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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2009 Dec 29;107(1):407–412. doi: 10.1073/pnas.0902006107

Isolated C-terminal tail of FGF23 alleviates hypophosphatemia by inhibiting FGF23-FGFR-Klotho complex formation

Regina Goetz a,1, Yuji Nakada b,1, Ming Chang Hu c,1, Hiroshi Kurosu b, Lei Wang b, Teruyo Nakatani d, Mingjun Shi c, Anna V Eliseenkova a, Mohammed S Razzaque d,e, Orson W Moe c,2, Makoto Kuro-o b,2, Moosa Mohammadi a,2
PMCID: PMC2806769  PMID: 19966287

Abstract

Fibroblast growth factor (FGF) 23 inhibits renal phosphate reabsorption by activating FGF receptor (FGFR) 1c in a Klotho-dependent fashion. The phosphaturic activity of FGF23 is abrogated by proteolytic cleavage at the RXXR motif that lies at the boundary between the FGF core homology domain and the 72-residue-long C-terminal tail of FGF23. Here, we show that the soluble ectodomains of FGFR1c and Klotho are sufficient to form a ternary complex with FGF23 in vitro. The C-terminal tail of FGF23 mediates binding of FGF23 to a de novo site generated at the composite FGFR1c-Klotho interface. Consistent with this finding, the isolated 72-residue-long C-terminal tail of FGF23 impairs FGF23 signaling by competing with full-length ligand for binding to the binary FGFR-Klotho complex. Injection of the FGF23 C-terminal tail peptide into healthy rats inhibits renal phosphate excretion and induces hyperphosphatemia. In a mouse model of renal phosphate wasting attributable to high FGF23, the FGF23 C-terminal peptide reduces phosphate excretion, leading to an increase in serum phosphate concentration. Our data indicate that proteolytic cleavage at the RXXR motif abrogates FGF23 activity by a dual mechanism: by removing the binding site for the binary FGFR-Klotho complex that resides in the C-terminal region of FGF23, and by generating an endogenous inhibitor of FGF23. We propose that peptides derived from the C-terminal tail of FGF23 or peptidomimetics and small-molecule organomimetics of the C-terminal tail can be used as therapeutics to treat renal phosphate wasting.

Keywords: FGF23 antagonist, endogenous inhibitor of FGF23, FGF23 C-terminal peptide, binary FGF receptor 1c-Klotho complex, composite FGF receptor 1c-Klotho interface

Inorganic phosphate plays a key role in a myriad of biological processes, including bone mineralization, reversible regulation of protein function by phosphorylation, and production of adenosine triphosphate. Plasma levels of phosphate range between 2.2 and 4.9 mg/dL (1, 2), and are primarily regulated by modifying renal tubular reabsorption. Because of phosphate’s pleiotropic activity, imbalances in phosphate homeostasis adversely affect essentially every major tissue/organ. Hypophosphatemia is a common clinical condition with an incidence ranging from 0.2% to 3.1% in all hospital admissions to 21.5% to 80% in specific subgroups of hospitalized patients (3, 4). Clinical manifestations of hypophosphatemia include respiratory failure, cardiac arrhythmia, hemolysis, rhabdomyolysis, seizures, and coma acutely and myalgia and osteomalacia chronically (3). Hypophosphatemia originates from diverse pathophysiologic mechanisms, most importantly from renal phosphate wasting, an inherited or acquired condition in which renal tubular reabsorption of phosphate is impaired (5, 6).

Oral or i.v. administration of inorganic phosphate salts currently is the mainstay for the management of hypophosphatemia. Oral phosphate therapy requires high doses, which frequently lead to diarrhea or gastric irritation (7). For i.v. phosphate therapy, the response to any given dose is sometimes unpredictable (8–10), and complications include “overshoot” hyperphosphatemia, hypocalcemia, and metastatic calcification (3, 7). In addition, parenteral regimens are not practical for chronic disorders. Most importantly, replacement therapy alone is never adequate when there is significant renal phosphate wasting. Therefore, novel strategies for the treatment of hypophosphatemia are needed.

