Klotho has profound effects on phosphate metabolism, but the mechanisms of how Klotho affects phosphate homeostasis is unknown. We detected Klotho in the proximal tubule cell, brush border, and urinary lumen, where phosphate homeostasis resides. Increasing Klotho in the kidney and urine chronically by transgenic overexpression or acutely by intravenous infusion caused hypophosphatemia, phosphaturia from decreased proximal phosphate reabsorption, and decreased activity and protein of the principal renal phosphate transporter NaPi-2a. The phosphaturic effect was present in FGF23-null mice, indicating a direct action distinct from Klotho's known role as a coreceptor for FGF23. Direct inhibition of NaPi-2a by Klotho was confirmed in cultured cells and in cell-free membrane vesicles characterized by acute inhibition of transport activity followed by decreased cell surface protein. Transport inhibition can be mimicked by recombinant beta-glucuronidase and is associated with proteolytic degradation and reduced surface NaPi-2a. The inhibitory effect of Klotho on NaPi-2a was blocked by beta-glucuronidase inhibitor but not by protease inhibitor. Klotho is a novel phosphaturic substance that acts as an enzyme in the proximal tubule urinary lumen by modifying glycans, which cause decreased transporter activity, followed by proteolytic degradation and possibly internalization of NaPi-2a from the apical membrane.

The mammalian fibroblast growth factor (FGF) family comprises 18 polypeptides (FGF1 to FGF10 and FGF16 to FGF23) which participate in a myriad of biological processes during embryogenesis, including but not limited to gastrulation, body plan formation, somitogenesis, and morphogenesis of essentially every tissue/organ such as limb, lung, brain and kidney (3,30). FGFs execute their biological actions by binding to, dimerizing, and activating FGF receptor (FGFR) tyrosine kinases, which are encoded by four distinct genes (Fgfr1 to Fgfr4). Prototypical FGFRs consist of an extracellular domain composed of three immunoglobulin-like domains, a single-pass transmembrane domain, and an intracellular domain responsible for the tyrosine kinase activity (16). The number of principal FGFRs is increased from four to seven due to a major tissue-specific alternative splicing event in the second half of the immunoglobulin-like domain 3 of FGFR1 to FGFR3, which creates epithelial lineage-specific b and mesenchymal lineage-specific c isoforms (16, 21). Generally, the receptor-binding specificity of FGFs is divided along this major alternative splicing of receptors whereby FGFRb-interacting FGFs are produced by mesenchymal cells and FGFRc-interacting FGFs are produced by epithelial cells (21). These reciprocal expression patterns of FGFs and FGFRs result in the establishment of a paracrine epithelial-mesenchymal signaling which is essential for proper organogenesis and patterning during development as well as tissue homeostasis in the adult organism.Based on phylogeny and sequence identity, FGFs are grouped into seven subfamilies (21). The FGF core homology domain (approximately 120 amino acids long) is flanked by Nand C-terminal sequences that are highly variable in both length and primary sequence, particularly among different FGF subfamilies. The core region of FGF19 shares the highest sequence identity with FGF21 (38%) and FGF23 (36%), and therefore, these ligands are considered to form a subfamily. However, the degree of identity within the FGF19 subfamily is only 2 to 3% greater than that between FGF19 subfamily members and members of other FGF subfamilies, making this subfamily the most divergent one. FGF19 subfamily members regulate diverse physiological processes uncommon to classical FGFs, namely, energy (32) and bile acid homeostasis (FGF19) (5,8,13), glucose and lipid metabolism (FGF21) (10), and phosphate and vitamin D homeostasis (FGF23) (27). Moreover, unlike classical FGFs, FGF19 subfamily members achieve their unconventional activities in an endocrine fashion.To date, only a single structure from the endocrine-acting FGF19 subfamily has been reported (4), whereas there are crystal structures available for eight classical, paracrine-acting FGFs (2,20,22,37,38,40). The structures from the paracrine class of FGFs (FGF1, show that the core homology region folds into a globular domain composed of 12 antiparallel ␤-strands (␤1 to ␤12) known as the * Corresponding author. Mailing address:

Fibroblast growth factor-23 (FGF-23), a recently identified molecule that is mutated in patients with autosomal dominant hypophosphatemic rickets (ADHR), appears to be involved in the regulation of phosphate homeostasis. Although increased levels of circulating FGF-23 were detected in patients with different phosphate-wasting disorders such as oncogenic osteomalacia (OOM) and X-linked hypophosphatemia (XLH), it is not yet clear whether FGF-23 is directly responsible for the abnormal regulation of mineral ion homeostasis and consequently bone development. To address some of these unresolved questions, we generated a mouse model, in which the entire Fgf-23 gene was replaced with the lacZ gene. Fgf-23 null (Fgf-23-/-) mice showed signs of growth retardation by day 17, developed severe hyperphosphatemia with elevated serum 1,25(OH)2D3 levels, and died by 13 weeks of age. Hyperphosphatemia in Fgf-23-/- mice was accompanied by skeletal abnormalities, as demonstrated by histological, molecular, and various other morphometric analyses. Fgf-23-/-) mice had increased total-body bone mineral content (BMC) but decreased bone mineral density (BMD) of the limbs. Overall, Fgf-23-/- mice exhibited increased mineralization, but also accumulation of unmineralized osteoid leading to marked limb deformities. Moreover, Fgf-23-/- mice showed excessive mineralization in soft tissues, including heart and kidney. To further expand our understanding regarding the role of Fgf-23 in phosphate homeostasis and skeletal mineralization, we crossed Fgf-23-/- animals with Hyp mice, the murine equivalent of XLH. Interestingly, Hyp males lacking both Fgf-23 alleles were indistinguishable from Fgf-23/-/ mice, both in terms of serum phosphate levels and skeletal changes, suggesting that Fgf-23 is upstream of the phosphate regulating gene with homologies to endopeptidases on the X chromosome (Phex) and that the increased plasma Fgf-23 levels in Hyp mice (and in XLH patients) may be at least partially responsible for the phosphate imbalance in this disorder.

