Genetic Disorders Of Phosphate Homeostasis
Regulation of Phosphate Homeostasis
Phosphate in serum exists almost exclusively as the free ion or in association with cations. Unlike calcium, only 12% of phosphate is protein bound.1 Also in contrast to calcium, serum phosphate concentrations may vary substantially throughout the day. Carbohydrate ingestion may markedly reduce serum phosphate by moving serum phosphate from the extracellular to the intracellular space. Moreover, serum phosphate undergoes diurnal variation of as much as 1.5 mg/dL (0.5 mmol/L), with a nadir between 8 and 11 am.2 Interference with the measurement of phosphate in serum may occur during hypertriglyceridemia,3 hypergammaglobulinemia,4 or mannitol therapy,5 depending on the method of analysis.
Fasting serum phosphate remains stable throughout the menstrual cycle and during pregnancy.6–10 The placenta actively transports phosphate into the fetus, as reflected in the higher phosphate concentrations of newborn cord arterial and venous blood compared with maternal blood levels.8 Lactating women, who may lose 100 to 500 mg of phosphorus daily in milk, nevertheless maintain normal levels of serum phosphate.11,12 Serum phosphate concentrations are relatively high in the newborn (5 to 7 mg/dL),8,13 decrease gradually thereafter, and then increase again briefly at puberty before reaching adult levels by age 18 to 20 years. Serum phosphate typically increases in women after menopause but decreases in the elderly.14–16
Phosphate is a ubiquitous constituent of a vast array of biomolecules. Of particular importance is the fundamental role of inorganic phosphate as a substrate for intracellular enzymes involved in glycolysis and respiration that synthesize high-energy phosphate bonds for storage of chemical energy in organophosphate compounds such as ATP, creatine phosphate, diphosphoglycerate (DPG), phosphoenolpyruvate, and others. Severe phosphate depletion leads to a concentration-dependent inhibition of glycolysis, accumulation of “triose phosphates” immediately proximal to glyceraldehyde 3-phosphate dehydrogenase, and decreased production of ATP. Adequate extracellular phosphate is required for normal mineralization of bone and cartilage,17,18 and chronic hypophosphatemia of any cause may therefore lead to osteomalacia or, in children, rickets.
The average dietary intake of phosphate—derived largely from dairy products, cereals, and meats—is roughly twice the estimated minimum requirement of 400 mg/day.19 Absorptive efficiency is high, averaging about 70%, and may increase further (=90%) if the intake of dietary phosphate decreases to less than 2 mg/kg/day.20 Phosphate is avidly absorbed throughout the small intestine, but especially in the jejunum in animals and humans.21–23
In the jejunum, overall phosphate uptake consists of two components: a saturable, sodium-dependent process that is responsive to vitamin D (see later) and a nonsaturable, sodium-independent mechanism thought to represent paracellular diffusional transport.21,24 The saturable mechanism reflects active transport via the transcellular route, the energy for which is derived from the transmembrane sodium gradient. In animal tissues and in human jejunal biopsies, the sodium phosphate cotransporter exhibits a Km of approximately 0.05 mmol/L, half-maximal stimulation by 30 to 50 mmol/L sodium, and a ratio of two sodium molecules per molecule of phosphate transported.21,25,26 Sodium-dependent active phosphate absorption is mediated by the NPT2b cotransporters present in the luminal brush-border membranes of enterocytes.27,28 Sodium phosphate transporter NPT2b is the product of a different gene from that which encodes the predominant forms expressed in the renal proximal tubule (NPT2a and NPT2c).27,28 For example, mice lacking the renal cotransporter manifest striking hyperphosphaturia because of continued intestinal phosphate absorption.29 NPT2b is fully saturated at intraluminal phosphate concentrations of 1 to 2 mmol/L, which are easily achieved after most typical meals. Subsequent transport across the basolateral membrane does not require active transport and is thought to proceed via facilitated diffusion, although the transporter or channel involved has not been characterized.30
Regulation Of Phosphate Absorption