Fibroblast growth factor (FGF) 23, originally identified as the mutated gene in patients with the phosphate wasting disorder autosomal dominant hypophosphatemic rickets (ADHR) (11) and as the causative factor of tumor-induced osteomalacia (12, 13), is an endocrine regulator of phosphate homeostasis. FGF23 inhibits reabsorption of phosphate in the renal proximal tubule by decreasing the abundance of the type II sodium-dependent phosphate transporters NaPi-2A and NaPi-2C in the apical brush border membrane (14–16). FGF23 activity is regulated by a proteolytic cleavage at the 176RXXR179 motif, located at the boundary between the FGF core homology domain and the 72-residue-long C-terminal tail of FGF23 (17, 18). The proteolytic cleavage generates an inactive N-terminal fragment (Y25 to R179, the FGF core homology domain) and a C-terminal fragment (S180 to I251) (19) (Fig. 1A). Missense mutations of either R176 or R179 of the 176RXXR179 motif inhibit this proteolytic cleavage (17, 18) and lead to accumulation of full-length, bioactive FGF23, thereby inducing renal phosphate wasting in ADHR patients (11). To exert its phosphaturic activity, FGF23 requires Klotho as an obligate coreceptor (20, 21), a protein first described as an aging suppressor (22). The dependency on Klotho compensates for the poor binding affinity of FGF23 to both FGF receptor (FGFR) and heparan sulfate (19). Klotho constitutively binds the cognate FGFRs of FGF23, and the binary FGFR-Klotho complexes exhibit enhanced binding affinity for FGF23 (20, 21).

Fig. 1.

Fig. 1.

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FGF23 C-terminal tail mediates binding of FGF23 to the binary FGFR-Klotho complex, and the isolated FGF23 C-terminal tail peptide competes with FGF23 for binding to FGFR-Klotho. (A) FGF23 proteins and peptides used in this study. Amino acid boundaries of each protein/peptide are labeled with residue letter and number. The FGF23 core region is shaded gray, and the position of the proteolytic cleavage site RXXR is indicated. (B) Representative SPR sensorgram of FGFR1c binding to Klotho, and fitted saturation binding curve. Klotho ectodomain was immobilized on a biosensor chip, and increasing concentrations of FGFR1c ectodomain were passed over the chip. The dissociation constant (KD) was calculated from the saturation binding curve. (C and D) Representative SPR sensorgrams illustrating binding of FGF2328-251 (C) and FGF23180-251 (D) to the binary FGFR1c-Klotho complex. FGF2328-251 and FGF23180-251 were immobilized on a biosensor chip, and increasing concentrations of FGFR1c-Klotho complex were passed over the chip. (E and F) Representative SPR sensorgrams illustrating inhibition by FGF23180-251 (E) or FGF23180-205 (F) of FGFR1c-Klotho binding to FGF2328-251 immobilized on a biosensor chip. Increasing concentrations of either FGF23180-251 or FGF23180-205 were mixed with a fixed concentration of FGFR1c-Klotho complex, and the mixtures were passed over a FGF23 chip.

We have recently shown that in contrast to full-length FGF23, the inactive N-terminal fragment of proteolytic cleavage fails to coimmunoprecipitate with binary FGFR-Klotho complexes, suggesting that the 72-residue-long C-terminal tail of FGF23 mediates binding of FGF23 to its cognate FGFR-Klotho complexes (19). Here, we demonstrate that the C-terminal tail of FGF23 mediates binding of FGF23 to a de novo site at the composite FGFR-Klotho interface. We exploit this finding for therapeutic purposes and show that the isolated C-terminal tail of FGF23 can compete with full-length ligand for binding to the FGFR-Klotho complex, and hence can antagonize the phosphaturic activity of FGF23 in vivo, both in healthy rats and in a mouse model of phosphate wasting disorders.

Results

The Ternary FGF23-FGFR1c-Klotho Complex Can Be Reconstituted in Solution by Using Recombinant Soluble Ectodomains of FGFR1c and Klotho.

To understand how FGF23, FGFR, and Klotho interact to form a ternary complex, we decided to reconstitute the ternary complex in solution by using bioactive, full-length FGF23 (FGF2328-251) (Fig. 1A) and the soluble ectodomains of FGFR1c and Klotho. The binary complex of FGFR1c ectodomain with Klotho ectodomain was formed by capturing the Klotho ectodomain onto an FGFR1c affinity column from conditioned media of a HEK293 cell line ectopically expressing the Klotho ectodomain (20). The FGFR1c-Klotho complex was further purified by size-exclusion chromatography to remove excess FGFR1c (Fig. S1A). Next, the FGFR1c-Klotho complex was mixed with FGF2328-251, and ternary complex formation was examined by size-exclusion chromatography. As shown in Fig. S1B, FGF23 coeluted with the FGFR1c-Klotho complex, demonstrating that the ectodomains of FGFR1c and Klotho are sufficient to form a stable ternary complex with FGF23.

