The mutant mice lacking FGF23 exhibited severe hyperphosphatemia, with enhanced renal phosphate reabsorption and high serum 1,25(OH)2
D. In addition, these phenotypes are the mirror images of those in previously reported animal studies in which administration of FGF23 caused hypophosphatemia and low serum 1,25(OH)2
). In contrast, heterozygotes showed no abnormality in any of the parameters examined, including serum FGF23. These results clearly indicate that production and serum concentrations of FGF23 are tightly regulated and that this regulatory mechanism can compensate for the absence of one allele of the Fgf23
gene, although the precise source and mechanism of regulation of FGF23 production are not sufficiently clear. Therefore, it is suggested that FGF23 is acting physiologically to reduce serum phosphate and 1,25(OH)2
Renal production of 1,25(OH)2
D is stimulated by PTH and hypophosphatemia, and inhibited by 1,25(OH)2
D and hypercalcemia (16
). The Fgf23–/–
mice showed hyperphosphatemia, hypercalcemia, high serum 1,25(OH)2
D, and suppressed PTH, all of which have been described to suppress 1,25(OH)2
D production. However, the KO mice showed sustained elevation of 1α-OHase expression and high 1,25(OH)2
D levels at least from 10 days of age. On the contrary, our preliminary experiment demonstrated that injecting recombinant FGF23 into the Fgf23–/–
mice resulted in a significant reduction in serum 1,25(OH)2
D within 8 hours (untreated WT mice, 152.1 ± 17.9 pg/ml; untreated homozygotes, 707.6 ± 258.2 pg/ml; FGF23-treated homozygotes, 79.9 ± 9.0 pg/ml). Therefore, it is likely that FGF23 is physiologically suppressing the renal 1α-OHase expression cooperating or competing with several humoral factors such as PTH and 1,25(OH)2
D itself. Although we were unable to evaluate the serum 1,25(OH)2
D in homozygotes at 6 days of age because there were insufficient sera available for the measurement, we could detect circulatory FGF23 in 6-day-old WT mice (154.2 ± 22.3 pg/ml, n
= 4). Because hyperphosphatemia of the KO mice first appeared at 10 days after birth and other phenotypes at later times, FGF23 seems to be less important in newborn mice and the FGF23-independent phosphate-handling mechanism is probably dominant at this stage.
One possibility is that the regulatory mechanism of intestinal and renal phosphate handling is immature and rather insensitive to hormone-dependent changes of phosphate influx and efflux. The other possibility is that another phosphate-regulatory factor present in milk plays a dominant role in regulating serum phosphate.
Because excess actions of FGF23 result in rickets/osteomalacia, it was surprising that the Fgf23–/–
mice also exhibited the disorganized growth plate and accumulation of osteoid in cortical and calvarial bones. Although the precise mechanism of this abnormal bone development is not clear at the moment, the elevated serum 1,25(OH)2
D may have contributed to these changes. The increase of osteoid has been reported in the 24-OHase–deficient mice that demonstrated marked elevation of 1,25(OH)2
). The other possible reason for the abnormal bone development may be a decreased PTH level. PTH is a potent anabolic factor for bone formation, and its absence is known to suppress bone turnover (22
). The significant reductions in both osteoblast and osteoclast surface areas in the KO mice may be caused by the deficient PTH action. However, increased osteoid cannot be explained by suppressed turnover of bone. In contrast, it is possible that FGF23 has a direct action on bone metabolism. Actually, a recent report showed expression of FGF23 in bone, suggesting an unknown function of FGF23 in bone metabolism (23
). Additional studies, such as an investigation of the receptor and the signaling pathway, will be necessary to clarify these issues.
It has been reported that administration of 1,25(OH)2
induces a positive balance of phosphate and calcium (16
). A recent report has shown that expression of intestinal type IIb sodium-dependent phosphate cotransporter, which is thought to be involved in active phosphate transport in the intestine, can be regulated by 1,25(OH)2
). Thus, increased serum 1,25(OH)2
D may have contributed to hyperphosphatemia in the Fgf23–/–
mice, at least in part. In addition, the FGF23-null mice demonstrated enhanced renal phosphate-uptake activities and more restricted distribution of NaPi-2a on the apical side of the proximal tubule, which should be downregulated in the presence of hyperphosphatemia in normal animals (25
). Bai et al. have reported that continuous administration of FGF23 induced the downregulation of NaPi-2a (10
), and we have also observed the decreased NaPi-2a expression at 8–13 hours after a single injection of FGF23 into WT mice (26
). Furthermore, 2-week-old Fgf23–/–
mutant mice showed normal BUN levels compared with those of control littermates (WT, 18.6 ± 1.6 mg/dl, n
= 3; heterozygotes, 19.9 ± 1.3 mg/dl, n
= 3; homozygotes, 22.6 ± 2.2 mg/dl, n
= 4), while homozygous mice by that age had already demonstrated significant hyperphosphatemia (Figure ). Therefore, these results indicate that physiological response to hyperphosphatemia is abolished in the Fgf23–/–
mice and demonstrate the essential role of FGF23 in the regulation of renal phosphate reabsorption. Although the extremely small body size of Fgf23–/–
mice did not allow us to analyze the effect of parathyroidectomy on serum phosphate, our previous study demonstrated that FGF23 is able to reduce serum phosphate levels in parathyroidectomized animals (26
). In addition, hyperphosphatemia of the KO mice observed at 10 days and 2 weeks after birth was associated with normal serum PTH levels. These results indicate that hyperphosphatemia of the KO mice is induced by a PTH-independent mechanism.
In contrast, it remains unclear whether FGF23 plays important roles in other physiological events besides mineral homeostasis. When we fed the Fgf23–/– mice with a low-phosphate diet (Pi 0.2%, Ca 0.5%) from just after weaning (4 weeks, n = 6), 50% of homozygous mice survived more than 13 weeks. However, these mice with prolonged life span still demonstrated severe growth retardation (7.4–12.4 g body weight), hypoglycemia, and elevated 1,25(OH)2D (670.1 ± 108.8 pg/ml), although serum phosphate was successfully decreased (5.8 ± 2.3 mg/dl). Thus, hyperphosphatemia itself is not sufficient to result in these phenotypes of the null mice. Atrophy of thymus and spleen became apparent only after weaning in FGF23-null mice. These results suggest that metabolic changes such as hyperphosphatemia and high serum 1,25(OH)2D, rather than the deficiency of FGF23 itself, are responsible for abnormalities in lymphocytes. Actually, 1,25(OH)2D has been reported to be a potent regulator of the immune system. Additional studies are necessary to clarify the abnormalities in these tissues of FGF23-null mice.
In summary, here we have clarified the essential roles of FGF23 in the physiological regulation of phosphate and vitamin D metabolism. These findings provide new insights for the understanding of bone and mineral metabolism. Additional studies using these Fgf23–/– mice, together with exploration of the receptor for FGF23 and of the tissues responsible for FGF23 production should more precisely decipher the physiological significance of FGF23.