The central role of vitamin D in the regulation of intestinal phosphate transport has been recognized for many years. Absorption of phosphate, like that of calcium, is strikingly augmented by 1,25(OH)2D.21,22,31–34 Basal fractional phosphate absorption in the absence of 1,25(OH)2D is much higher than that of calcium, however. The action of 1,25(OH)2D on phosphate transport has been studied in vitro by using intact intestinal segments, isolated enterocytes, and brush-border membrane vesicles (BBMVs).24,35–39 In each case, stimulation by 1,25(OH)2D was shown to result from activation of the sodium-dependent active transport mechanism and not the passive diffusional component. Specifically, 1,25(OH)2D stimulates the maximal velocity of sodium-dependent phosphate cotransport by increasing expression of NPT2b, mainly or exclusively via a posttranscriptional mechanism.30,40–43 Other studies have pointed to an additional, very rapid (minutes), nongenomic mechanism of 1,25(OH)2D-dependent stimulation of intestinal phosphate transport, analogous to its nongenomic effect on duodenal calcium transport.44,45
Restriction of dietary phosphate increases intestinal NPT2b expression41,43 and thus, like 1,25(OH)2D, enhances the Vmax of the saturable component of intestinal phosphate absorption.46–48 This response also involves a posttranscriptional mechanism,41,43 results in part from augmented renal synthesis of 1,25(OH)2D,30 but can be seen also in vitamin D–deficient or vitamin D receptor–null animals.43,49–51
Phosphate Excretion
Eighty percent of filtered phosphate is reabsorbed by the proximal tubule, and the capacity for phosphate transport appears to diminish between the early convoluted and the straight (pars recta) portions of the proximal tubule.52–54 Additional phosphate may be reabsorbed in the distal tubule or cortical collecting tubule or both.55–60 Phosphate must be actively transported across the luminal brush-border membrane against a steep electrochemical gradient. Consistent with this, renal phosphate transport requires luminal sodium ions and is blocked by inhibitors of Na+/K+-ATPase.54,61–64 Dibasic phosphate is preferentially transported by the rat proximal tubule,65–67 and studies with isolated perfused tubules have shown that increased intraluminal pH and decreased intracellular pH accelerate phosphate reabsorption by the intact cell.68
The two primary transport proteins responsible for Pi reabsorption in the kidney are the type II sodium-phosphate cotransporters NPT2a and NPT2c, expressed in the apical membrane of the proximal tubule. In the mouse, NPT2a is a critical phosphate cotransporter in the renal proximal tubule. Ablation of the NPT2a gene in the mouse results in a decrease in proximal tubular sodium phosphate cotransport, hypophosphatemia, and loss of regulation of phosphate reabsorption by both parathyroid hormone (PTH) and dietary phosphate.29,69,70 Ablation of NPT2c in mice does not result in a phosphate phenotype,71 thereby indicating that it may play a less central role in phosphate transport in the mouse. However, mutations in the gene coding NPT2c, SLC34A3, result in hereditary hypophosphatemic rickets with hypercalciuria (HHRH, see later), indicating that this transporter plays an important role in maintenance of phosphate homeostasis in humans.
Measurement of Renal Phosphate Transport
For clinical purposes, a relatively convenient way of measuring a patient’s ability to reabsorb phosphate is to calculate the tubular maximum reabsorption of phosphate divided by the glomerular filtration rate (TMP/GFR). A simple nomogram has been developed by Walton and Bijvoet72 that works well in most situations (Fig. 6-1).
FIGURE 6-1 Nomogram for calculating TmP/GFR. Calculations are done from fasting samples obtained in the morning. The urine is collected for 1 to 2 hours (the time period is not critical). The CPO4/Ccreat = (UPO4 × [Creat])/(Ucreat × [Pi]). To use the nomogram to calculate TMP/GFR, a straight line is passed through the appropriate plasma phosphate concentration and the CPO4/Ccreat. The line will intersect with the corresponding TMP/GFR value. For SI units, use the inside scales. For metric (mg/dL), use the outside scales. (From Walton RJ, Bijvoet OL: Nomogram for derivation of renal threshold phosphate concentration. Lancet 2:309–310, 1975.)
