Calciprotein particles regulate fibroblast growth factor-23 expression in osteoblasts
Authors: Ken-ichi Akiyama, Yutaka Miura, Hirosaka Hayashi, Asuka Sakata, Yoshitaka Matsumura, Masaki Kojima, Ken Tsuchiya, Kosaku Nitta, Kazuhiro Shiizaki, Hiroshi Kurosu, Makoto Kuro-o
Journal: Kidney International (2019)

Fibroblast growth factor-23 (FGF23) is a hormone indispensable for maintaining phosphate homeostasis. In response to phosphate intake, FGF23 is secreted from osteocytes/osteoblasts and acts on the kidney to increase urinary phosphate excretion. However, the mechanism by which these cells sense phosphate intake remains elusive. Calciprotein particles are nanoparticles of calcium-phosphate precipitates bound to serum protein fetuin-A and are generated spontaneously in solution containing calcium, phosphate, and fetuin-A to be dispersed as colloids. In cultured osteoblastic cells, increase in either calcium or phosphate concentration in the medium induced FGF23 expression, which was dependent on calciprotein particle formation. When transition of calcium-phosphate precipitates from the amorphous phase to the crystalline phase was blocked by bisphosphonate, the calciprotein particle size was reduced and FGF23 expression was augmented, suggesting that small calciprotein particles containing amorphous calcium-phosphate precipitates function as a more potent FGF23 inducer than larger calciprotein particles containing crystalline calcium-phosphate precipitates. In mice, bolus phosphate administration by oral gavage transiently increased circulating calciprotein particle levels followed by a modest increase in FGF23 expression and serum FGF23 levels. However, continuous dietary phosphate load induced robust and persistent increase in circulating calciprotein particles and FGF23 levels. We confirmed by in vivo imaging that calciprotein particles injected intravenously extravasated into the bone marrow and were deposited on the inner surface of the bone, indicating that these particles have direct access to osteoblasts. Thus, we propose that osteoblasts induce FGF23 expression and secretion when they sense an increase in extracellular calciprotein particles following phosphate ingestion. Fibroblast growth factor-23 (FGF23) is a hormone indispensable for maintaining phosphate homeostasis. In response to phosphate intake, FGF23 is secreted from osteocytes/osteoblasts and acts on the kidney to increase urinary phosphate excretion. However, the mechanism by which these cells sense phosphate intake remains elusive. Calciprotein particles are nanoparticles of calcium-phosphate precipitates bound to serum protein fetuin-A and are generated spontaneously in solution containing calcium, phosphate, and fetuin-A to be dispersed as colloids. In cultured osteoblastic cells, increase in either calcium or phosphate concentration in the medium induced FGF23 expression, which was dependent on calciprotein particle formation. When transition of calcium-phosphate precipitates from the amorphous phase to the crystalline phase was blocked by bisphosphonate, the calciprotein particle size was reduced and FGF23 expression was augmented, suggesting that small calciprotein particles containing amorphous calcium-phosphate precipitates function as a more potent FGF23 inducer than larger calciprotein particles containing crystalline calcium-phosphate precipitates. In mice, bolus phosphate administration by oral gavage transiently increased circulating calciprotein particle levels followed by a modest increase in FGF23 expression and serum FGF23 levels. However, continuous dietary phosphate load induced robust and persistent increase in circulating calciprotein particles and FGF23 levels. We confirmed by in vivo imaging that calciprotein particles injected intravenously extravasated into the bone marrow and were deposited on the inner surface of the bone, indicating that these particles have direct access to osteoblasts. Thus, we propose that osteoblasts induce FGF23 expression and secretion when they sense an increase in extracellular calciprotein particles following phosphate ingestion. see commentary on page 648 see commentary on page 648 Translational StatementCalciprotein particles (CPPs) are colloidal nanoparticles of calcium-phosphate dispersed in the blood. CPPs