The size-exclusion data showing that Klotho and FGFR1c ectodomains form a stable binary complex (Fig. S1A) indicate that Klotho must harbor a high-affinity binding site for FGFR1c. To further confirm this, we used surface plasmon resonance (SPR) spectroscopy to determine the dissociation constant of the FGFR1c-Klotho interaction. Klotho ectodomain was immobilized on a biosensor chip, and increasing concentrations FGFR1c ectodomain were passed over the chip. Consistent with the results obtained by using size-exclusion chromatography (Fig. S1A), Klotho bound FGFR1c with high affinity (KD = 72 nM; Fig. 1B). Because Klotho harbors a high-affinity binding site for FGFR1c, we reasoned that Klotho might also possess a distinct high-affinity binding site for FGF23 and promote FGF23-FGFR1c binding by engaging FGF23 and FGFR1c simultaneously. To test this, FGF2328-251 was coupled to a biosensor chip, and increasing concentrations of Klotho ectodomain were passed over the chip. As shown in Fig. S1C, Klotho bound poorly to FGF2328-251. These data demonstrate that the Klotho ectodomain contains a high-affinity binding site for FGFR1c but not for FGF23.

Next, we measured binding of FGF23 to FGFR1c by injecting increasing concentrations of FGFR1c over the FGF23 chip. As shown in Fig. S1D, FGF2328-251 exhibited poor binding to FGFR1c. Thus, the SPR data show that FGF23 exhibits poor binding affinity for both the Klotho ectodomain alone and the FGFR1c ectodomain alone. Together with the size-exclusion chromatography data showing that FGF23 binds stably to the purified binary FGFR1c-Klotho complex, the data raised the question of whether FGF23 binds to a de novo site generated at the composite FGFR1c-Klotho interface. To test this, we purified FGFR1c-Klotho complex as described above and passed increasing concentrations of the binary complex over the FGF23 chip. As shown in Fig. 1C, FGF2328-251 bound to the FGFR1c-Klotho complex demonstrating that FGF23 interacts with a de novo site generated at the composite FGFR1c-Klotho interface.

C-Terminal Tail of FGF23 Mediates Binding of FGF23 to a de Novo Site at the Composite FGFR1c-Klotho Interface.

We then examined whether the C-terminal tail of FGF23 mediates binding of FGF23 to the FGFR1c-Klotho complex. To test this, the C-terminal tail peptide of FGF23 (FGF23180-251; Fig. 1A) was coupled to a biosensor chip, and increasing concentrations of FGFR1c-Klotho complex were passed over the chip. As shown in Fig. 1D, FGF23180-251 avidly bound to the binary complex. Size-exclusion chromatography and coimmunoprecipitation experiments yielded similar results supporting the SPR data (Fig. S2A–C).

To fully nail down that the C-terminal tail of FGF23 mediates FGF23 binding to the binary FGFR1c-Klotho complex, a fixed concentration of FGFR1c-Klotho was mixed with increasing concentrations of FGF23180-251, and the mixtures were passed over the FGF23 chip. Mixtures of FGF2328-251 with FGFR1c-Klotho were used as a control. As shown in Fig. 1E and Fig. S2D, FGF23180-251 competed, in a dose-dependent fashion, with FGF2328-251 for binding to the FGFR1c-Klotho complex. Half-maximum inhibition of FGFR1c-Klotho binding to FGF2328-251 was reached with a 3.3-fold molar excess of FGF23180-251 over FGFR1c-Klotho complex (Fig. S2D). As expected, less than an equimolar amount of FGF2328-251 relative to FGFR1c-Klotho complex already yielded 50% inhibition of binding of the binary complex to immobilized FGF2328-251 (Fig. S2D and E). A coimmunoprecipitation-based competition assay also confirmed that the C-terminal tail peptide of FGF23 can inhibit binding of FGF23 to its binary cognate FGFR-Klotho complex (Fig. S2F). Together, the data unambiguously demonstrate that the C-terminal tail of FGF23 harbors the binding site for the binary FGFR-Klotho complex and hence is essential for formation of the ternary FGF23-FGFR-Klotho complex. Importantly, the binding data unveil that proteolytic cleavage at the RXXR motif abrogates FGF23 activity by removing the binding site for the binary FGFR-Klotho complex that resides in the C-terminal tail of FGF23.