Regulation of Phosphate Reabsorption
PTH has a major effect on serum phosphate. The clinical relevance of this effect is demonstrated in patients with hyperparathyroidism, who develop phosphaturia and hypophosphatemia, as well as those with hypoparathyroidism, who have increased phosphate reabsorption and hyperphosphatemia. PTH rapidly (15 to 60 minutes) reduces the number of NPT2a cotransporters on the apical surface of the cells in the renal proximal tubule. The effect appears to result from microtubule-dependent internalization into endocytic vesicles and subsequent destruction of the transporters.73,74 This acute down-regulation of NPT2a cotransporters does not involve reduction in NPT2a gene transcription,75 although transcriptional suppression is seen after more prolonged PTH exposure.76 After parathyroidectomy, rats manifest a two- to threefold increase in both protein and messenger RNA (mRNA) levels of NPT2a, which correlates with a striking increase in phosphate reabsorption.75 Evidence exists for involvement of both PKA and protein kinase C (PKC), as well as MAPK signaling via PTH/PTHrP receptors in bringing about these responses.77,78 Despite the fact that PTH clearly has a major effect on phosphate homeostasis, it should be kept in mind that the primary role of PTH is to regulate serum calcium level and not phosphate homeostasis. Recent studies of the role of FGF23 further illuminate this issue (see later).
Dietary Phosphate
Renal phosphate excretion is extremely sensitive to changes in dietary phosphate availability. Thus, dietary phosphate deprivation79–86 or supplementation80,82,87 rapidly evokes a compensatory increase or decrease, respectively, in renal phosphate reabsorption ([Pi]Th). Compelling clinical and experimental evidence has established that these adaptations to dietary phosphate occur quite independently of PTH.74,75,79–82,88
Increased sodium-dependent phosphate transport by isolated brush-border membrane vesicles occurs within a few hours of phosphate deprivation86,89 and reflects increased maximal velocity (rather than affinity) of the phosphate carrier,89–91 consistent with an increased number of membrane transporters. This has been corroborated by direct immunohistologic demonstration that institution of a low-phosphate diet causes rapid (within 2 hours) insertion of NPT2a cotransporters into the apical plasma membrane of rat proximal tubular cells by a microtubule-dependent mechanism.92–94 Up-regulation of NPT2a gene transcription occurs subsequently during more prolonged phosphate restriction (i.e., several days).74 Similarly, high dietary phosphate rapidly reduces apical membrane NPT2a protein levels, with no change in NPT2a gene transcription for at least several hours.74,95
Fibroblast Growth Factor-23
Fibroblast growth factor-23 (FGF23), the gene identified as causative for autosomal dominant hypophosphatemic rickets (see later) and some forms of familial hyperphosphatemic tumoral calcinosis, does not appear to play a role in the rapid changes in phosphate reabsorption due to acute changes in intestinal phosphate.96 However, there is substantial evidence that FGF23 plays a role in maintenance of normal phosphate homeostasis over the course of days.
Fibroblast Growth Factor-23 Activity
FGF23 has similar functions as PTH to reduce renal Pi reabsorption but has opposite effects on 1,25(OH)2D production. FGF23 delivery leads to renal Pi wasting through the down-regulation of both NPT2a and NPT2c.97 Under normal circumstances, hypophosphatemia is a strong positive stimulator for increasing serum 1,25(OH)2D production. However, patients with ADHR, TIO, XLH, and ARHR manifest hypophosphatemia with paradoxically low or inappropriately normal serum 1,25(OH)2D concentrations. In mice, the expression of the 1α(OH)ase enzyme and the catabolic 24(OH)ase are reduced and elevated, respectively, when the animals are exposed to FGF23 by injection or by transgenic approaches.98 Thus, the effects of FGF23 on the renal vitamin D metabolic enzymes is most likely responsible for the reductions in 1,25(OH)2D in the setting of often marked hypophosphatemia in ADHR, XLH, TIO, and ARHR patients.
