicating that mouse FGF23 is quantitatively detectable by this ELISA. Results were expressed as values calculated from the standard curve using human FGF23. Urine samples were collected by rearing mice in metabolic cages (Sugiyamagen Co., Tokyo, Japan) for 24 hours. Serum alkaline phosphatase (ALP) activity, glucose, blood urea nitrogen (BUN), creatinine, total cholesterol, and triglycerides were determined by using the Hitachi 7180 autoanalyser (Hitachi High-Technologies Corp., Tokyo, Japan).
Measurement of sodium-dependent phosphate uptake activity of brush border membrane vesicles (BBMV). The BBMV were prepared by the method previously reported (14). The sodium-dependent phosphate uptake of the BBMV was measured by a rapid filtration method with several modifications (15).
Immunohistochemistry of type IIa sodium-phosphate cotransporter (NaPi-2a) protein. The kidneys were fixed in 10% paraformaldehyde, and embedded in paraffin. Sections of 3 μm thickness were incubated with a polyclonal rabbit anti–mouse NaPi-2a antibody produced by immunizing rabbits with the C-terminal peptide of NaPi-2a (LALPAHHNATRL). The signals were detected by ENVISION+ HRP system (DakoCytomation, Kyoto, Japan).
Northern blotting. The DNA fragments used as probes for all experiments were prepared from the mouse kidney cDNA by PCR with the following primer pairs. For 25-hydroxyvitamin D-1α-hydroxylase (1α-OHase): 5′-CAGACAGAGACATCCGTGTAG-3′ and 5′-CCACATGGTCCAGGTTCAGTC-3′; for 25-hydroxyvitamin D-24-hydroxylase (24-OHase): 5′-CTCGGAACGTCACCTCCTTA-3′ and 5′-CAGGCTGCTGGGAATATCTC-3′; and for GAPDH: 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′. Total RNA was isolated from the frozen kidneys using ISOGEN reagent (Nippon Gene Co., Tokyo, Japan). A 20-μg sample of each of the RNA samples was electrophoresed and transferred to Hybond N+ (Amersham Biosciences Corp., Piscataway, New Jersey, USA). Radiolabeled probe was prepared using Megaprime labeling system (Amersham Biosciences Corp.). The membrane was hybridized with the 32P-labeled probe in PerfectHyb reagent (TOYOBO Co., Osaka, Japan) overnight at 65°C. Then the blot was washed with a solution of 0.1 × SSC and 0.5% SDS for 30 minutes at 65°C. The signals were visualized by the BAS system (Fuji Photo Film Co.).
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Results
Generation of the FGF23-null mice. Targeted ablation of the Fgf23 gene generated heterozygotes (Fgf23+/–) and homozygotes (Fgf23–/–) (Figure 1, a and b). Mice with each genotype were born at the expected mendelian frequency. The heterozygotes did not show significant differences from WT mice in general appearance and growth (Figure 1, c and d). These phenotypes are consistent with the same serum level of FGF23 in heterozygotes (120.1 ± 4.4 pg/ml, n = 3) at 9 weeks of age as that of age-matched WT mice (126.0 ± 2.8 pg/ml, n = 13). Serum FGF23 could not be detected in homozygotes at this age, confirming a successful ablation of the Fgf23 gene (n = 7). However, marked growth retardation of homozygotes was observed by 13 days of age and thereafter, although at birth the body size and appearance of the Fgf23–/– mice were not different from those of WT or heterozygous littermates (Figure 1d). Moreover, the life span of the Fgf23–/– mice was markedly shorter than that of either WT mice or heterozygotes (Figure 1e). The ablation of FGF23 did not affect the developmental process of any organs by birth. However, the KO mice began to die at various ages after weaning, and no homozygotes survived more than 13 weeks. The survival ratios between male and female homozygous mice were not statistically different (P = 0.1455, log rank test). An autopsy of a 12-week-old homozygous mouse demonstrated sparse skeletal muscle and fat tissues. Furthermore, atrophy of glomeruli and marked vascular calcification in the kidneys, accompanied by elevated serum BUN (88.0 mg/dl, obtained at 2 days before death) were observed. Therefore, the impaired renal function, as one possible reason for the short life span of the Fgf23–/– mice, was probably due to the constantly higher serum levels of both calcium and phosphate almost throughout the life span, as discussed later. In addition, that the KO mice were hypoglycemic for unknown reasons (as discussed later) may also have contributed to the short life span.