are increased in patients with chronic kidney disease and are regarded as pathological agents that induce inflammatory responses and vascular calcification. On the other hand, circulating levels of a bone-derived phosphaturic hormone fibroblast growth factor-23 are also increased with chronic kidney disease progression and are associated with mineral bone disorder. In this study, we show that CPPs function as a physiological agent that delivers calcium-phosphate to the bone and induces fibroblast growth factor-23 expression in osteoblasts. The measurement of blood CPP levels is expected to be useful not only for a better understanding on how fibroblast growth factor-23 production/secretion is regulated but also for risk evaluation of chronic kidney disease–mineral bone disorder. Calciprotein particles (CPPs) are colloidal nanoparticles of calcium-phosphate dispersed in the blood. CPPs are increased in patients with chronic kidney disease and are regarded as pathological agents that induce inflammatory responses and vascular calcification. On the other hand, circulating levels of a bone-derived phosphaturic hormone fibroblast growth factor-23 are also increased with chronic kidney disease progression and are associated with mineral bone disorder. In this study, we show that CPPs function as a physiological agent that delivers calcium-phosphate to the bone and induces fibroblast growth factor-23 expression in osteoblasts. The measurement of blood CPP levels is expected to be useful not only for a better understanding on how fibroblast growth factor-23 production/secretion is regulated but also for risk evaluation of chronic kidney disease–mineral bone disorder. Phosphorus homeostasis at the organismal level is maintained by balancing phosphate intake and excretion. Specifically, the amount of phosphate excreted into urine is regulated so as to become equal to the amount of phosphate absorbed from the digestive tract.1Schiavi S.C. Kumar R. The phosphatonin pathway: new insights in phosphate homeostasis.Kidney Int. 2004; 65: 1-14Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar The amount of urinary phosphate excretion is primarily regulated by the endocrine axis consisting of fibroblast growth factor-23 (FGF23) and its obligate coreceptor klotho. FGF23 is a hormone secreted from osteoblasts and osteocytes in response to phosphate intake. FGF23 binds to the binary complex of FGF receptor and klotho expressed in renal tubules and suppresses phosphate reabsorption, thereby promoting urinary phosphate excretion.2Hu M.C. Shiizaki K. Kuro-o M. et al.Fibroblast growth factor 23 and klotho: physiology and pathophysiology of an endocrine network of mineral metabolism.Annu Rev Physiol. 2013; 75: 503-533Crossref PubMed Scopus (336) Google Scholar However, it is not clear how osteocytes/osteoblasts sense phosphate intake to induce expression and secretion of FGF23. A possible mechanism is that osteoblasts/osteocytes secrete FGF23 when they sense postprandial increase in the blood phosphate level through a putative "phosphate-sensing receptor." This hypothesis is analogous to the fact that parathyroid cells secrete parathyroid hormone (PTH) when they sense decrease in the blood calcium level through the calcium-sensing receptor.3Brown E.M. Gamba G. Riccardi D. et al.Cloning and characterization of an extracellular Ca(2+)-sensing receptor from bovine parathyroid.Nature. 1993; 366: 575-580Crossref PubMed Scopus (2258) Google Scholar However, several lines of evidence argue against this hypothesis. First, serum FGF23 levels were correlated not only with serum phosphate but also with calcium levels.4Quinn S.J. Thomsen A.R. Pang J.L. et al.Interactions between calcium and phosphorus in the regulation of the production of fibroblast growth factor 23 in vivo.Am J Physiol Endocrinol Metab. 2013; 304: E310-E320Crossref PubMed Scopus (78) Google Scholar Second, increase in serum phosphate failed to raise serum FGF23 when the serum calcium level was low5Rodriguez-Ortiz M.E. Lopez I. Munoz-Castaneda J.R. et al.Calcium deficiency reduces circulating levels of FGF23.J Am Soc Nephrol. 