Regulation of FGF23 Production
In humans, dietary Pi supplementation increased FGF23, whereas Pi restriction and the addition of Pi binders suppressed serum FGF23 (Fig. 6-2),99 indicating that FGF23 plays a role in maintenance of Pi homeostasis. In animal studies, the FGF23 response to serum Pi appears to be much more dramatic than in the human studies. Mice given high and low Pi diets have increased and decreased serum FGF23 levels, respectively.100
FIGURE 6-2 FGF23 levels in response to diet. Percent change in FGF23 during the intervention period (
, phosphate depletion intervention as measured with the intact FGF23 assay; 
, phosphate loading intervention as measured with the intact FGF23 assay). The intervention, dietary phosphate depletion, or loading was started after the day 5 samples were collected. Values are presented as mean ± SE. (Data from Burnett SM, Gunawardene SC, Bringhurst FR et al: Regulation of C-terminal and intact FGF-23 by dietary phosphate in men and women, J Bone Miner Res 21:1187–1196, 2006.)
Vitamin D has important regulatory effects on FGF23 in vivo. In mice, injections of 20 to 200 ng 1,25(OH)2D led to dose-dependent increases in serum FGF23 concentrations.98 These changes in FGF23 occurred before detectable changes in serum Pi, indicating that FGF23 may be directly regulated by vitamin D. Physiologically, this would be consistent with results examining the role of FGF23 in vitamin D metabolism, in that FGF23 has been shown to down-regulate the 1α(OH)ase mRNA.97,98 Thus as 1,25(OH)2D is elevated in the blood as a product of 1α(OH)ase activity, vitamin D would then increase FGF23 production, which would complete the negative-feedback loop and down-regulate 1α(OH)ase.
Serum Assays
FGF23 can be measured in the bloodstream via several assays. One widely used assay is a “C-terminal” FGF23 enzyme-linked immunosorbent assay (ELISA), with both the capture and detection antibodies binding C-terminal to the FGF23 176RXXR179/S cleavage site.101 This assay thus recognizes full-length FGF23 as well as C-terminal fragments that could arise through proteolytic processing. The C-terminal assay is quantified relative to standards composed of FGF23-conditioned media produced from stable cell lines expressing the human protein, and it only recognizes the human FGF23 isoform. The normal mean for this assay is 55 ± 50 reference units (RU)/mL, and the upper limit of normal is 150 RU/mL. In a study with a large number of controls and TIO patients, this ELISA was used to examine FGF23 concentrations in TIO and XLH101 and showed that serum FGF23 is detectable in normal individuals. The mean FGF23 was greater than 10-fold elevated in TIO patients, which rapidly fell after surgical removal of the tumor. Importantly, most XLH patients (13 out of 21) had elevated FGF23 compared to controls,101 and in those with “normal” FGF23, these levels may be “inappropriately normal” in the setting of hypophosphatemia.