FGF23 Residues S180 to T200 Comprise the Minimal Binding Epitope for the FGFR-Klotho Complex.

In follow-up studies, we found that FGF2328-200, which lacks the last 51 C-terminal amino acids, still retains the ability to coimmunoprecipitate with the binary FGFR-Klotho complex (Fig. S2C). The finding suggested that FGF2328-200 may have similar biological activity as the full-length protein. To test this, we compared the ability of FGF2328-200 and FGF2328-251 to induce tyrosine phosphorylation of FGF receptor substrate 2α (FRS2α) and downstream activation of MAP kinase cascade in Klotho-expressing cultured cells, and to induce phosphaturia in mice. As shown in Fig. S3A, FGF2328-200 induced phosphorylation of FRS2α and downstream activation of MAP kinase cascade at a dose comparable to that of FGF2328-251. The truncated FGF23 was also nearly as effective as the full-length ligand in reducing serum phosphate concentration in healthy C57BL/6 mice (Fig. S3B). These data show that deletion of the last 51 amino acids from the FGF23 C terminus has little effect on FGF23 biological activity, narrowing down the epitope on the FGF23 C-terminal tail for the composite FGFR-Klotho interface to residues S180 and T200. Indeed, a FGF23 peptide comprising the minimal binding epitope for FGFR-Klotho (FGF23180-205; Fig. 1A) was able to compete, in a dose-dependent fashion, with FGF2328-251 for binding to the binary FGFR1c-Klotho complex (Fig. 1F). Half-maximum inhibition of FGFR1c-Klotho binding to FGF2328-251 was reached with a 5.7-fold molar excess of FGF23180-205 over FGFR1c-Klotho complex (Fig. S2D). Similarly, in a coimmunoprecipitation-based competition assay, the FGF23180-205 peptide was able to inhibit binding of FGF23 to the binary complexes of its cognate FGFR and Klotho (Fig. S3C). Our data also explain the finding by Garringer and colleagues (23) showing that residues P189 to P203 are required for FGF23 signaling.

FGF23 C-Terminal Peptides Block FGF23 Signaling.

Based on these data, we postulated that FGF23180-251 and FGF23180-205 should antagonize FGF23 signaling by competing with full-length FGF23 for binding to the FGFR-Klotho complex. To test this, we stimulated cells stably overexpressing Klotho with FGF2328-251 alone or FGF2328-251 mixed with increasing concentrations of either FGF23180-251 or FGF23180-205. As shown in Fig. 2 and Fig. S4A, both peptides inhibited, in a dose-dependent fashion, FGF23-induced tyrosine phosphorylation of FRS2α and downstream activation of MAP kinase cascade. To test the specificity of the FGF23 antagonists, we examined the ability of the FGF23180-251 peptide to inhibit signaling of FGF2, a prototypical paracrine FGF, which does not require Klotho for signaling. As shown in Fig. S4B, the FGF23 antagonist failed to inhibit tyrosine phosphorylation of FRS2α and downstream activation of MAP kinase cascade induced by FGF2. These data show that FGF23 C-terminal peptides specifically block FGF23 signaling.

Fig. 2.

Fig. 2.

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FGF23 C-terminal tail peptide inhibits tyrosine phosphorylation of FRS2α and downstream activation of MAP kinase cascade induced by FGF23. Shown is an immunoblot analysis for phosphorylation of FRS2α (pFRS2α) and 44/42 MAP kinase (p44/42 MAPK) in a HEK293 Klotho cell line, which had been stimulated with FGF proteins/peptide as denoted. Numbers above the lanes give the amounts of protein/peptide added in nanomolar. To control for equal sample loading, the protein blots were probed with an antibody to Klotho.

In renal proximal tubule epithelium, FGF23 signaling leads to inhibition of phosphate uptake. To establish further that FGF23 C-terminal peptides block FGF23 action, we studied the effects of the peptides on sodium-coupled phosphate uptake in a proximal tubular cell model. As shown in Fig. S5A, FGF23180-251 antagonized the inhibition of phosphate uptake by FGF2328-251 in a dose-dependent fashion, with an IC50 of ∼21 nM. FGF23180-205 exhibited a similar, albeit less potent antagonistic effect (Fig. S5B). As expected, neither of the two FGF23 C-terminal peptides altered phosphate uptake when applied alone (Fig. S5).