2012; 23: 1190-1197Crossref PubMed Scopus (159) Google Scholar and vice versa. Namely, in the presence of hypophosphatemia, FGF23 was not increased in response to increase in serum calcium.4Quinn S.J. Thomsen A.R. Pang J.L. et al.Interactions between calcium and phosphorus in the regulation of the production of fibroblast growth factor 23 in vivo.Am J Physiol Endocrinol Metab. 2013; 304: E310-E320Crossref PubMed Scopus (78) Google Scholar These observations indicate that both serum calcium and phosphate concentration must be above a certain level to induce FGF23 secretion. Calciprotein particles (CPPs) are colloidal nanoparticles composed of serum protein fetuin-A laden with calcium-phosphate precipitates (CaPis) and dispersed in the blood.6Heiss A. DuChesne A. Denecke B. et al.Structural basis of calcification inhibition by alpha 2-HS glycoprotein/fetuin-A: formation of colloidal calciprotein particles.J Biol Chem. 2003; 278: 13333-13341Crossref PubMed Scopus (363) Google Scholar,7Kuro-o M. Klotho, phosphate and FGF-23 in ageing and disturbed mineral metabolism.Nat Rev Nephrol. 2013; 9: 650-660Crossref PubMed Scopus (165) Google Scholar We recently established a method for quantification of CPPs in the blood and found that plasma CPP levels were correlated with serum FGF23 and phosphate levels.8Miura Y. Iwazu Y. Shiizaki K. et al.Identification and quantification of plasma calciprotein particles with distinct physical properties in patients with chronic kidney disease.Sci Rep. 2018; 8: 1256Crossref PubMed Scopus (44) Google Scholar Thus, we hypothesize that it is not phosphate or calcium per se but CPPs that may induce FGF23 secretion and/or production in osteoblasts/osteocytes. The purpose of this study is to test this hypothesis. To determine whether increase in the extracellular calcium and phosphate can directly induce FGF23 expression in osteoblasts, we cultured rat osteoblastic cells (UMR-106), increased calcium and phosphate concentration in the medium, and measured cellular FGF23 mRNA levels by quantitative reverse transcriptase polymerase chain reaction (qPCR). FGF23 expression was increased not only with increase in phosphate but also with increase in calcium (Figure 1a). We measured CPP levels in the medium by the gel filtration method8Miura Y. Iwazu Y. Shiizaki K. et al.Identification and quantification of plasma calciprotein particles with distinct physical properties in patients with chronic kidney disease.Sci Rep. 2018; 8: 1256Crossref PubMed Scopus (44) Google Scholar and observed a linear correlation between the CPP and FGF23 mRNA levels (Figure 1b). However, when formation of CaPis was inhibited by adding citrate to the medium, both CPP and FGF23 mRNA levels were decreased (Figure 1c and d), whereas mRNA levels encoded by other genes, including cyclophilin and cytochrome P450 family 24, were not decreased (data not shown). These results are consistent with the hypothesis that formation of CPPs in the medium is necessary for osteoblastic cells to induce FGF23 expression. Next, we tested whether CPPs could increase FGF23 expression without increasing the calcium and phosphate concentration in the medium. We added synthesized CPPs to the medium of UMR-106 cells and observed a dose-dependent increase in FGF23 expression (Figure 2a). The FGF23 mRNA levels were significantly increased within 4 hours after addition of synthesized CPPs (Figure 2b), followed by increase in the FGF23 protein levels in the conditioned medium (Figure 2c). Although to a significantly lesser extent than the CPP treatment, the vehicle treatment also induced FGF23 over time (Figure 2b and c), which may reflect the effect of 1,25-dihydroxyvitamin D3 added to the medium. We confirmed that addition of synthesized CPPs never increased but rather reduced the levels of phosphate, calcium, and calcium phosphate product in the medium before FGF23 expression was induced (Figure 2d–f). We also confirmed that the CPP levels in the medium stayed constant during the experimental period (Figure 2g). Hence, we concluded that persistent increase in the extracellular CPPs, but not increase in the phosphate or calcium concentration, is necessary and sufficient for osteoblastic cells to induce FGF23 expression. Amorphous CaPis undergo spontaneous transition into the thermodynamically stable crystalline phase over time.9Dorozhkin S.V. Amorphous calcium (ortho)phosphates.Acta Biomater. 