An “intact” FGF23 assay has been developed that uses conformation-specific monoclonal antibodies that span the 176RXXR179/S180 SPC cleavage site (see later) and thus recognize N-terminal and C-terminal regions of the FGF23 polypeptide.102 In normal individuals, this assay has a mean circulating concentration of 29 pg/mL. The published upper limit of normal is 54 pg/mL.102 The results of these two assays generally agree with regard to the relative ranges of FGF23 concentrations in XLH and in TIO patients, and that FGF23 is elevated in most XLH patients. Based upon limited data from two TIO patients undergoing resection, the intact assay was used to determine that the half-life of FGF23 is between 20 and 50 minutes.103,104
FGF23-Associated Syndromes
FGF23-associated syndromes, summarized in Table 6-1, can be divided into three groups:
1. Disorders associated with increased FGF23 bioactivity
2. Disorders associated with reduced FGF23 bioactivity
3. Genetic hypophosphatemia not associated with elevated FGF23
Disorders Associated With Increased FGF23 Bioactivity
Autosomal-Dominant Hypophosphatemic Rickets (OMIM No. 193100)
ADHR is a rare disorder characterized by low serum Pi concentrations due to decreased TmP/GFR and inappropriately low or normal circulating vitamin D concentrations.105 ADHR was first described in a small family,106 and subsequently, a large ADHR kindred with many affected individuals was evaluated.105 This kindred provided an opportunity to test the phenotypic variability of ADHR in a large number of individuals with the same mutation. There was no evidence of genetic anticipation or imprinting. In contrast to the other genetic renal phosphate-wasting disorders, ADHR displays incomplete penetrance and variable age of onset. Important to the diagnosis and clinical management of ADHR, it was observed that this expanded ADHR family contains two subgroups of affected individuals. One subgroup consists of patients who presented during childhood with Pi wasting, rickets, and lower-extremity deformity in a pattern similar to the classic presentation of XLH. The second group consists of individuals who presented clinically during adolescence or adulthood. These individuals had bone pain, weakness, and insufficiency fractures but did not have lower extremity deformities.105 The patients with adult-onset ADHR had clinical presentations essentially identical to patients who present with TIO (none of the ADHR patients developed tumors). The molecular mechanisms for early-onset ADHR resembling XLH and late-onset ADHR resembling TIO are currently unknown. To date, all patients that have been described with delayed onset of clinically evident disease are female. In addition to these two groups, we found unaffected individuals who are carriers for the ADHR mutation and two patients who were treated for hypophosphatemia and rickets but later lost the Pi wasting defect.105 Thus, the clinical manifestations of ADHR are more variable than those observed in XLH.
To identify the gene for ADHR, the ADHR Consortium undertook a family-based positional cloning approach. A genomewide linkage scan in the large ADHR kindred described earlier demonstrated linkage to chromosome 12p13.3 (homologous to mouse chromosome 6).107 FGF23 was identified using exon prediction programs on genomic DNA sequence from the Human Genome Project.108 The FGF23 gene is composed of three coding exons and contains an open reading frame of 251 residues.108 The tissue with the highest FGF23 expression is bone, where FGF23 mRNA is observed in osteoblasts, osteocytes, flattened bone-lining cells, and osteoprogenitor cells.109 Quantitative RT-PCR showed that FGF23 mRNA was most highly expressed in long bone, followed by thymus, brain, and heart.110
Western blot analysis has demonstrated that wild-type FGF23 is secreted as a full-length 32-kD protein, as well as cleavage products of 20 kD (N-terminal) and 12 kD (C-terminal).110–112 Cleavage of FGF23 occurs within a subtilisin-like proprotein convertase (SPC) proteolytic site (176RXXR179/S180) that separates the conserved FGF-like N-terminal domain from the variable C-terminal tail (Fig. 6-3).
FIGURE 6-3 Model of FGF23 protein domains and effect of the ADHR mutations. FGF23 has a 24-residue signal peptide followed by residues 25 to 179 that comprise the conserved N-terminal FGF-like domain. The SPC-like site is interrupted by the ADHR mutations at R176 and R179 and divides the FGF-like domain from the variable C-terminal tail region. FGF23 undergoes glycosylation at three regions (denoted *), with the T178 most likely being the glycosylated residue that protects the mature protein from SPC degradation between R179 and S180 and is therefore critical for maintaining intact, active FGF23.
