Hypophosphatemic rickets: Revealing Novel Control Points for Phosphate Homeostasis

Rapid and somewhat surprising advances have recently been made towards understanding the molecular mechanisms causing heritable disorders of hypophosphatemia. The results of clinical, genetic, and translational studies have interwoven novel concepts underlying the endocrine control of phosphate metabolism, with far-reaching implications for treatment of both rare, Mendelian diseases as well as common disorders of blood phosphate excess such as chronic kidney disease (CKD). In particular, diseases caused by changes in the expression and proteolytic control of the phosphaturic hormone Fibroblast growth factor-23 (FGF23) have come to the forefront in terms of directing new models explaining mineral metabolism. These hypophosphatemic disorders, as well as others resulting from independent defects in phosphate transport or metabolism, will be reviewed herein, and implications for emerging therapeutic strategies based upon these new findings will be discussed

Heritable and acquired disorders of phosphate metabolism: Etiologies involving FGF23 and current therapeutics

Phosphate is critical for many cellular processes and structural functions, including as a key molecule for nucleic acid synthesis and energy metabolism, as well as hydroxyapatite formation in bone. Therefore it is critical to maintain tight regulation of systemic phosphate levels. Based upon its broad biological importance, disruption of normal phosphate homeostasis has detrimental effects on skeletal integrity and overall health. Investigating heritable diseases of altered phosphate metabolism has led to key discoveries underlying the regulation and systemic actions of the phosphaturic hormone Fibroblast growth factor-23 (FGF23). Both molecular and clinical studies have revealed novel targets for the development and optimization of therapies for disorders of phosphate handling. This review will focus upon the bridge between genetic discoveries involving disorders of altered FGF23 bioactivity, as well as describe how these findings have translated into pharmacologic application

Characterization of native FGF23 protein and mutant forms causing autosomal dominant hypophosphatemic rickets and familial tumoral calcinosis

The regulation of phosphate metabolism is a complex process that is still only partly understood. At the end of the eighties, studies in a mouse model for hypophosphatemic rickets provided evidence that phosphate wasting could not be explained by a primary defect of the kidney but rather by an unknown circulating factor with phosphaturic properties. X-linked hypophosphatemia (XLH), autosomal dominant hypophosphatemic rickets (ADHR), and tumor induced osteomalacia (TIO) are three well defined human disorders of isolated renal phosphate wasting. XLH and ADHR are mendelian diseases while TIO is caused by rare, mostly benign tumors. The opposite phenotype, hyperphosphatemia due to increased renal phosphate reabsorption is associated to the recessive disorder familial tumoral calcinosis (FTC). At the beginning of this work the genes mutated in XLH and ADHR were cloned. One gene codes for the endopeptidase PHEX, the other for the fibroblast growth factor FGF23. Both proteins are probably involved in a novel common pathway of the regulation of phosphate homeostasis. Missense mutations in FGF23 causing phosphate wasting in patients with ADHR, overexpression of FGF23 in tumors from patients with TIO, and the observation that FGF23 plasma levels are elevated in most patients with XLH provided strong evidence that FGF23 is a hormone with phosphaturic activity. However, the function of FGF23 in the regulation of phosphate metabolism is far from understood. The intention of this study was to investigate the molecular properties of native FGF23 and its mutant forms. I conducted protein expression experiments in HEK293 cells which showed that native FGF23 is a secreted protein partially processed into an N-terminal fragment and a C-terminal fragment. I provided evidence that this cleavage occurs during protein secretion and it is performed by subtilisin like-proprotein convertases (SPCs). In addition, I determined that native FGF23 undergoes O-linked glycosylation before secretion by using a deglycosylation assay. Further, RT-PCR analysis of human tissues showed FGF23 expression in whole fetus, heart, liver, thyroid/parathyroid, small intestine, testis, skeletal muscle, differentiated chondrocytes and TIO tumor tissue. In mouse, FGF23 was expressed in day 17 embryo and spleen. The FGF23 ADHR mutations replace arginine residues at the SPC cleavage site (RXXR motif). By expression of the FGF23-R176Q and –R179Q mutant proteins in HEK293 cells I showed that ADHR mutations prevent cleavage at the RXXR site and stabilize FGF23. This alteration in the FGF23 cleavage enhances FGF23 phosphaturic activity in ADHR. Familial tumoral calcinosis (FTC) with hyperphosphatemia is a disease considered the mirror image of the hypophosphatemic condition. It is known that FTC is caused by mutations in the GALNT3 gene. By performing mutation analysis in two families with FTC, I could show that FTC can also be caused by inactivating mutations in the FGF23 gene. To characterize the FGF23-S71G mutant protein I conducted in vitro expression assays, immunocytochemistry and ELISA to measure the FGF23 plasma levels in the patient with FTC. Taken together the results of these experiments showed that intact FGF23-S71G mutant protein remained inside the cells and only the C-terminal FGF23 fragment was secreted. These investigations demonstrate that FGF23 mutations in ADHR and FTC have opposite effects on phosphate homeostasis. There is evidence that the endopeptidase PHEX which is mutated in patients with XLH and FGF23 act in the same pathway. PHEX function resides upstream of FGF23 and may be involved in the degradation of FGF23 thereby regulating its phosphaturic activity. I designed an assay with a recombinant secreted form of PHEX (secPHEX) to prove whether FGF23 is a substrate of PHEX. Although secPHEX activity could be demonstrated by degradation of PTHrP107-139, secPHEX failed to degrade FGF23 in this assay. These results provided evidence against a direct interaction of PHEX and FGF23