2010; 6: 4457-4475Crossref PubMed Scopus (317) Google Scholar Because bisphosphonate interferes with the amorphous-to-crystalline phase transition of CaPis,10Fleisch H. Diphosphonates: history and mechanisms of action.Metab Bone Dis Relat Res. 1981; 3: 279-287Abstract Full Text PDF PubMed Scopus (121) Google Scholar we speculated that bisphosphonate might inhibit growth of crystalline CaPis and thus formation of CPPs, thereby attenuating the effect of increase in the extracellular calcium and phosphate concentration on FGF23 induction in osteoblasts. Contrary to our prediction, addition of alendronate to the high calcium (3 mmol/l [mM]), high phosphate (5 mM) medium augmented FGF23 expression (Figure 3a). These unexpected results led us to hypothesize that CPPs generated in the presence or absence of alendronate could be different in quality, which might have affected the ability of CPPs to induce FGF23 expression. To test this hypothesis, we performed small angle X-ray scattering analysis and characterized physical properties of CPPs generated in the high-phosphate, high-calcium medium in the presence and absence of alendronate. Because CPPs can be dissolved by chelating calcium in the medium with ethylenediamine tetraacetic acid (EDTA),8Miura Y. Iwazu Y. Shiizaki K. et al.Identification and quantification of plasma calciprotein particles with distinct physical properties in patients with chronic kidney disease.Sci Rep. 2018; 8: 1256Crossref PubMed Scopus (44) Google Scholar we extracted the X-ray scattering profile of CPPs by taking the difference before and after addition of EDTA (Figure 3b and c). In the absence of alendronate, we identified CPPs with a hydrostatic diameter of approximately 35 nm or over. By contrast, these CPPs disappeared in the presence of alendronate. Instead, smaller CPPs with a diameter of around 9.2 nm were detected, which appeared as flat particles with platelike shapes (Figure 3d). Because the size of a single fetuin-A molecule is ∼7.1 nm in diameter,11Heiss A. Pipich V. Jahnen-Dechent W. et al.Fetuin-A is a mineral carrier protein: small angle neutron scattering provides new insight on fetuin-a controlled calcification inhibition.Biophys J. 2010; 99: 3986-3995Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar these small particles are considered to be CPPs composed of a single fetuin-A molecule that adsorbs amorphous CaPis. In fact, the size of these particles is comparable with that of calciprotein monomers, described previously.11Heiss A. Pipich V. Jahnen-Dechent W. et al.Fetuin-A is a mineral carrier protein: small angle neutron scattering provides new insight on fetuin-a controlled calcification inhibition.Biophys J. 2010; 99: 3986-3995Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Calciprotein monomers represent fetuin-A monomers laden with Posner clusters, or Ca9(PO4)6, which are around 0.9 nm in diameter and deemed as precursors of amorphous CaPis.11Heiss A. Pipich V. Jahnen-Dechent W. et al.Fetuin-A is a mineral carrier protein: small angle neutron scattering provides new insight on fetuin-a controlled calcification inhibition.Biophys J. 2010; 99: 3986-3995Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar These observations imply that the 9.2-nm CPPs containing amorphous CaPis can induce FGF23 expression more robustly than the >35-nm CPPs containing crystalline CaPis. For simplicity, we designate CPPs as any colloidal particles of CaPis bound to fetuin-A, regardless of whether the fetuin-A is a monomer or multimers, although significant difference may exist between CPPs and calciprotein monomers in terms of function and clearance. Provided that osteoblasts produce/secrete FGF23 in response to phosphate intake by sensing CPPs in the blood, the following findings should be observed. First, blood CPP levels should be increased after dietary phosphate intake, followed by increase in FGF23 mRNA levels in the bone and FGF23 protein levels in the blood. Second, blood CPPs should extravasate in the bone marrow and have direct access to osteoblasts and/or osteocytes. To verify these predictions, we first administered phosphate in mice (6-week-old male animals, C57BL/6) by oral gavage and determined how FGF23 mRNA levels in the bone and blood levels of phosphate, calcium, CPPs, PTH, 1,25-dihydroxyvitamin D3, and FGF23 would be changed over time. Serum FGF23 levels were measured in 2 distinct assays—c-terminal FGF23 (cFGF23) enzyme-linked immunosorbent assay (ELISA) and intact FGF23 (iFGF23) ELISA.12Smith E.R. Cai M.M. McMahon L.P. et al.Biological variability of plasma intact and C-terminal FGF23 measurements.J Clin Endocrinol Metab. 