The ADHR mutations replace arginine (R) residues at FGF23 positions 176 or 179 with glutamine (Q) or tryptophan (W) within the SPC cleavage site, 176RXXR179/S180 (Table 6-2 and see Fig. 6-3).108,111,112 The SPCs are a family of serine proteases that process a wide variety of proteins including neuropeptides, peptide hormones, growth factors, membrane-bound receptors, blood coagulation factors, and plasma proteins.113 SPC substrates are cleaved C-terminal to the basic motif K/R-Xn-K/R, where Xn = 2, 4, or 6 residues of any amino acid.114,115 The SPCs, such as the furin protease, are largely expressed in the trans-Golgi network and possess similar but not exact substrate specificities. Following insertion of these mutations into wild-type FGF23, FGF23 secreted from mammalian cells was primarily produced as the full-length protein (32 kD) active polypeptide, as opposed to the 32-kD cleavage products typically observed for wild-type FGF23 expression.111 Peptide sequencing demonstrated that the 32-kD FGF23 form corresponded to full-length FGF23 after cleavage of the signal peptide (residues 25 to 251) and that the 12-kD isoform was the C-terminal portion of FGF23 downstream from the SPC cleavage site after R179 (residues 180 to 251) (see Fig. 6-3).112 As further evidence that FGF23 is processed intracellularly, the cleavage of wild-type FGF23 between R179/S180 is inhibited by a nonspecific SPC competitive inhibitor, Dec-RVKR-CMK, at concentrations between 25 and 50 µM.110,116 These studies show that the RXXR motif in FGF23 is central to its intracellular processing.
The SPC family is usually associated with the production of the active form of their substrate polypeptides. However, cleavage of FGF23 at the RXXR motif appears to be inactivating. In this regard, when full-length FGF23 or N-terminal (residues 25 to 179) and C-terminal (residues 180 to 251) fragments were injected into rodents, only the full-length protein lowered circulating phosphate concentrations.112 Since the full-length form of FGF23 induces hypophosphatemia, it is likely that the ADHR mutations increase the biological activity of FGF23 by stabilizing the full-length form and increasing its concentrations in the serum. Indeed, severely affected ADHR patients have increased circulating levels of FGF23.117
Tumor-Induced Osteomalacia
TIO is an acquired disorder of renal Pi wasting that is associated with tumors. Patients with TIO present with hypophosphatemia with inappropriately suppressed 1,25(OH)2D concentrations and elevated alkaline phosphatase levels.118 Osteomalacia is observed in bone biopsies. Clinical symptoms include gradual onset of muscle weakness, fatigue, and bone pain, especially from ankles, legs, hips, and back.118,119 Insufficiency fractures are common, and proximal muscle weakness can become severe enough for patients to require a wheelchair or become bed bound.118
The study of TIO introduced the idea for the existence of possible tumor-produced circulating factors, referred to as phosphatonins, that act upon the kidney to decrease Pi reabsorption.120,121 Support for these factors primarily comes from the knowledge that if the responsible tumor is surgically removed, the abnormalities in Pi wasting and vitamin D metabolism are rapidly corrected, as well as the fact that PTH, which decreases renal Pi reabsorption, is usually within normal ranges in TIO patients. Other studies have supported this hypothesis by showing that implantation of tumor tissue into nude mice resulted in increased urinary Pi excretion.122 To determine whether FGF23 could be involved in TIO as phosphatonin, five TIO tumors and several control tissues were tested by Northern blot for the presence of FGF23 transcripts, and it was determined that FGF23 mRNA was highly expressed in all of these tumors.123 Furthermore, FGF23 was present in a tumor lysate by Western blot analysis, with an anti–human FGF23 antibody.123
FGF23 was also independently identified in RNA from TIO tumors. Transcripts from tumors were isolated by differential selection using comparisons to mRNAs present in normal bone.124 The highly expressed transcripts were then subcloned, and the individual mRNAs were stably expressed in Chinese hamster ovary (CHO) cells, then injected into nude mice to form tumor masses.124 The cells that produced FGF23 recapitulated the TIO phenotype in vivo by causing hypophosphatemia, elevated alkaline phosphatase, and inappropriately low 1,25(OH)2D concentrations.124 In addition, the mice that received implanted cells also showed growth retardation, kyphosis, and osteomalacia. Further, there was marked decrease in the renal 1α(OH)ase. Both the biochemical and metabolic bone profiles were remarkably similar to those observed in TIO and ADHR patients. These experiments provided evidence that FGF23 was a phosphaturic substance and had dramatic effects on enzymes involved in vitamin D metabolism, and increased circulating FGF23 concentrations were consistent with the idea that FGF23 was at least in part responsible for the TIO phenotype. Serum FGF23 is elevated in patients with TIO,101,102 and tumors that cause TIO have a dramatic overexpression of FGF23 mRNA.123 Surgical resection of the tumor results in rapid decreases in serum FGF23.101