FGF23 C-Terminal Peptides Antagonize Phosphaturic Activity of FGF23 in Vivo.

These findings motivated us to conduct in vivo studies and investigate whether the FGF23 C-terminal peptides antagonize the phosphaturic effects of endogenous FGF23. I.v. injection of FGF23180-251 into healthy Sprague–Dawley rats led to renal phosphate retention and hyperphosphatemia (Fig. 3), suggesting that FGF23 C-terminal peptides antagonize the phosphaturic action of endogenous FGF23. As expected, injection of FGF2328-251 induced increases in excretion rate and fractional excretion of phosphate, and led to a significant decrease in plasma phosphate compared to vehicle-treated animals (Fig. 3).

Fig. 3.

Fig. 3.

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FGF23 C-terminal tail peptide antagonizes phosphaturic activity of FGF23 in vivo. FGF2328-251 (0.1 μg·kg body weight−1) or FGF23180-251 (0.1 μg·kg body weight−1) were injected i.v. into Sprague–Dawley rats. Serum and urine parameters were measured and calculated before and 3 h after injection. FE Pi, fractional excretion of phosphate; UPiV, phosphate excretion rate; ClCr, creatinine clearance.

FGF23 exerts its phosphaturic activity by inhibiting phosphate uptake by renal proximal tubule epithelium. The effect has been attributed to reduced transport activity of NaPi-2A and NaPi-2C, down-regulation of NaPi-2A and NaPi-2C proteins in the apical brush border membrane, and upon more chronic exposure to FGF23, repression of NaPi-2A and NaPi-2C genes (14–16, 24, 25). We examined the abundance of NaPi-2A and NaPi-2C proteins in brush border membrane vesicles isolated from the kidneys of rats. I.v. injection of FGF23180-251 into healthy rats led to an increase in NaPi-2A protein expression in the apical brush border membrane compared to vehicle treatment (Fig. S6A and B). The peptide exhibited similar effects on the NaPi-2C protein (Fig. S6C). As expected, injection of FGF2328-251 led to a decrease in NaPi-2A protein expression (Fig. S6A and B). These findings establish that FGF23 C-terminal peptides counteract or cancel out FGF23’s phosphaturic action mediated through NaPi-2A and NaPi-2C.

Therapeutic Potential of FGF23 C-Terminal Peptides.

To evaluate the therapeutic potential of FGF23180-251 for treating renal phosphate wasting, we analyzed the peptide’s efficacy in Hyp mice, a mouse model of human X-linked hypophosphatemia (XLH) (26–29). XLH is an inherited phosphate wasting disorder associated with high FGF23, which is thought to be due to reduced clearance of FGF23 from the circulation. Excess FGF23 causes increased phosphate excretion resulting in hypophosphatemia. As shown in Fig. 4, i.p. injection of FGF23180-251 induced a decrease in renal phosphate excretion in Hyp mice compared to vehicle treatment. The effect persisted for at least 4 h after injection. Concomitantly, serum phosphate levels were elevated by the FGF23 antagonist treatment (Fig. 4). Likewise, i.p. injection of the FGF23180-205 peptide, which comprises the minimal binding epitope for the composite FGFR-Klotho interface, caused an increase in serum phosphate in Hyp mice compared to vehicle-treated animals (Fig. 4). These results show that FGF23 C-terminal peptides are effective in reducing renal phosphate wasting caused by excess FGF23.

Fig. 4.

Fig. 4.

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FGF23 C-terminal peptides alleviate renal phosphate wasting in Hyp mice. FGF23180-251 (1 mg), FGF23180-205 (860 μg), or vehicle were injected i.p. into Hyp mice. Urine phosphate (urinary Pi) and creatinine levels and serum phosphate levels (serum Pi) were measured before and at the indicated time points after the injection. Urinary Pi of Hyp mice treated with FGF23180-205 was not determined (ND). Bars and error bars are mean + SE. *, P < 0.05 by ANOVA.

Discussion

Our study provides firm evidence that proteolytic cleavage at the RXXR motif abrogates FGF23 activity by a dual mechanism: by removing FGF23’s binding site for the binary FGFR-Klotho complex, and by generating an endogenous inhibitor of FGF23. We exploited this regulatory mechanism to develop an FGF23 antagonist with therapeutic potential for renal phosphate wasting.