2012; 97: 3357-3365Crossref PubMed Scopus (128) Google Scholar In the cFGF23 ELISA, both the capture and detection antibodies recognize the C-terminal portion of FGF23. On the other hand, the iFGF23 ELISA consists of the capture antibody that binds to the N-terminal portion and the detection antibody that binds to the C-terminal portion of FGF23. Because FGF23 is inactivated by proteolytic cleavage to produce the N-terminal and C-terminal fragments,13Shimada T. Muto T. Urakawa I. et al.Mutant FGF-23 responsible for autosomal dominant hypophosphatemic rickets is resistant to proteolytic cleavage and causes hypophosphatemia in vivo.Endocrinology. 2002; 143: 3179-3182Crossref PubMed Scopus (354) Google Scholar iFGF23 represents the active fraction of FGF23, whereas cFGF23 represents the total amount of secreted FGF23 protein including both active and inactive FGF23. We observed increase in serum CPP, phosphate, and PTH levels and decrease in serum calcium levels within 2 hours, which restored to the basal levels within 4 to 6 hours (Figure 4a–d). In contrast, FGF23 mRNA levels and serum FGF23 levels were not increased significantly until 4 hours after the phosphate ingestion (Figure 4e–g), indicating that increase in plasma CPP levels preceded the increase in FGF23. The similar time course was observed in the serum 1,25-dihydroxyvitamin D3 levels (Figure 4h). Although 1,25-dihydroxyvitamin D3 has the ability to induce FGF23 expression in osteoblasts,14Shimada T. Hasegawa H. Yamazaki Y. et al.FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis.J Bone Miner Res. 2004; 19: 429-435Crossref PubMed Scopus (1243) Google Scholar the increase in 1,25-dihydroxyvitamin D3 cannot explain the increase in serum FGF23 levels, because the FGF23 mRNA levels showed a decreasing trend when the 1,25-dihydroxyvitamin D3 levels were increased. These observations suggest that CPPs, but not 1,25-dihydroxyvitamin D3, may primarily contribute to FGF23 induction on bolus phosphate ingestion. Blood CPP and phosphate levels started increasing within 2 hours after the phosphate gavage and returned to the basal level within 4 hours, indicating that CPPs and phosphates were cleared from the blood. Because CPPs are nanoparticles, it is implausible that such large particles are filtrated through glomerulus and excreted into urine like phosphate is. However, it is possible that CPPs may exit from blood vessels through sinusoids, which are capillary beds composed of endothelial cells with large fenestrations. Sinusoids exist in the liver and the bone marrow. To determine whether CPPs could extravasate in the liver and the bone marrow, we administered synthesized CPPs fluorescently labeled with fluorescein isothiocyanate (FITC)–conjugated alendronate into mice by tail vein injection and performed in vivo imaging. We confirmed that injected CPPs were phagocytosed by Kupffer cells in the liver as previously reported15Herrmann M. Schafer C. Heiss A. et al.Clearance of fetuin-A–containing calciprotein particles is mediated by scavenger receptor-A.Circ Res. 2012; 111: 575-584Crossref PubMed Scopus (102) Google Scholar (Figure 5a– c, Supplementary Movies S1 and S2). By in vivo imaging of the calvaria, we found that injected CPPs were deposited on the bone surface facing the marrow cavity where osteoblasts reside (Figure 5d and e, Supplementary Movies S3–S5). We conclude that blood CPPs can exit from blood vessels in the bone and reach osteoblasts directly. Because increase in dietary phosphate intake was reported to elevate serum FGF23 levels,4Quinn S.J. Thomsen A.R. Pang J.L. et al.Interactions between calcium and phosphorus in the regulation of the production of fibroblast growth factor 23 in vivo.Am J Physiol Endocrinol Metab. 