X-Linked Hypophosphatemic Rickets (OMIM No. 307800)
XLH is an X-linked dominant disorder and the most common form of heritable rickets.125 XLH is fully penetrant with variable severity. XLH patients present with laboratory findings that include hypophosphatemia with normocalcemia and inappropriately normal or low 1,25(OH)2D concentrations.125 Skeletal defects include lower-extremity deformities from rickets, bone pain, osteomalacia, fracture, and enthesopathy (calcification of the tendons and ligaments).125 It was determined by the Hyp Consortium that XLH is caused by inactivating mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome).126 Based upon sequence homology, PHEX encodes a protein that is a member of the M13 family of membrane-bound metalloproteases. Other members of this enzyme class include neutral endopeptidase (NEP) and endothelin-converting enzymes 1 and 2 (ECE-1 and ECE-2).126,127 This protease family is known to cleave small peptide hormones, therefore it is likely that PHEX has similar activity. Over 160 inactivating PHEX mutations have been described in XLH patients, including genomic variations that cause missense, nonsense, frameshift, and splicing changes (see PHEX Locus database: www.phexdb.mcgill.ca). Of note, although XLH is a renal Pi wasting disorder, PHEX shows the highest expression in bone cells such as osteoblasts, osteocytes, and odontoblasts in teeth, as well as lower expression in the parathyroid glands, lung, brain, and skeletal muscle but no expression in kidney.128 Taken together with the biochemical phenotype of XLH, PHEX protein homology and tissue expression are consistent with the hypothesis that PHEX interacts with small circulating factors outside of the kidney.
A valuable tool for the study of the pathophysiology of XLH has been the Hyp mouse, which has 3′ deletion in the Phex gene from intron 15 through the 3′ UTR129 and does not make a stable Phex transcript.128 This rodent model parallels the XLH phenotype, characterized by hypophosphatemia with inappropriately normal 1,25(OH)2D levels and normal serum calcium, as well as growth retardation and bone mineralization defects.130 Parabiosis studies between the Hyp mouse and a normal mouse pointed to the presence of a humoral factor, a phosphatonin, being transferred through the circulation of the Hyp mouse to the normal mouse to cause isolated renal Pi wasting.131 After the identification of PHEX/Phex, it was logically postulated that the enzyme may directly degrade a phosphaturic substance; however, recent studies suggest a more complex pathophysiology.
X-Linked Hypophosphatemic Rickets and Fibroblast Growth Factor-23: As described earlier, patients with XLH have overlapping phenotypes with ADHR patients. Because XLH results from a mutation in PHEX, which shares homology to a family of extracellular proteases, and ADHR arises from mutations in a protease cleavage site, it was logically hypothesized that circulating FGF23 would be cleaved and inactivated by PHEX. Thus, by mutational inactivation of PHEX in XLH, serum FGF23 concentrations would then elevate and cause renal Pi wasting. As described earlier, lending further support to this hypothesis were parabiosis studies between Hyp and normal mice, which pointed to the presence of a humoral phosphaturic factor in the Hyp mouse being transferred to the normal mouse. However, evidence to date has not supported a direct enzyme-substrate relationship between FGF23 and PHEX. In this regard, it was shown that recombinant PHEX did not cleave FGF23 but did cleave a positive control substrate.110 Furthermore, another report provided evidence that recombinant FGF23 was not cleaved by PHEX in cultured HEK293 cells coexpressing the proteins.116 This latter study expressed native FGF23 that was not epitope tagged to ensure that the additional residues did not cause conformational changes within FGF23 and interfere with potential PHEX activity.116
Several reports have established that FGF23 is elevated in many XLH patients.101,102,132 To understand the possible relationship between PHEX and FGF23, quantitative real-time RT-PCR was used to test Hyp bone for FGF23 mRNA concentrations versus wild-type bone. Interestingly, FGF23 mRNA in bone tissue from Hyp mice was elevated compared to levels present in control mice,110 and serum concentrations of FGF23 have been reported to be 10-fold higher in Hyp mice when compared to normal mice (our unpublished results and Ref. 133). This finding provides support for the idea that there is a cellular connection between inactive PHEX mutants (or lack of Phex expression in Hyp mice) and the up-regulation of FGF23 mRNA in bone cells. The elevated FGF23 mRNA levels may indicate that the increase in serum FGF23 in XLH patients is due to overproduction and secretion of FGF23 by skeletal cells, as opposed to the alternative hypothesis of a decreased rate of FGF23 degradation by cell surface proteases after secretion into the circulation. Although the interactions between FGF23 and PHEX are most likely indirect, the encoded proteins are coexpressed in osteoblasts and osteocytes.109,110,128 At present, the PHEX substrate and the mechanisms for phosphate sensing are unknown.
