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Circulating FGF-23 Is Regulated by 1α,25-Dihydroxyvitamin D3 and Phosphorus in Vivo

2004; Elsevier BV; Volume: 280; Issue: 4 Linguagem: Inglês

10.1074/jbc.m408903200

1083-351X

Hitoshi Saitô, Akira Maeda, Shuichi Ohtomo, Michinori Hirata, Kenichiro Kusano, Shigeaki Kato, Etsuro Ogata, Hiroko Segawa, Ken–ichi Miyamoto, Naoshi Fukushima,

Heterotopic Ossification and Related Conditions

Fibroblast growth factor-23 (FGF-23), a novel phosphate-regulating factor, was elevated in hypophosphatemic patients with X-linked hypophosphatemic rickets/osteomalacia and also in patients with chronic kidney disease. These observations suggested the pathophysiological importance of FGF-23 on phosphate homeostasis. However, regulation of FGF-23 production is still unclear. We investigated effects of both dietary phosphorus and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) on circulating FGF-23 in vivo Administration of. 1α,25(OH)2D3 dose-dependently increased serum FGF-23 in thyroparathyroidectomized rats without correlating with serum inorganic phosphorus or serum parathyroid hormone. On the other hand, vitamin D receptor null mice had very low serum FGF-23 and did not respond to the 1α,25(OH)2D3 administration. These observations suggested 1α,25(OH)2D3 directly or indirectly regulates circulating FGF-23. Serum FGF-23 had a strong correlation with serum inorganic phosphorus controlled by dietary phosphorus in 5/6 nephrectomized rats. High phosphate diet elicited a 5-fold increase in serum FGF-23 compared with sham-operated rats, whereas serum FGF-23 did not correlate with serum calcium or serum creatinine in 5/6 nephrectomized rats. Administration of 1α,25-dihydroxyvitamin D3 also elicited a severalfold increase in serum FGF-23 in the uremic rats. Taken together, this shows that both serum phosphorus and 1α,25(OH)2D3 regulate circulating FGF-23 independent of each other. Therefore, we proposed there was a feedback loop existing among serum phosphorus, 1α,25(OH)2D3, and FGF-23, in which the novel phosphate-regulating bone-kidney axis integrated with the parathyroid hormone-vitamin D3 axis in regulating phosphate homeostasis. Fibroblast growth factor-23 (FGF-23), a novel phosphate-regulating factor, was elevated in hypophosphatemic patients with X-linked hypophosphatemic rickets/osteomalacia and also in patients with chronic kidney disease. These observations suggested the pathophysiological importance of FGF-23 on phosphate homeostasis. However, regulation of FGF-23 production is still unclear. We investigated effects of both dietary phosphorus and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) on circulating FGF-23 in vivo Administration of. 1α,25(OH)2D3 dose-dependently increased serum FGF-23 in thyroparathyroidectomized rats without correlating with serum inorganic phosphorus or serum parathyroid hormone. On the other hand, vitamin D receptor null mice had very low serum FGF-23 and did not respond to the 1α,25(OH)2D3 administration. These observations suggested 1α,25(OH)2D3 directly or indirectly regulates circulating FGF-23. Serum FGF-23 had a strong correlation with serum inorganic phosphorus controlled by dietary phosphorus in 5/6 nephrectomized rats. High phosphate diet elicited a 5-fold increase in serum FGF-23 compared with sham-operated rats, whereas serum FGF-23 did not correlate with serum calcium or serum creatinine in 5/6 nephrectomized