2013; 304: E310-E320Crossref PubMed Scopus (78) Google Scholar we asked whether plasma CPP levels would be increased after dietary phosphate load. We placed mice (8-week-old male, C57BL/6) on a high phosphate diet containing 2.0% inorganic phosphate for 2 days and observed that these mice had significantly higher plasma CPP levels than did mice placed on a regular diet containing 0.35% inorganic phosphate (Figure 6a). We also found that the dietary phosphate load caused significant increase in serum FGF23 (both cFGF23 and iFGF23), phosphate, PTH, and 1,25-dihydroxyvitamin D3 levels and marginal decrease in calcium levels in the blood (Figure 6b–g). FGF23 mRNA levels in the bone were also significantly increased by feeding the high phosphate diet (Figure 6h). Thus, unlike bolus phosphate ingestion, continuous dietary phosphate load causes persistent increase in plasma CPPs and robust increase in serum FGF23. Plasma CPP levels were increased after phosphate ingestion (Figures 4a and 6a). Because FGF23 functions as a phosphaturic hormone as well as a counterregulatory hormone for 1,25-dihydroxyvitamin D3,2 FGF23 should exert negative impact on CPP formation through lowering phosphate concentration in the extracellular fluid by inducing phosphaturia and through inhibiting calcium absorption in the digestive tract by lowering circulating 1,25-dihydroxyvitamin D3 levels. Thus, the ability of CPPs to induce FGF23 may be required for a negative feedback loop to prevent increase in the circulating CPP level, which is associated with vascular calcification, aortic stiffness, and inflammation in patients with chronic kidney disease (CKD).16Hamano T. Matsui I. Mikami S. et al.Fetuin-mineral complex reflects extraosseous calcification stress in CKD.J Am Soc Nephrol. 2010; 21: 1998-2007Crossref PubMed Scopus (146) Google Scholar,17Smith E.R. Ford M.L. Tomlinson L.A. et al.Phosphorylated fetuin-A-containing calciprotein particles are associated with aortic stiffness and a procalcific milieu in patients with pre-dialysis CKD.Nephrol Dial Transplant. 2012; 27: 1957-1966Crossref PubMed Scopus (112) Google Scholar Koppert et al.18Koppert S. Buscher A. Babler A. et al.Cellular clearance and biological activity of calciprotein particles depend on their maturation state and crystallinity.Front Immunol. 2018; 9: 1991Crossref PubMed Scopus (37) Google Scholar recently reported that synthesized secondary CPPs were endocytosed by Kupffer cells by intravital 2-photon imaging, which was consistent with the finding of our in vivo imaging. However, they did not describe deposition of CPP to the bone. This may be simply because they did not look at the bone or because the CPPs used in their studies were different from the CPPs used in our present study. They used high-density secondary CPPs precipitated by centrifugation at 20,000g for 20 minutes. In contrast, we used CPPs that passed through a gel-filtration spin column with the size-exclusion limit at 40 kDa,8Miura Y. Iwazu Y. Shiizaki K. et al.Identification and quantification of plasma calciprotein particles with distinct physical properties in patients with chronic kidney disease.Sci Rep. 2018; 8: 1256Crossref PubMed Scopus (44) Google Scholar which contained not only high-density CPPs equivalent to the secondary CPPs they used but also low-density CPPs that were not precipitated by the centrifugation.8Miura Y. Iwazu Y. Shiizaki K. et al.Identification and quantification of plasma calciprotein particles with distinct physical properties in patients with chronic kidney disease.Sci Rep. 2018; 8: 1256Crossref PubMed Scopus (44) Google Scholar It remains to be determined whether the low-density CPPs may be efficiently deposited to the bone when compared with high-density secondary CPPs. In the present mouse study, bolus phosphate ingestion increased phosphate and CPPs but reciprocally decreased calcium within 2 hours, which was accompanied by increase in PTH. These changes were restored to the basal levels within 4 to 6 hours (Figure 4a–d). In contrast to these quick responses, significant increase in bone FGF23 mRNA and serum FGF23 levels did not become evident until 4 hours after the phosphate ingestion when serum phosphate levels had already started decreasing (Figure 4e–g). This slow response of FGF23 indicates that the phosphaturic activity of FGF23 did not contribute to the restoration of serum phosphate levels after the bolus phosphate ingestion. This is also applicable to humans.19Nishida Y. Taketani Y. Yamanaka-Okumura H. et al.Acute effect of oral phosphate loading on serum