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 FGFRKlotho 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.

Appropriate levels of phosphate in the body are maintained by the coordinated regulation of the bone-derived growth factor FGF23 and the membrane-bound protein Klotho. The endocrine actions of FGF23, in association with parathyroid hormone and vitamin D, mobilize sodium–phosphate cotransporters that control renal phosphate transport in proximal tubular epithelial cells. The availability of an adequate amount of Klotho is essential for FGF23 to exert its phosphaturic effects in the kidney. In the presence of Klotho, FGF23 activates downstream signaling components that influence the homeostasis of phosphate, whereas in the absence of this membrane protein, it is unable to exert such regulatory effects, as demonstrated convincingly in animal models. Several factors, including phosphate and vitamin D, can regulate the production of both FGF23 and Klotho and influence their functions. In various acquired and genetic human diseases, dysregulation of FGF23 and Klotho is associated with vascular and skeletal anomalies owing to altered phosphate turnover. In this Review, I summarize how the endocrine effects of bone-derived FGF23, in coordination with Klotho, can regulate systemic phosphate homeostasis, and how an inadequate balance of these molecules can lead to complications that are caused by abnormal mineral ion metabolism.

Fibroblast growth factor 23 null mice (Fgf-23 −/− ) have a short lifespan and show numerous biochemical and morphological features consistent with premature aging-like phenotypes, including kyphosis, severe muscle wasting, hypogonadism, osteopenia, emphysema, uncoordinated movement, T cell dysregulation, and atrophy of the intestinal villi, skin, thymus, and spleen. Aging is a complex biological process controlled by multiple genetic and environmental factors (1-4). Studies involving molecular mechanisms of human aging and its progression are challenging, as it takes decades to develop some of the age-related features. Since extensive subsets of age-associated phenotypes define human aging, the availability of animal models exhibiting multiple aging features are useful, not only to analyze the molecular mechanisms of age-related changes in various organs, but also for the in vivo screening of molecules that counteract age-associated syndromes including anti-oxidant agents and hormones (5,6). DNA damage through oxidative stress, among others, is thought to be an important contributing factor in aging, and has been extensively studied in animals (1,3,(7)(8)(9)(10)(11). However, the potential role of humoral factor(s) regulating the aging process has not been studied in similar depth and detail. In this study, we show that genetic ablation of Fgf-23 results in a syndrome that resembles premature aging. (12,19,20).In this study, using in vivo genetic manipulation approaches, we present a novel role of Fgf-23 in premature aging and show that the premature aging-like phenotype in Fgf-23 −/− mice is partly mediated through increased vitamin D activities. MATERIALS AND METHODS Experimental miceWe recently generated . Animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were employed using protocols approved by the institution's subcommittee on animal care (IACUC). Macroscopic phenotypeThe total body weight of all mice was taken every 3-5 days starting at 2.5 wk of age until death. Survival of various groups of animals was recorded until death of control, Fgf-23 −/− , and double mutant Fgf-23 −/− /1α(OH)ase −/− mice. Biochemical measurementsBlood was obtained either by retro-orbital or tail bleeding of 3-, 6-, 9-and 11-wk-old wildtype, Fgf-23 −/− , and Fgf-23 −/− /1α(OH)ase −/− littermates. Serum was isolated by centrifugation at 3000 g for 10 min and stored at −80°C. Serum phosphorus and serum calcium were determined by colorometric measurements using the Stanbio Phosphorus Liqui-UV Test and Calcium (Arsenazo) LiquiColor Test, respectively. Total blood of 4-wk-old mice was used to determine routine hematological parameters such as cell counts. Skeletal analysesSkeletal changes in Fgf-23 −/− mice and their control littermates were analyzed by X-ray, quantitative CT (pQCT) and PIXImus measurements. Alizarin red S staining of total body skeletons, routine histology, and von Kossa staining were executed as described in our earlier studies (22). Immunohistochemica...

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A major breakthrough in systemic phosphate homeostasis regulation was achieved by the demonstration of strikingly similar physical, morphological, and biochemical phenotypes of fibroblast growth factor 23 (Fgf23) and klotho ablated mice, which led to identification of klotho as an Fgf23 signaling cofactor. Here, we generated Fgf23 and klotho double-knockout (Fgf23(-/-)/klotho(-/-)) mice to test the hypothesis whether Fgf23 has a klotho-independent function. Fgf23(-/-)/klotho(-/-) mice are viable and have high serum phosphate levels, similar to Fgf23(-/-) and klotho(-/-) single-knockout mice. In addition, the Fgf23(-/-)/klotho(-/-) mice have increased renal expression of the sodium/phosphate cotransporter NaP(i)2a and of 1- alpha-hydroxylase concomitant with increased serum levels of 1,25-dihydroxyvitamin-D, as also observed in the Fgf23(-/-) and klotho(-/-) mice. Moreover, Fgf23(-/-)/klotho(-/-) mice show soft tissue and vascular calcification, severe muscle wasting, hypogonadism, pulmonary emphysema, distention of intestinal wall, and skin atrophy, all of which are also seen in Fgf23(-/-) and klotho(-/-) mice. Notably, injection of bioactive FGF23 protein into Fgf23(-/-)/klotho(-/-) and klotho(-/-) mice does not lower serum phosphate, whereas in wild-type and Fgf23(-/-) mice, it reduces serum phosphate. Together, these results provide compelling evidence that Fgf23 does not have a klotho-independent role in the regulation of systemic phosphate and vitamin D homeostasis.