rats. Administration of 1α,25-dihydroxyvitamin D3 also elicited a severalfold increase in serum FGF-23 in the uremic rats. Taken together, this shows that both serum phosphorus and 1α,25(OH)2D3 regulate circulating FGF-23 independent of each other. Therefore, we proposed there was a feedback loop existing among serum phosphorus, 1α,25(OH)2D3, and FGF-23, in which the novel phosphate-regulating bone-kidney axis integrated with the parathyroid hormone-vitamin D3 axis in regulating phosphate homeostasis. The parathyroid hormone-vitamin D3 endocrine system, as well as dietary phosphorus, plays an important role in regulating renal and gastrointestinal absorption of phosphate. Recently, emerging evidence suggests that other systemic and/or paracrine/autocrine factors are present in bones for maintaining phosphate homeostasis, such as fibroblast growth factor-23 (FGF-23), 1The abbreviations used are: FGF-23, fibroblast growth factor-23; Pi, phosphate; IP, inorganic phosphorus; TPTX, thyroparathyroidectomy; XLH, X-linked hypophosphatemia; PHEX, phosphate-regulating gene with homologies to endopeptidases on the X chromosome; 1α,25(OH)2D3, 1α,25-dihydroxyvitamin D3; 1α-OHase, 25-hydroxyvitamin D3 1α-hydroxylase; PTH, parathyroid hormone; ELISA, enzymelinked immunosorbent assay; VDRKO, vitamin D receptor null(–/–). frizzled-related protein-4 (FRP-4), and matrix extracellular phosphoglycoprotein (MEPE) (1The ADHR, ConsortiumNat. 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Among these factors, FGF-23 strongly suppressed 1α,25(OH)2D3 production and elicited hypophosphatemia. Administration of the recombinant FGF-23 protein reduced serum phosphorus without affecting serum calcium, as well as increasing renal phosphorus excretion in mice (12Shimada T. Muto T. Urakawa I. Yoneya T. Yamazaki Y. Okawa K. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Endocrinology. 2002; 143: 3179-3182Crossref PubMed Scopus (387) Google Scholar). Mice bearing FGF-23-expressing Chinese hamster ovary cells showed suppressed 25-hydroxyvitamin D3 1α-hydroxylase mRNA expression in the kidney (3Shimada T. Mizutani S. Muto T. Yoneya T. Hino R. Takeda S. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6500-6505Crossref PubMed Scopus (1231) Google Scholar). FGF-23 mRNA is expressed in a variety of tissues such as thymus, brain, bone, thyroid/parathyroid gland, and heart (2Yamashita T. Yoshioka M. Ito N. Biochem. Biophys. Res. Commun. 2000; 277: 494-498Crossref PubMed Scopus (451) Google Scholar, 3Shimada T. Mizutani S. Muto T. Yoneya T. Hino R. Takeda S. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6500-6505Crossref PubMed Scopus (1231) Google Scholar, 13Liu S. Guo R. Simpson L.G. Xian Z-S. Burnham C.E. Quarles L.D. J. Biol. Chem. 2003; 278: 37419-37426Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). Recent studies (13Liu S. Guo R. Simpson L.G. Xian Z-S. Burnham C.E. Quarles L.D. J. Biol. Chem. 2003; 278: 37419-37426Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar, 14Riminucci M. Collins M.T. Fedaeko N.S. Cherman N. Corsi A. White K.E. Waguespack S. Gupta A. Hannon T. Econs M.J. Bianco P. Robey P.G. J. Clin. Invest. 2003; 112: 683-692Crossref PubMed Scopus (553) Google Scholar) indicated FGF-23 mRNA as well as FGF-23 protein was elevated in bones from patients with McCune-Albright syndrome and also in bones from HYP mouse, mouse homologue to X-linked hypophosphatemic (XLH) rickets. However, the level of serum FGF-23 in hypophosphatemic patients with XLH is still controversial (15Yamazaki Y. Okazaki R. Shibata M. Hasegawa Y. Satoh K. Tajima T. Takeuchi Y. Fujita T. Nakahara K. Yamashita T. Fukumoto S. J. Clin. Endocrinol. Metab. 