fibroblast growth factor 23 levels in healthy men.Kidney Int. 2006; 70: 2141-2147Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar In addition, the ratio of cFGF23 to iFGF23 (c/iFGF23 ratio), which represents the activity of proteolytic inactivation of FGF23, was increased followed by the increase in the FGF23 mRNA level (Supplementary Figure S1A), suggesting an attempt to attenuate the FGF23 activity. These observations suggest that phosphaturic activity of PTH may be primarily responsible for the quick restoration of the serum phosphate level after bolus phosphate ingestion. Hence, in this context, physiological significance of the delayed increase in FGF23 is unclear. In contrast to bolus phosphate ingestion, continuous dietary phosphate load by placing mice on high phosphate diet for 2 days caused persistent and robust increase in CPP and FGF23 levels (Figure 4a–c). This is consistent with the results of in vitro experiment showing that induction of FGF23 secretion in cultured osteoblastic cells required incubation with CPPs for 8 hours or longer (Figure 2c). Additionally, unlike bolus phosphate ingestion, continuous dietary phosphate load significantly decreased the c/iFGF23 ratio (Supplementary Figure S1B), suggesting an attempt to limit proteolytic inactivation of FGF23. Reduction of c/iFGF23 ratio is observed under the condition where phosphaturic activity of FGF23 is required to maintain phosphate homeostasis as in CKD.12Smith E.R. Cai M.M. McMahon L.P. et al.Biological variability of plasma intact and C-terminal FGF23 measurements.J Clin Endocrinol Metab. 2012; 97: 3357-3365Crossref PubMed Scopus (128) Google Scholar In fact, injection of a neutralizing antibody against FGF23 to CKD mice raised serum phosphate levels before PTH started decreasing,20Hasegawa H. Nagano N. Urakawa I. et al.Direct evidence for a causative role of FGF23 in the abnormal renal phosphate handling and vitamin D metabolism in rats with early-stage chronic kidney disease.Kidney Int. 2010; 78: 975-980Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar indicating that it is not PTH but FGF23 that had primarily contributed to maintaining phosphate homeostasis in CKD. It appears that PTH and FGF23 are primarily responsible for maintaining phosphate homeostasis in response to acute and chronic phosphate load, respectively. In contrast, increase in c/iFGF23 ratio is observed under the condition where FGF23 is produced more than is necessary. For example, administration of bisphosphonate into mice fed high phosphate diet elevated serum levels of cFGF23, but not iFGF23, resulting in increase in the c/iFGF23 ratio (Supplementary Figure S2). This is consistent with the in vitro finding that treatment with bisphosphonate increased FGF23 expression in osteoblastic cells cultured in high phosphate medium (Figure 3a). The mechanism by which CPPs induce FGF23 and inflammatory responses including upregulation of the osteopontin21Lund S.A. Giachelli C.M. Scatena M. The role of osteopontin in inflammatory processes.J Cell Commun Signal. 2009; 3: 311-322Crossref PubMed Scopus (282) Google Scholar and dentin matrix protein-122Dussold C. Gerber C. White S. et al.DMP1 prevents osteocyte alterations, FGF23 elevation and left ventricular hypertrophy in mice with chronic kidney disease.Bone Res. 2019; 7: 12Crossref PubMed Scopus (21) Google Scholar (Supplementary Figure S3) in osteoblasts remains to be determined. Hori et al.23Hori M. Kinoshita Y. Taguchi M. et al.Phosphate enhances Fgf23 expression through reactive oxygen species in UMR-106 cells.J Bone Miner Metab. 2016; 34: 132-139Crossref PubMed Scopus (30) Google Scholar reported that induction of FGF23 expression in UMR-106 cells by increasing phosphate in the medium was dependent on production of reactive oxygen species (ROS) and that increase in FGF23 expression induced by hydrogen peroxide was blocked by a mitogen-activated protein kinase kinase inhibitor PD98059. Because CPPs were reported to stimulate ROS production in cultured macrophages24Smith E.R. Hanssen E. McMahon L.P. et al.Fetuin-A-containing calciprotein particles reduce mineral stress in the macrophage.PLoS One. 2013; 8e60904Crossref PubMed Scopus (98) Google Scholar and vascular smooth muscle cells,25Aghagolzadeh P. Bachtler M. Bijarnia R. et al