Neonatal iron deficiency causes abnormal phosphate metabolism by elevating FGF23 in normal and ADHR mice.

Fibroblast growth factor 23 (FGF23) gain of function mutations can lead to autosomal dominant hypophosphatemic rickets (ADHR) disease onset at birth, or delayed onset following puberty or pregnancy. We previously demonstrated that the combination of iron deficiency and a knock-in R176Q FGF23 mutation in mature mice induced FGF23 expression and hypophosphatemia that paralleled the late-onset ADHR phenotype. Because anemia in pregnancy and in premature infants is common, the goal of this study was to test whether iron deficiency alters phosphate handling in neonatal life. Wild-type (WT) and ADHR female breeder mice were provided control or iron-deficient diets during pregnancy and nursing. Iron-deficient breeders were also made iron replete. Iron-deficient WT and ADHR pups were hypophosphatemic, with ADHR pups having significantly lower serum phosphate (p 50 fold; p < 0.01). WT and ADHR pups receiving low iron had elevated intact serum FGF23; ADHR mice were affected to a greater degree (p < 0.01). Iron-deficient mice also showed increased Cyp24a1 and reduced Cyp27b1, and low serum 1,25-dihydroxyvitamin D (1,25D). Iron repletion normalized most abnormalities. Because iron deficiency can induce tissue hypoxia, oxygen deprivation was tested as a regulator of FGF23, and was shown to stimulate FGF23 mRNA in vitro and serum C-terminal FGF23 in normal rats in vivo. These studies demonstrate that FGF23 is modulated by iron status in young WT and ADHR mice and that hypoxia independently controls FGF23 expression in situations of normal iron. Therefore, disturbed iron and oxygen metabolism in neonatal life may have important effects on skeletal function and structure through FGF23 activity on phosphate regulation

Hormone-like (endocrine) Fgfs: their evolutionary history and roles in development, metabolism, and disease

Fibroblast growth factors (Fgfs) are proteins with diverse functions in development, repair, and metabolism. The human Fgf gene family with 22 members can be classified into three groups, canonical, intracellular, and hormone-like Fgf genes. In contrast to canonical and intracellular Fgfs identified in invertebrates and vertebrates, hormone-like Fgfs, Fgf15/19, Fgf21, and Fgf23, are vertebrate-specific. The ancestral gene of hormone-like Fgfs was generated from the ancestral gene of canonical Fgfs by gene duplication early in vertebrate evolution. Later, Fgf15/19, Fgf21, and Fgf23 were generated from the ancestral gene by genome duplication events. Canonical Fgfs act as autocrine/paracrine factors in an Fgf receptor (Fgfr)-dependent manner. In contrast, hormone-like Fgfs act as endocrine factors in an Fgfr-dependent manner. Canonical Fgfs have a heparin-binding site necessary for the stable binding of Fgfrs and local signaling. In contrast, hormone-like Fgfs acquired endocrine functions by reducing their heparin-binding affinity during their evolution. Fgf15/19 and Fgf23 require βKlotho and αKlotho as cofactors, respectively. However, Fgf21 might physiologically require neither. Hormone-like Fgfs play roles in metabolism at postnatal stages, although they also play roles in development at embryonic stages. Fgf15/19 regulates bile acid metabolism in the liver. Fgf21 regulates lipid metabolism in the white adipose tissue. Fgf23 regulates serum phosphate and active vitamin D levels. Fgf23 signaling disorders caused by hereditary diseases or tumors result in metabolic disorders. In addition, serum Fgf19 or Fgf21 levels are significantly increased by metabolic disorders. Hormone-like Fgfs are newly emerging and quite unique in their evolution and function