2002; 87: 4957-4960Crossref PubMed Scopus (582) Google Scholar, 16Webber T.J. Liu S. Indridason O.S. Quarles L.D. J. Bone Miner. Res. 2003; 18: 1227-1234Crossref PubMed Scopus (310) Google Scholar, 17Jonsson K.B. Zahradnik R. Larsson T. White K.E. Sugimoto T. Imanishi Y. Yamamoto T. Hampson G. Koshiyama H. Ljuggren Ö. Oba K. Yang I.M. Miyauchi A. Econs M.J. Lavigne J. Jüppner H. N. Eng. J. Med. 2003; 348: 1656-1663Crossref PubMed Scopus (784) Google Scholar). Hyperphosphatemic patients with chronic kidney disease showed significant elevation in circulating FGF-23, which correlated with serum phosphorus and creatinine (16Webber T.J. Liu S. Indridason O.S. Quarles L.D. J. Bone Miner. Res. 2003; 18: 1227-1234Crossref PubMed Scopus (310) Google Scholar, 18Larsson T. Nisbeth U. Ljunggren Ö. Jüppner H. Jonsson K.B. Kidney Int. 2003; 64: 2272-2279Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar, 19Imanishi Y. Inaba M. Nakatsuka K. Nagasue K. Okuno S. Yoshihara A. Miura M. Miyauchi A. Kobayashi K. Miki T. Shoji T. Ishimura E. Nishizawa Y. Kidney Int. 2004; 65: 1943-1946Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 20Shigematsu T. Kazama J.J. Yamashita T. Fukumoto S. Hosoya T. Geyjo F. Fukagawa M. Am. J. Kidney Dis. 2004; 44: 250-256Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar), suggesting (a) serum phosphorus was a possible regulator of FGF-23 production or (b) circulating FGF-23 accumulated in chronic renal failure. The purpose of this study was to evaluate the effects of dietary phosphorus and 1α,25(OH)2D3 on FGF-23 production. Administration of FGF-23 protein or overexpression of Fgf23 gene in rodent suppressed 1α,25(OH)2D3 production by reducing 25-hydroxyvitamin D3 1α-hydroxylase in the proximal tubules (12Shimada T. Muto T. Urakawa I. Yoneya T. Yamazaki Y. Okawa K. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Endocrinology. 2002; 143: 3179-3182Crossref PubMed Scopus (387) Google Scholar, 21Saito H. Kusano K. Kinosaki M. Ito H. Hirata M. Segawa H. Miyamoto K. Fukushima N. J. Biol. Chem. 2003; 278: 2206-2211Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 22Bai X.Y. Miao D. Goltzman D. Karaplis A.C. J. Biol. Chem. 2003; 278: 9843-9849Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 23Shimada T. Urakawa I. Yamazaki Y. Hasegawa H. Hino R. Yoneya T. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Biochem. Biophys. Res. Comm. 2004; 314: 409-414Crossref PubMed Scopus (378) Google Scholar). On the contrary, Fgf23-null mice reported increased circulating 1α,25(OH)2D3 despite hyperphosphatemia, hypercalcemia, and low PTH levels (24Shimada T. Kakitani M. Yamazaki Y. Hasegawa H. Takeuchi Y. Fujita T. Fukumoto S. Tomizuka K. Yamashita T. J. Clin. Invest. 2004; 113: 561-568Crossref PubMed Scopus (1287) Google Scholar). Administration of 1α,25(OH)2D3 increased serum FGF-23 in normal mice (25Shimada T. Hasegawa H. Yamazaki Y. Muto T. Hino R. Takeuchi Y. Fujita T. Nakahara K. Fukumoto S. Yamashita T. J. Bone Miner. Res. 2004; 19: 429-435Crossref PubMed Scopus (1442) Google Scholar). These observations suggested mutual regulation between FGF-23 and 1α,25(OH)2D3; however, 1α,25(OH)2D3 administration also increases intestinal phosphate uptake and suppresses PTH. Thus, we used