The FGF23-null mice exhibited an obvious immaturity of the reproductive organs that resulted in infertility. Severe and rapid atrophy of the thymus and reduction of the spleen cells were observed after weaning, although these organs appeared to be normal at 3 weeks of age (data not shown). The Fgf23–/– mice also demonstrated marked decrease in peripheral lymphocytes (heterozygotes, 5,455 ± 1,424 cells/μl, n = 4; homozygotes, 833 ± 140 cells/μl, n = 4, P < 0.05).
Abnormal bone of Fgf23–/– mice. The Fgf23–/– mice showed abnormal bone development. As shown in Figure 2a, the femurs isolated from 7-week-old Fgf23–/– mice were very short, and the soft X-ray images of cortical bone demonstrated a marked reduction in mineral content. However, bone diameter was disproportionately wide and the epiphysis relatively large. Histological analysis revealed that the femurs isolated from 2-week-old homozygous mice appeared to be normal when the growth retardation became obvious (Figure 2, b and c). In contrast, in 7-week-old Fgf23–/– mice, the growth plate was severely disorganized and hypertrophic chondrocytes were not observed. In addition, a marked accumulation of unmineralized osteoids was seen in cortical bone and secondary spongiosa (Figure 2, b and c). Similar accumulation of osteoid was seen in tibia and calvarial bone (data not shown). Table 1 summarizes the results of bone histomorphometry in femoral secondary cancellous bones at 7 weeks of age. The mineralized bone volume/tissue volume ratio (mBV/TV) significantly decreased in the KO mice, although the bone volume/tissue volume ratio (BV/TV) was not statistically changed. There were fewer trabeculae present than in normal littermates, while the trabecular thickness was relatively greater as a result of osteoid accumulation. The osteoid volume, thickness, and surface area were markedly increased in the Fgf23–/– mice. Both osteoblast and osteoclast surface areas were markedly reduced in the null mice, indicating that bone turnover was suppressed. The double-labeling analysis failed to determine the parameters for mineralization because of an absence of the labeled mineralization surface in the null mice. None of the parameters that we examined were statistically different between WT and heterozygous mice.
Figure 2Histological analysis of bone. (a) Soft X-rays of femurs prepared from 7-week-old mice. (b and c) Villanueva-Goldner stain of undecalcified femurs prepared from 2- and 7-week-old mice. (b) Distal metaphysis of femurs isolated at 2 weeks (upper) and 7 weeks (lower) of age. Original magnification, ×4. (c) Growth plates of femurs at 2 weeks (upper) and 7 weeks (lower) of age. Mineralized bone tissues are colored in green, and unmineralized osteoid tissues are shown in orange. Original magnification, ×20.
Serum parameters in the Fgf23–/– mice. We had previously reported that administration of FGF23 reproduced the typical features of TIO including hypophosphatemia and low serum 1,25(OH)2D (6). In contrast, the Fgf23–/– mice showed significant elevations of serum phosphate, calcium, and 1,25(OH)2D (Figure 3). Serum phosphate concentrations were significantly elevated from day 10 after birth, and those elevations were maintained at least up to 9 weeks of age. Serum 1,25(OH)2D of the KO mice also began to increase at day 10. At this point, serum FGF23 was detected in WT and in heterozygous mice (WT, 186.4 ± 14.1 pg/ml, n = 10; heterozygotes; 205.5 ± 21.3 pg/ml, n = 9) but not in KO mice (n = 5). Although FGF23 protein (10–61 pg/ml) was detected in breast milk collected from the female heterozygous mice nursing homozygous pups, it was not sufficient to compensate for the lack of serum FGF23 to rescue the phenotypes in the Fgf23–/– mice. Elevation of serum calcium observed from 2 weeks after birth was modest but significant. Serum PTH of the FGF23-null mice were significantly decreased at 6 or 9 weeks of age, but not at 10 days or 2 weeks. This decrease in PTH may be at least in part due to the increased serum calcium and 1,25(OH)2D. The FGF23-null mice exhibited significant increases in serum ALP activities. The marked elevation was already observed at 2 weeks and was maintained at 6 weeks (Table 2). Hypoglycemia was observed from 2 weeks of age and became remarkable at 3 weeks and afterward (Table 2). In addition, serum total cholesterol was increased and triglycerides were decreased (Table 2). Heterozygotes did not show any changes in serum parameters examined in comparison with those of WT mice.