FGF-23 in bone biology

Recent studies have demonstrated that levels of fibroblast growth factor 23 (FGF-23), a key regulator of phosphorus and vitamin D metabolism, rise dramatically as renal function declines and may play a key initiating role in disordered mineral and bone metabolism in patients with chronic kidney disease (CKD). The physiologic importance of FGF-23 in mineral metabolism was first identified in human genetic and acquired rachitic diseases and further characterized in animal models. FGF-23 and its regulators, including phosphate regulating endopeptidase homolog, dentin matrix 1 (DMP1), and matrix extracellular phosphoglycoprotein, are made primarily in bone, specifically in osteocytes. Dysregulation of these proteins results in osteomalacia, implicating the osteocyte in the regulation of skeletal mineralization. Studies in pediatric patients with CKD, the majority of whom have altered skeletal mineralization in early stages of CKD, have demonstrated that skeletal expression of both FGF-23 and its regulator, DMP1, are increased in early stages of CKD and that expression of these proteins is associated with alterations in skeletal mineralization. Thus, dysregulation of osteocytic proteins occur very early in the course of CKD and appear to be central to altered bone and mineral metabolism in this patient population

Conditional Deletion of Murine Fgf23: Interruption of the Normal Skeletal Responses to Phosphate Challenge and Rescue of Genetic Hypophosphatemia

The transgenic and knockout (KO) animals involving Fgf23 have been highly informative in defining novel aspects of mineral metabolism, but are limited by shortened lifespan, inability of spatial/temporal FGF23 control, and infertility of the global KO. To more finely test the role of systemic and genetic influences in FGF23 production, a mouse was developed that carried a floxed ("f")-Fgf23 allele (exon 2 floxed) which demonstrated in vivo recombination when bred to global-Cre transgenic mice (eIIa-cre). Mice homozygous for the recombined allele ("Δ") had undetectable serum intact FGF23, elevated serum phosphate (p < 0.05), and increased kidney Cyp27b1 mRNA (p < 0.05), similar to global Fgf23-KO mice. To isolate cellular FGF23 responses during phosphate challenge, Fgf23(Δ/f) mice were mated with early osteoblast type Iα1 collagen 2.3-kb promoter-cre mice (Col2.3-cre) and the late osteoblast/early osteocyte Dentin matrix protein-1-cre (Dmp1-cre). Fgf23(Δ/f) /Col2.3-cre(+) and Fgf23(Δ/f) /Dmp1-cre(+) exhibited reduced baseline serum intact FGF23 versus controls. After challenge with high-phosphate diet Cre(-) mice had 2.1-fold to 2.5-fold increased serum FGF23 (p < 0.01), but Col2.3-cre(+) mice had no significant increase, and Dmp1-cre(+) mice had only a 37% increase (p < 0.01) despite prevailing hyperphosphatemia in both models. The Fgf23(Δ/f) /Col2.3-cre was bred onto the Hyp (murine X-linked hypophosphatemia [XLH] model) genetic background to test the contribution of osteoblasts and osteocytes to elevated FGF23 and Hyp disease phenotypes. Whereas Hyp mice maintained inappropriately elevated FGF23 considering their marked hypophosphatemia, Hyp/Fgf23(Δ/f) /Col2.3-cre(+) mice had serum FGF23 <4% of Hyp (p < 0.01), and this targeted restriction normalized serum phosphorus and ricketic bone disease. In summary, deleting FGF23 within early osteoblasts and osteocytes demonstrated that both cell types contribute to baseline circulating FGF23 concentrations, and that targeting osteoblasts/osteocytes for FGF23 production can modify systemic responses to changes in serum phosphate concentrations and rescue the Hyp genetic syndrome