thyroparathyroidectomized rats as well as 5/6 nephrectomized rats fed a diet with various kinds of phosphorus content to examine the direct effect of 1α,25(OH)2D3 administration on serum FGF-23. Dietary phosphate deprivation or loading rapidly induces activation or repression of phosphate absorption in kidney and in intestine, mainly by inducing or suppressing type II sodium-dependent phosphate (Na/Pi) cotransporter expression (26Katai K. Miyamoto K. Kishida S. Segawa H. Nii T. Tanaka H. Tani Y. Arai H. Tatsumi S. Morita K. Taketani Y. Takeda E. Biochem. J. 1999; 343: 705-714Crossref PubMed Scopus (161) Google Scholar, 27Katai K. Segawa H. Haga H. Morita K. Arai H. Tatsumi S. Taketani Y. Miyamoto K. Hisano S. Fukui Y. Takeda E. J. Biochem. (Tokyo). 1997; 121: 50-55Crossref PubMed Scopus (49) Google Scholar). Therefore, the serum phosphorus level is not susceptible to the change in dietary phosphorus content in normal animals in vivo. We investigated the effects of dietary phosphorus on FGF-23 production in vivo using 5/6 nephrectomized uremic rats, in which the serum phosphorus level can be easily manipulated by dietary phosphorus due to reduced kidney function. We also examined the correlations between serum FGF-23 and (a) serum inorganic phosphorus, (b) serum calcium, (c) serum creatinine, and (d) serum PTH in 5/6 nephrectomized rats. Normal Rat—Eight-week-old male Sprague-Dawley rats (CREA Japan, Inc., Shizuoka, Japan) were given either vehicle, 10, 30, 100, or 300 ng of 1α,25(OH)2D3/kg of bodyweight intravenously, three times a week for 2 weeks. Blood samples were obtained from the vena cava under ether anesthesia on day 14. Serum calcium, serum inorganic phosphorus, and serum creatinine were measured by an automatic analyzer (Type 7170E, Hitachi Corp.). Serum FGF-23 was determined by the Human FGF-23 ELISA kit (Kinos Inc., Tokyo, Japan). Thyroparathyroidectomized (TPTX) Rats and PTH-infused TPTX Rats—Eight-week-old male Sprague-Dawley rats purchased from Charles River (Tokyo, Japan) were TPTX under ether anesthesia. After confirming hypocalcemia had been induced, rats were divided to two groups, the TPTX group and the TPTX + PTH group. Rats in the TPTX + PTH group were subcutaneously implanted with ALZET® osmotic pumps (model 2ML2, Durect Corp., Cupertino, CA) and administered human PTH-(1–34) at a constant rate of 2.4 μg/day for 14 days. Rats in the TPTX group were intravenously injected with vehicle, 50 ng/kg or 300 ng/kg 1α,25(OH)2D3, three times a week for 2 weeks; rats in the TPTX + PTH group were injected with either vehicle or 50 ng/kg 1α,25(OH)2D3 three times a week for 2 weeks. Blood samples were collected and various parameters analyzed as described above. Vitamin D Receptor Null(–/–) (VDRKO) Mice—VDRKO mice and their littermates kept on a high calcium and high phosphorus diet (28Yoshizawa T. Handa Y. Uematsu Y. Takeda S. Sekine K. Yoshihara Y. Kawakami T. Arioka K. Sato H. Uchiyama Y. Masushige S. Fukamizu A. Matsumoto T. Kato S. Nat. Genet. 1997; 6: 391-396Crossref Scopus (1001) Google Scholar) were administered vehicle or 300 ng/kg 1α,25(OH)2D3 three times a week for 2 weeks. Blood samples were collected and analyzed as described above. 