Figure 3Comparison of serum parameters. Blood samples were collected from carotid artery (day 1 and 6) or heart (day 10 and thereafter) under conditions of anesthesia. Because sufficient amounts of blood could not be obtained from mice aged younger than 10 days, sera were pooled in each genotype for measurement of 1,25(OH)2D. Other results represent mean ± SEM. The numbers of mice killed at each age were variable (average; WT n = 5, heterozygotes n = 9, and homozygotes n = 6). Statistical analysis was carried out by Dunnett’s method for comparison of multiple means (*P < 0.05; **P < 0.01). ND, not detected.
Altered expressions of key molecules for mineral homeostasis. Serum 1,25(OH)2D is mainly determined by activities of the renal enzymes 1α−OHase, which increases the serum concentrations of 1,25(OH)2D, and 24-OHase, which metabolizes and decreases serum 1,25(OH)2D (16). To seek an explanation for the high serum 1,25(OH)2D in the FGF23-null mice, we examined the expression of these enzymes by Northern blot. The expression of 1α-OHase was increased in the Fgf23–/– mice from at least day 10, when the serum 1,25(OH)2D also started to rise, and the increased 1α-OHase mRNA was maintained thereafter (Figure 4a). In addition, the enhanced mRNA level of 24-OHase was also observed at 3 weeks and thereafter in the Fgf23–/– mice (Figure 4a). Since the increase of 24-OHase mRNA occurred at 3 weeks, when serum 1,25(OH)2D levels had already increased, it is probable that this enhanced 24-OHase expression was promoted by the increased 1,25(OH)2D levels in the Fgf23–/– mice. Thus, upregulated 1α-OHase expression seems to be the main reason for the significant elevation of serum 1,25(OH)2D in the KO mice.
Figure 4Key molecules for serum phosphate and 1,25(OH)2D levels. (a) Northern blot analysis of renal 1α-OHase and 24-OHase mRNAs. The blots were reprobed with a GAPDH fragment to confirm integrity of the electrophoresed RNAs. (b) TmP/GFR. Mice (6 weeks old) were reared in metabolic cages to collect urine samples for 24 hours. The results represent mean ± SEM. WT, n = 6; heterozygotes, n = 6; homozygotes, n = 6. **P < 0.01, evaluated by Dunnett’s method. (c) Immunohistochemistry of renal NaPi-2a protein at 6 weeks of age.
Serum phosphate level is mainly maintained by renal phosphate reabsorption, which is regulated by the amount of NaPi-2a in brush border membrane of the renal proximal tubular cells (17). Actually, 6-week-old FGF23-KO mice demonstrated the increased tubular maximum transport of phosphate/GFR (TmP/GFR), suggesting that hyperphosphatemia in the Fgf23–/– mice was due to the increased renal phosphate reabsorption (Figure 4b). Therefore, we analyzed the expression of NaPi-2a to clarify the mechanism of this enhanced renal phosphate reabsorption. Figure 4c shows the results of immunohistochemical analysis for NaPi-2a with kidney sections prepared from 6-week-old mice. Despite remarkable hyperphosphatemia, NaPi-2a protein in the KO mice was more localized to the apical surface of proximal tubules, confirming there was the enhanced phosphate reabsorption in those mice. This finding was further confirmed by a significant increase in sodium-dependent phosphate-uptake activity of kidney BBMV in the KO mice (heterozygotes, 396.4 ± 88.2 pmol/mg/min, n = 5; homozygotes, 632.6 ± 105.8 pmol/mg/min, n = 6, P < 0.05, at 9 weeks). PTH is known to induce renal phosphate excretion by rapidly decreasing NaPi-2a on the apical surface of proximal tubules (18). Therefore, it is possible that the decreased PTH level in the Fgf23–/– mice at 6–9 weeks might have contributed to the disordered response of NaPi-2a. However, decreased PTH levels were not observed at 10 days and 2 weeks, when hyperphosphatemia was already present, indicating that suppression of PTH is not required for hyperphosphatemia in the FGF23-null mice. Thus, enhanced renal phosphate reabsorption via NaPi-2a is likely to be one of the major reasons for severe hyperphosphatemia in the FGF23-null mice.
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