The current therapy for XLH, ADHR, and TIO includes oral replacement of phosphorus in combination with high-dose 1,25(OH)2D. This regimen “treats” XLH by increasing serum Pi concentrations and ameliorates much of the metabolic bone disease, but it does not directly “cure” the disorder by reversing the underlying molecular defects in kidney and in bone. In this regard, several studies have attempted to reverse the XLH phenotype. Transgenic expression of wild-type PHEX under the control of the bone-specific mouse pro-alpha(I) collagen gene134 and the osteocalcin (OG2)135 promoters on the Hyp background was undertaken. Interestingly, the defective mineralization of bone and teeth in the Hyp mice was partially resolved with PHEX under the regulation of the collagen promoter, and dry ash weight increased with the OG2 PHEX, indicating improved mineralization. However, the hypophosphatemia was not normalized in either study, indicating that expression of PHEX under the temporal regulation of an osteoblast-specific promoter is not sufficient to rescue the Hyp phenotype. Furthermore, expression of PHEX to levels observed in wild-type animals was not obtained in all studies. Importantly, a recent report of a transgenic model overexpressing PHEX in the Hyp mouse—using the human beta-actin promoter for directing expression in a wider tissue distribution (bone, skin, lung, muscle, heart)—resulted in similar results as the bone-specific promoter studies,136 further demonstrating that proper spatial-temporal expression of Phex is critical for normal mineral metabolism.
Treatment of X-Linked Hypophosphatemic Rickets: The current standard of care is combination therapy with phosphate and 1,25(OH)2D (calcitriol) or 1-hydroxyvitamin D3.137–139 Therapy is labor intensive, and patients and caregivers should understand this before initiating treatment. As in any medical encounter, patients should be fully informed about potential side effects of therapy.
The indications for treatment of adult patients are more controversial. There are no data to suggest that current treatment regimens prevent enthesopathy (calcifications of tendons and ligaments). Pseudofractures are common in moderately to severely affected adult XLH patients. Since pseudofractures are often painful, may lead to fracture, and generally respond well to treatment, we generally recommend medical therapy for patients with pseudofractures. XLH patients frequently complain of bone pain,121 presumably due to osteomalacia. Treatment lessens osteomalacia and bone pain, and it is therefore reasonable to treat patients with this complaint. Additionally, it is advisable to treat patients who have nonunion after fractures or osteotomies, because treatment may improve fracture healing. In light of the complexity of therapy, potential side effects (see later), and lack of increased risk of fracture in patients without pseudofractures,121 therapy is not recommended in asymptomatic patients who do not have pseudofractures.
Related posts:
Hereditary Disorders of The Skeleton
Disorders Of Calcification: Osteomalacia and Rickets
Bone Development and Remodeling
Malignancy-Associated Hypercalcemia and Medical Management
Parathyroid Hormone and Parathyroid Hormone–Related Peptide In The Regulation of Calcium Homeostasis and Bone Development
Bone Density and Imaging of Osteoporosis