5/6 Subtotally Nephrectomized Uremic Rat Models and Diets—Male Sprague-Dawley rats weighing 180–200 g were purchased from CREA Japan and maintained under specific pathogen-free conditions with a 12-h light/dark cycle. After acclimating for 1 week, the rats were 5/6 nephrectomized and then allowed unlimited access to normal rodent chow (CE-2, CREA Japan Inc.) and tap water. The phosphate (Pi)-controlled diets and 1α,25(OH)2D3 injection started when rat serum creatinine reached within the range of 1.1–1.5 mg/dl. Three Pi-controlled diets (Oriental Yeast Co., Ltd., Osaka, Japan) were used in the present study: a high Pi diet, containing 0.9% phosphorus, 0.6% calcium; a midrange Pi diet, containing 0.6% phosphorus, 0.6% calcium; and a low Pi diet, containing 0.2% phosphorus, 0.6% calcium (26Katai K. Miyamoto K. Kishida S. Segawa H. Nii T. Tanaka H. Tani Y. Arai H. Tatsumi S. Morita K. Taketani Y. Takeda E. Biochem. J. 1999; 343: 705-714Crossref PubMed Scopus (161) Google Scholar, 27Katai K. Segawa H. Haga H. Morita K. Arai H. Tatsumi S. Taketani Y. Miyamoto K. Hisano S. Fukui Y. Takeda E. J. Biochem. (Tokyo). 1997; 121: 50-55Crossref PubMed Scopus (49) Google Scholar). The following groups of rats were studied: 1) 5/6 nephrectomized rats fed either the high, midrange, or low Pi diet for 4 weeks, 2) 5/6 nephrectomized rats fed the high, midrange, or low Pi diet and injected with 50 ng of 1α,25(OH)2D3/kg of bodyweight intravenously twice weekly for 4 weeks, and 3) age-matched sham-operated rats fed normal rat chow (CE-2, CREA Japan) used as a control. Blood samples were obtained from the vena cava under ether anesthesia at day 28 and various parameters analyzed as described above. Serum PTH was determined using a Rat Intact PTH ELISA kit (Immutopics, Inc., San Clemente, CA). All animal procedures were conducted in accordance with Chugai Pharmaceutical's ethical guidelines for animal care, and all experimental protocols were approved by the Animal Care Committee of the institution. Statistical Analysis—Data were expressed as means ± S.E., and statistical significance was determined using Student's t test or Dunnett's t test (SAS Preclinical Package, Version 5.0, SAS Institute Japan, Tokyo) unless otherwise indicated. A p value of <0.05 was considered statistically significant. Effect of 1α,25(OH)2D3 on Serum FGF-23 in Normal Rats— Intravenous administration of 1α,25(OH)2D3, three times a week for 2 weeks, dose-dependently increased serum FGF-23 (Fig. 1A); however, it also increased serum inorganic phosphorus (Fig. 1B). Therefore, 1α,25(OH)2D3 administration increased both serum FGF-23 and serum phosphorus in normal rats. Effects of 1α,25(OH)2D3 on Serum FGF-23 in TPTX Rats—As shown in Fig. 2, A–C, Thyroparathyroidectomized rats induced hyperphosphatemia, hypocalcemia, and also a slight but significant decrease in serum FGF-23 in comparison with normal rats. Administration of 1α,25(OH)2D3 dose-dependently increased serum FGF-23 as well as serum calcium in TPTX rats. Also 1α,25(OH)2D3 suppressed serum inorganic phosphorus in TPTX rats. PTH infusion to TPTX rats normalized serum calcium, serum inorganic phosphorus, and serum FGF-23. 1α,25(OH)2D3 increased serum FGF-23; however, unlike TPTX rats, no significant change was observed in serum inorganic phosphorus and serum calcium in PTH-infused TPTX rats. These observations suggested that 1α,25(OH)2D3 increased serum FGF-23 independent of serum inorganic phosphorus and PTH. Effect of 1α,25(OH)2D3 on Serum FGF-23 in VDRKO Mice— VDRKO mice kept on high calcium (2%) and high phosphorus (1.25%) diets showed considerably low serum FGF-23 (<3 pg/ml, below detection limit) and low serum calcium (5.8 ± 0.6 mg/dl) in comparison with the wild-type littermates (FGF-23, 254 ± 35.6 pg/ml; calcium, 9.5 ± 0.1 mg/dl). 