Genetic diseases of renal phosphate handling

UNLABELLED: Renal control of systemic phosphate homeostasis is critical as evident from inborn and acquired diseases causing renal phosphate wasting. At least three transport proteins are responsible for renal phosphate reabsorption: NAPI-IIa (SLC34A1), NAPI-IIc (SLC34A3) and PIT-2 (SLC20A2). These transporters are highly regulated by various cellular mechanisms and factors including acid-base status, electrolyte balance and hormones such as dopamine, glucocorticoids, growth factors, vitamin D3, parathyroid hormone and fibroblast growth factor 23 (FGF23). Whether renal phosphate wasting is caused by inactivating mutations in the NAPI-IIa transporter is controversial. Mutations in the NAPI-IIc transporter cause hereditary hypophosphatemic rickets with hypercalciuria. Besides the primary inherited defects, there are also inherited defects in major regulators of phosphate homeostasis that lead to alterations in phosphate handling. Autosomal dominant hypophosphatemic rickets is due to FGF23 mutations leading to resistance against its own degradation. Similarly, inactivating mutations in the PHEX gene, which causes FGF23 inactivation, cause X-linked hypophosphatemia due to renal phosphate losses. In contrast, mutations in galactosamine:polypeptide N-acetyl-galactosaminyltransferase, responsible for O-glycosylation of FGF23, or in klotho, a cofactor for FGF23 signalling result in hyperphosphatemia. Acquired syndromes of renal phosphate wasting, hypophosphatemia and osteomalacia (tumour-associated osteomalacia) can be due to the excessive synthesis or release of phosphaturic factors (FGF23, FGF-7, MEPE and sFRP4) from mesenchymal tumour

Genetic diseases of renal phosphate handling

UNLABELLED: Renal control of systemic phosphate homeostasis is critical as evident from inborn and acquired diseases causing renal phosphate wasting. At least three transport proteins are responsible for renal phosphate reabsorption: NAPI-IIa (SLC34A1), NAPI-IIc (SLC34A3) and PIT-2 (SLC20A2). These transporters are highly regulated by various cellular mechanisms and factors including acid-base status, electrolyte balance and hormones such as dopamine, glucocorticoids, growth factors, vitamin D3, parathyroid hormone and fibroblast growth factor 23 (FGF23). Whether renal phosphate wasting is caused by inactivating mutations in the NAPI-IIa transporter is controversial. Mutations in the NAPI-IIc transporter cause hereditary hypophosphatemic rickets with hypercalciuria. Besides the primary inherited defects, there are also inherited defects in major regulators of phosphate homeostasis that lead to alterations in phosphate handling. Autosomal dominant hypophosphatemic rickets is due to FGF23 mutations leading to resistance against its own degradation. Similarly, inactivating mutations in the PHEX gene, which causes FGF23 inactivation, cause X-linked hypophosphatemia due to renal phosphate losses. In contrast, mutations in galactosamine:polypeptide N-acetyl-galactosaminyltransferase, responsible for O-glycosylation of FGF23, or in klotho, a cofactor for FGF23 signalling result in hyperphosphatemia. Acquired syndromes of renal phosphate wasting, hypophosphatemia and osteomalacia (tumour-associated osteomalacia) can be due to the excessive synthesis or release of phosphaturic factors (FGF23, FGF-7, MEPE and sFRP4) from mesenchymal tumour

A Novel Nonsense Mutation in the DMP1 Gene Identified by a Genome-Wide Association Study Is Responsible for Inherited Rickets in Corriedale Sheep

Inherited rickets of Corriedale sheep is characterized by decreased growth rate, thoracic lordosis and angular limb deformities. Previous outcross and backcross studies implicate inheritance as a simple autosomal recessive disorder. A genome wide association study was conducted using the Illumina OvineSNP50 BeadChip on 20 related sheep comprising 17 affected and 3 carriers. A homozygous region of 125 consecutive single-nucleotide polymorphism (SNP) loci was identified in all affected sheep, covering a region of 6 Mb on ovine chromosome 6. Among 35 candidate genes in this region, the dentin matrix protein 1 gene (DMP1) was sequenced to reveal a nonsense mutation 250C/T on exon 6. This mutation introduced a stop codon (R145X) and could truncate C-terminal amino acids. Genotyping by PCR-RFLP for this mutation showed all 17 affected sheep were “T T” genotypes; the 3 carriers were “C T”; 24 phenotypically normal related sheep were either “C T” or “C C”; and 46 unrelated normal control sheep from other breeds were all “C C”. The other SNPs in DMP1 were not concordant with the disease and can all be ruled out as candidates. Previous research has shown that mutations in the DMP1 gene are responsible for autosomal recessive hypophosphatemic rickets in humans. Dmp1_knockout mice exhibit rickets phenotypes. We believe the R145X mutation to be responsible for the inherited rickets found in Corriedale sheep. A simple diagnostic test can be designed to identify carriers with the defective “T” allele. Affected sheep could be used as animal models for this form of human rickets, and for further investigation of the role of DMP1 in phosphate homeostasis