Patients with phosphate wasting disorders are, by and large, treated symptomatically, with oral phosphate supplementation and 1,25-dihydroxyvitamin D3/calcitriol. As alluded to in the introduction, oral phosphate therapy can be poorly tolerated, and in certain circumstances can induce hyperparathyroidism and poses risk of exacerbation of hypophosphatemia. In patients with XLH, the persistent and even exaggerated renal phosphate wasting during therapy can cause nephrocalcinosis and nephrolithiasis. For patients with renal phosphate wasting from tumor-induced osteomalacia, a causative treatment option exists, which is resection of the tumor producing excess amounts of phosphaturic hormone. These tumors are often difficult to locate, however, or the tumors are found in locations that are difficult to access, leaving most patients with tumor-induced osteomalacia also currently with no options other than symptomatic therapy (30, 31). Because excess FGF23 is the pathogenic factor in phosphate wasting disorders, blocking its action with FGF23 C-terminal peptides holds promise of providing the first causative pharmacotherapy. In a mouse model of phosphate wasting disorders, we have shown that FGF23 C-terminal peptides are effective in counteracting the phosphaturic action of FGF23. Our findings warrant further evaluation of the peptides’ efficacy in nonhuman primates, and eventually, in humans. Neutralizing FGF23 activity with antibody provides an alternative approach for treating renal phosphate wasting. Indeed, Aono, Yamazaki, and colleagues (32, 33) have explored this approach and developed antibodies against FGF23 that effectively neutralize FGF23 activity in both healthy mice and Hyp mice.

Although it has been conclusively demonstrated that the phosphaturic activity of FGF23 is Klotho-dependent (34), the possibility that FGF23 may have some Klotho-independent functions has not yet been ruled out experimentally. In this regard, our inhibitory peptide approach may offer a more targeted therapy for hypophosphatemia than anti-FGF23 antibodies because these peptides specifically target the binary FGFR-Klotho complex and hence only neutralize Klotho-dependent function of FGF23. In contrast, the antibody approach does not discriminate between Klotho-dependent and -independent functions of FGF23. These peptides can also serve as an experimental tool to dissect Klotho-dependent and -independent functions of FGF23. The ability of the FGF23 C-terminal peptides to specifically recognize the binary receptor complex makes them a powerful tool to image tissues that express the cognate FGFR-Klotho complexes of FGF23.

Hypophosphatemia complicating recovery from kidney transplantation and parenteral iron therapy has been associated with increased plasma levels of FGF23 (35, 36). Thus, the FGF23 antagonist discovered in this study may be of therapeutic value for a broader collection of patients than inherited or tumor-induced phosphate wasting disorders alone.

Another indication for therapy with FGF23 C-terminal peptides might be chronic kidney disease, a condition with a growing incidence, currently affecting ∼20 million people in the United States alone. In patients with chronic kidney disease, plasma levels of FGF23 increase as kidney function declines (37), and the gradual increases in plasma FGF23 correlate with disease progression (38). Although the precise role of FGF23 in the pathogenesis of chronic kidney disease and its sequelae remains to be determined, blocking FGF23 action with FGF23 C-terminal peptides might prove effective in preventing or attenuating the occurrence of disease complications such as hyperparathyroidism.

Our identification of the FGF23 C-terminal tail as an FGF23 antagonist suggests that proteolytic cleavage not only removes the binding site on FGF23 for the FGFR-Klotho complex, but also generates an endogenous inhibitor of FGF23. A pathophysiological role of the latter mechanism is indicated by familial tumoral calcinosis (FTC), an autosomal recessive metabolic disorder with clinical manifestations mirroring those of phosphate wasting disorders. Missense mutations in either the UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) gene (39) or the FGF23 gene (23, 40) have been associated with FTC. All FTC patients have abnormally high plasma levels of the C-terminal proteolytic fragment of FGF23 (39, 40). Our findings suggest that excess C-terminal FGF23 fragment may aggravate hyperphosphatemia and the resulting soft tissue calcification, by antagonizing the action of any residual, functional FGF23 ligand in these patients. Our findings also call for the need to interpret full-length and fragments of FGF23 separately in chronic kidney disease.