1α,25(OH)2D3 administration did not affect either serum FGF-23 (<3 pg/ml, below detection limit) or serum calcium (5.1 ± 0.2 mg/dl). Effects of Pi-controlled Diets on Serum FGF-23 and Serum Inorganic Phosphorus in 5/6 Nephrectomized Rats—Serum inorganic phosphorus correlated with the dietary phosphorus contents (Fig. 3A) in 5/6 nephrectomized rats. On the contrary, serum calcium correlated with serum phosphorus in a reciprocal fashion (Fig. 3B). The 5/6 nephrectomy induced a significant increase in serum FGF-23 in rats, regardless of the dietary phosphorus contents (Fig. 3C). Serum FGF-23 of sham-operated rats was 305 ± 23 pg/ml. Whereas, serum FGF-23 increased in high, midrange, and low Pi diet groups (5108 ± 989 pg/ml, 1815 ± 200 pg/ml, and 1133 ± 121 pg/ml, respectively). Serum FGF-23 showed a clear correlation with serum phosphorus (Fig. 4A) and a weak inverse correlation with serum calcium (Fig. 4B) in 5/6 nephrectomized rats. However, serum FGF-23 did not correlate with serum creatinine (Fig. 4C).Fig. 4Correlations between serum FGF-23 and serum inorganic phosphorus (A), serum calcium (B), and serum creatinine (C) in 5/6 nephrectomized rats fed with Pi-controlled diet. 5/6 nephrectomized rats were fed either high Pi, midrange Pi, or low Pi diet for 4 weeks. Serum creatinine, serum inorganic phosphorus, and serum FGF-23 were determined as described under "Experimental Procedures." Open circles represent the data from individual rats.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Effect of 1α,25(OH)2D3 on Serum FGF-23 and Serum Inorganic Phosphorus in 5/6 Nephrectomized Rats—Serum FGF-23 in the 5/6 nephrectomized rats on each Pi-controlled diet was augmented significantly by the administration of 1α,25(OH)2D3 (Fig. 5). 1α,25(OH)2D3 administration also increased serum inorganic phosphorus, serum calcium, and serum creatinine and decreased serum PTH in all diet groups (data not shown). Serum inorganic phosphorus weakly correlated with serum FGF-23 in the nephrectomized rats with or without 1α,25(OH)2D3 treatment (Fig. 6A). However, the other three parameters did not have a strong correlation with serum FGF-23 (Fig. 6, B–D).Fig. 6Correlations between serum FGF-23 and serum inorganic phosphorus (A), serum calcium (B), serum creatinine (C), and serum PTH (D) in 5/6 nephrectomized rats with or without 1α,25-dihydroxyvitamin D3 administration. 5/6 nephrectomized rats fed Pi-controlled diets were given intravenously either vehicle or 50 ng/kg 1α,25-dihydroxyvitamin D3 for 4 weeks. Serum creatinine, serum inorganic phosphorus, and serum FGF-23 were determined as described under "Experimental Procedures." Open circles represent the data from individual rats.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In normal rats, 30 and 100 ng/kg 1α,25(OH)2D3 injection increased serum FGF-23 only by 1.5- and 3-fold, respectively. Whereas, in the 5/6 nephrectomized rats, 50 ng/kg 1α,25(OH)2D3 increased serum FGF-23 by 3–9-fold, suggesting that 1α,25(OH)2D3 had a more profound effect on increasing serum FGF-23 in rats with chronic renal failure. Shimada et al. reported that a single injection of 1α,25(OH)2D3 increased serum FGF-23 and suggested the increase in FGF-23 by 1α,25(OH)2D3 was independent of serum inorganic phosphorus (25Shimada T. Hasegawa H. Yamazaki Y. Muto T. Hino R. Takeuchi Y. Fujita T. Nakahara K. Fukumoto S. Yamashita T. J. Bone Miner. Res. 2004; 19: 429-435Crossref PubMed Scopus (1442) Google Scholar). We confirmed their result that intravenous administration of 1α,25(OH)2D3 dose-dependently increased serum FGF-23 (Fig. 1A). However, we also observed an increase in serum inorganic phosphorus (Fig. 1B). There was a strong significant correlation between serum FGF-23 and serum phosphorus (Fig. 1C) in the normal rats given 1α,25(OH)2D3. Since 1α,25(OH)2D3 stimulates intestinal phosphate uptake and suppresses PTH production, this experiment indicated that 1α,25(OH)2D3 increased serum FGF-23 as well as serum phosphorus. To evaluate the effect of 1α,25(OH)2D3 apart from serum phosphorus on FGF-23 production, 1α,25(OH)2D3 was administered to thyroparathyroidectomized rats with or without PTH infusion. 1α,25(OH)2D3 also increased serum FGF-23 in thyroparathyroidectomized, and the effect was independent of serum phosphorus (Fig. 2, A–C). The direct effect of 1α,25(OH)2D3 on FGF-23 production was confirmed by the fact that VDRKO mice did not respond to the 1α,25(OH)2D3 administration. Larsson et al. (18Larsson T. Nisbeth U. Ljunggren Ö. Jüppner H. Jonsson K.B. Kidney Int. 2003; 64: 2272-2279Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar) reported phosphate deprivation and/or phosphate loading to normal subjects did not affect serum FGF-23; however, serum phosphorus weakly correlated with serum FGF-23 in predialysis patients with chronic kidney disease. Recent studies also revealed that serum FGF-23 was elevated in patients with end-stage renal disease (16Webber T.J. Liu S. Indridason O.S. Quarles L.D. J. Bone Miner. Res. 2003; 18: 1227-1234Crossref PubMed Scopus (310) Google Scholar, 20Shigematsu T. Kazama J.J. Yamashita T. Fukumoto S. Hosoya T. Geyjo F. Fukagawa M. Am. J. Kidney Dis. 2004; 44: 250-256Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 28Yoshizawa T. Handa Y. Uematsu Y. Takeda S. Sekine K. Yoshihara Y. Kawakami T. Arioka K. Sato H. Uchiyama Y. Masushige S. Fukamizu A. Matsumoto T. Kato S. Nat. Genet. 1997; 6: 391-396Crossref Scopus (1001) Google Scholar). In the present study, we investigated the effect of dietary phosphorus on FGF-23 production using 5/6 nephrectomized rats fed the diets with various kinds of phosphorus content. Serum FGF-23 was elevated in uremic rats; however, serum FGF-23 did not clearly correlate with serum creatinine in those rats as was observed in human subjects. Serum phosphorus was well controlled by the dietary phosphorus in 5/6 nephrectomized rats (Fig. 3A). Serum FGF-23 positively correlated with serum phosphorus in those rats (Fig. 3C). In the physiological condition, a high serum phosphorus suppresses 1α,25(OH)2D3 production in kidney. Thus, the elevation of serum FGF-23 induced by a high Pi diet was independent of serum 1α,25(OH)2D3. Moreover, serum FGF-23 was drastically elevated by 1α,25(OH)2D3 administration in 5/6 nephrectomized rats fed with various Pi-controlled diets (Fig. 5). However, serum FGF-23 did not correlate with serum calcium, serum creatinine, or serum PTH in those rats (Fig. 6, B–D). These observations suggested that FGF-23 production was mainly regulated by serum phosphorus and serum 1α,25(OH)2D3. Recent studies (16Webber T.J. Liu S. Indridason O.S. Quarles L.D. J. Bone Miner. Res. 2003; 18: 1227-1234Crossref PubMed Scopus (310) Google Scholar, 17Jonsson K.B. Zahradnik R. Larsson T. White K.E. Sugimoto T. Imanishi Y. Yamamoto T. Hampson G. Koshiyama