There has been a conundrum surrounding the mechanism of action of FGF23 in the kidney because Klotho is expressed in the distal convoluted tubule (41–43), whereas FGF23 inhibits phosphate reabsorption in the proximal tubule (14–16). A recent study suggested that FGF23 signaling initiates in the distal tubule and its effects are then transmitted to the proximal tubule through an unknown diffusible paracrine factor (44). In addition to the membrane-bound isoform of Klotho, alternative splicing and proteolytic cleavage give rise to two soluble isoforms of Klotho found in the circulation (45–48). Importantly, the recombinant Klotho ectodomain that we used to reconstitute the ternary FGF23-FGFR-Klotho complex corresponds to the complete ectodomain of Klotho that is shed into the circulation by a proteolytic cleavage at the juncture between the extracellular domain and transmembrane domain (45, 46). Thus, our data points to the possibility that it is the shed soluble isoform of Klotho that makes its way to the proximal tubule to promote formation of FGF23-FGFR-Klotho ternary complex, and inhibition of phosphate reabsorption.

Materials and Methods

Purification of FGF23, FGFR, and Klotho Proteins.

See SI Materials and Methods; Fig. S7.

Analysis of FGF23-FGFR1c-Klotho Interactions by SPR Spectroscopy.

See SI Materials and Methods.

Analysis of FGF23 Protein/Peptide Binding to FGFR1c-Klotho Complex by Size-Exclusion Chromatography.

Binding of FGF23 proteins/peptides to the 1:1 binary complex of FGFR1c and Klotho ectodomains was analyzed by using a HiLoad 16/60 Superdex 200 prep grade size-exclusion column on an ÄKTApurifier (GE Healthcare) (SI Materials and Methods).

Pull-Down Assays of FGF23 Protein/Peptide Binding to FGFR-Klotho Complex.

Binding of FGF23 proteins/peptides to FGFR-Klotho complexes isolated from lysate of a HEK293 cell line expressing the membrane-spanning form of murine Klotho was analyzed as described in ref. 19 (SI Materials and Methods).

Analysis of Phosphorylation of FRS2α and 44/42 MAP Kinase in Epithelial Cell Lines.

Phosphorylation of FRS2α and 44/42 MAP kinase in response to treatment with FGF23 proteins/peptides was analyzed in HEK293 and CHO cell lines expressing the membrane-spanning form of murine Klotho (20, 45) (SI Materials and Methods).

Measurement of Phosphate Uptake by Opossum Kidney Cells.

Phosphate uptake by the opossum kidney cell line OKP, which endogenously expresses FGFR1-4 and Klotho (Fig. S8), was measured in response to cell stimulation with FGF23 proteins/peptides (SI Materials and Methods).

Measurement of Phosphate in Serum and Urine of Rodents.

The phosphaturic activity of FGF2328-200 was examined in ∼6-week-old C57BL/6 mice by using a published protocol (19). The anti-phosphaturic activity of FGF23 C-terminal peptides was examined in normal Sprague–Dawley rats and in Hyp mice, a mouse model of XLH (27–29) (SI Materials and Methods). In addition, plasma clearance of recombinant full-length FGF23 and C-terminal tail peptide of FGF23 was analyzed in normal Sprague-Dawley rats (SI Materials and Methods; Fig. S9). The experiments in mice were approved by the Harvard University Animal Care and Research Committee board. The experiments in rats were approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center at Dallas.

Analysis of NaPi-2A and NaPi-2C Protein Abundance in the Apical Brush Border Membrane of Renal Proximal Tubule Epithelium.

See SI Materials and Methods.

Statistical Analysis.

Data are expressed as the mean ± SE (n ≥ 6 or more). Statistical analysis was performed by using Student’s unpaired or paired t test or by using analysis of variance (ANOVA) when applicable. A value of P ≤ 0.05 was considered as statistically significant.

Supplementary Material

Supporting Information
supp_107_1_407__index.html (779B, html)

Acknowledgments

This work was supported by National Institutes of Health Grants DE13686 (to M.M.), AG19712 (to M.K.), AG25326 (to M.K.), DK48482 (to O.W.M.), DK20543 (to O.W.M.), and DK077276 (to M.S.R.), and by the Irma T. Hirschl Fund (M.M.), the Eisai Research Fund (M.K.), the Ellison Medical Foundation (M.K.), Ted Nash Long Life Foundation (M.K.), the Simmons Family Foundation (O.W.M.), and a seed grant from the Pak Center of Mineral Metabolism and Clinical Research (to M.C.H.).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0902006107/DCSupplemental.

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