H. Ljuggren Ö. Oba K. Yang I.M. Miyauchi A. Econs M.J. Lavigne J. Jüppner H. N. Eng. J. Med. 2003; 348: 1656-1663Crossref PubMed Scopus (784) Google Scholar) reported that FGF-23 was elevated in some patients with XLH. Serum phosphorus concentrations were negatively correlated with circulating FGF-23 levels in patients with XLH. Moreover, FGF-23 mRNA expression was enhanced in the calvarial and mandible bones of Hyp-mouse, which is a homologue of human XLH (13Liu S. Guo R. Simpson L.G. Xian Z-S. Burnham C.E. Quarles L.D. J. Biol. Chem. 2003; 278: 37419-37426Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). Mutations in PHEX, a phosphate-regulating gene with homology to endopeptidase on the X-chromosome, are responsible for XLH. PHEX mRNA is predominantly expressed in bone and teeth. 1α,25(OH)2D3 decreased PHEX mRNA and PHEX protein in primary osteoblasts derived from newborn mouse calvaria as well as MC3T3-E1 cells, a mouse osteoblastic cell line, in vitro (29Ecarot B. Desbarats M. Endocrinology. 1999; 140: 1192-1199Crossref PubMed Google Scholar). In addition, PHEX mRNA expression in tibial bone was suppressed by 1α,25(OH)2D3 administration in 5/6 nephrectomized rats in vivo (30Brewer A.J. Canaff L. Hendy G.F. Tennenhouse H.S. Am. J. Physiol. 2004; 286: F739-F748Crossref PubMed Scopus (14) Google Scholar). It is plausible that administration of 1α,25(OH)2D3 up-regulated circulating FGF-23 levels in 5/6 nephrectomized rats at least partly by down-regulation of PHEX expression in bones. FGF-23 induces hypophosphatemia by inhibiting both renal and intestinal Pi absorption by suppressing NaPi-IIa and -IIb production (3Shimada T. Mizutani S. Muto T. Yoneya T. Hino R. Takeda S. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6500-6505Crossref PubMed Scopus (1231) Google Scholar, 12Shimada T. Muto T. Urakawa I. Yoneya T. Yamazaki Y. Okawa K. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Endocrinology. 2002; 143: 3179-3182Crossref PubMed Scopus (387) Google Scholar, 21Saito H. Kusano K. Kinosaki M. Ito H. Hirata M. Segawa H. Miyamoto K. Fukushima N. J. Biol. Chem. 2003; 278: 2206-2211Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 22Bai X.Y. Miao D. Goltzman D. Karaplis A.C. J. Biol. Chem. 2003; 278: 9843-9849Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 25Shimada T. Hasegawa H. Yamazaki Y. Muto T. Hino R. Takeuchi Y. Fujita T. Nakahara K. Fukumoto S. Yamashita T. J. Bone Miner. Res. 2004; 19: 429-435Crossref PubMed Scopus (1442) Google Scholar). FGF-23 also inhibits 1α,25(OH)2D3 production in renal proximal tubules, which results in the reduction of intestinal Pi absorption and PTH secretion. On the contrary, 1α,25(OH)2D3 induced an increase in circulating FGF-23, and also loss of vitamin D signaling in VDRKO mice led to very low serum FGF-23. In 5/6 nephrectomized rats, serum phosphorus controlled by dietary phosphorus content positively correlated with serum FGF-23, suggesting an increase in serum phosphorus induces FGF-23 production. We propose that a feedback loop exists between serum phosphorus, 1α,25(OH)2D3, and FGF-23, in which the novel phosphate-regulating bone-kidney axis would be integrated with the parathyroid hormone-vitamin D3 axis in regulating phosphate homeostasis. We thank Keiko Kuroiwa and Yuko Azabu (Pharmaceutical Research Dept. II, Chugai Pharmaceutical Co., Ltd.) for their technical assistance. We also thank Dr. Paul Langman for his assistance with English usage.