Hereditary hypophosphatemias: New genes in the bone–kidney axis (Review Article)
Dr Armando Luis Negri, Instituto de Investigaciones Metabólicas, Libertad 836 1 piso, Buenos Aires 1012, Argentina. Email: firstname.lastname@example.org
Hypophosphatemia due to isolated renal phosphate wasting is a genetically heterogeneous disease. Two new genes linked to two different forms of hereditary hypophosphatemias have recently been described. Autosomal recessive form of hypophosphatemic rickets was mapped to chromosome 4q21 and identified homozygous mutations in dentin matrix protein 1 (DMP1) gene, which encodes a non-collagenous bone matrix protein. Intact plasma levels of the phosphaturic protein FGF23 (fibroblast growth factor 23) were clearly elevated in some of the affected individuals, providing a possible explanation for the phosphaturia and inappropriately normal 1,25(OH)2D levels, and suggesting that DMP1 may regulate FGF23 expression. Hereditary hypophosphatemic rickets with hypercalciuria is another rare disorder of autosomal recessive inheritance. Affected individuals present with hypercalciuria due to increased serum 1,25-dihydroxyvitamin D levels and increased intestinal calcium absorption. The disease was mapped to a 1.6 Mbp region on chromosome 9q34, which contains SLC34A3, the gene encoding the renal sodium-phosphate cotransporter NaPi-IIc. This was the first demonstration that NaPi-IIc has a key role in the regulation of phosphate homeostasis. Thus, DMP1 and NaPi-IIc add two new members to the bone–kidney axis proposed since it was discovered that the first phosphatonin, FGF23, was of osteoblastic/osteocyte origin. This provides a mechanism for the skeleton to communicate with the kidney to coordinate the mineralization of extracelular matrix and the renal handling of phosphate.
Hypophosphatemia due to isolated renal phosphate wasting is a genetically heterogeneous disease. It may result from several hereditary disorders, including X-linked hypophosphatemic rickets (XLH),1 autosomal dominant hypophosphatemic rickets (ADHR),2–4 or a related disorder known as hypophosphatemic bone disease,5 or hereditary hypophosphatemic rickets with hypercalciuria (HHRH).6 XLH is the most common form, and is characterized by rachitic and osteomalacic bone disease, growth retardation hypophosphatemia with normal parathyroid hormone (PTH) levels and inappropriately normal 1,25-dihydroxyvitamin D concentrations for serum phosphorus levels.1,7 Inactivating mutations in the phosphate-regulating gene with homologies to endopeptidases on the X-chromosome (PHEX) has been found responsible for XLH.8 ADHR is a less common form of hypophosphatemic rickets with similar biochemical disturbances but, in contrast with XLH, with variable and incomplete penetrance.2–4 Fibroblast growth factor 23 (FGF23) missense mutations have been identified in several ADHR families.9
Mineralization of extracellular matrix and the renal handling of phosphate appear to be two interrelated processes, and some or a group of factors could provide a mechanism for the skeleton to communicate with the kidney to coordinate them. This bone–kidney axis originated in the phosphatonin concept:10 a phosphaturetic hormone that could control renal phosphate handling. When FGF23, the first phosphatonin, was discovered to be of osteoblastic/osteocyte origin, the bone–kidney axis was established. The search for new genes involved in phosphate regulation and bone matrix mineralization has been constant and has recently shed light on two new candidates in this bone–kidney axis.
DENTIN MATRIX PROTEIN 1
Lorenz-Depiereux et al. have recently mapped an autosomal recessive form of hypophosphatemia to chromosome 4q21 and identified homozygous mutations in dentin matrix protein 1 (DMP1), which encodes a non-collagenous bone matrix protein. Intact plasma levels of the phosphaturic protein FGF23 were clearly elevated in two of four affected individuals, providing a possible explanation for the phosphaturia and inappropriately normal 1,25(OH)2D levels, and suggesting that DMP1 may regulate FGF23 expression.11
Dentin matrix protein 1 is a member of the Small Integrin-Binding LIgand, N-linked Glycoprotein (SIBLING) family of secreted acidic extracellular glycophosphoproteins that also includes bone sialoprotein, dentin sialophosphoprotein, osteopontin and matrix extracellular phosphoglycoprotein (MEPE). It is highly expressed in mineralized tissues, specially in osteoblasts and osteocytes, the terminally differentiated cell comprising 90–95% of all bone cells. In vitro, DMP1 peptides can promote or inhibit mineralization, depending on the extent of phosphorylation, the peptide size and concentration. DMP1 deletion in mice results in a hypomineralized bone phenotype. There has been no demonstration that DMP1 affects renal phosphate handling directly. MEPE, another SIBLING related to DMP1, is elevated in Hypophophatemic mouse and other hypophosphatemic disorders. The administration of an acidic serine-aspartate-rich MEPE-associated motif (ASARM) peptide derived from MEPE causes phosphaturia and inhibits bone mineralization in mice, suggesting that MEPE also plays a role in phosphate homeostasis. Recent studies have found that MEPE binds specifically to PHEX in vitro, and inhibits PHEX enzyme activities in a dose-dependent manner. Long-term bone marrow stromal cell cultures supplemented with ASARM-PO peptide resulted in significant elevation of FGF23 transcripts and inhibition of mineralization. These findings suggest that MEPE inhibits mineralization and PHEX activity and leads to increased FGF23 production.12
Feng et al.13 investigated the potential of DMP1 not only to direct skeletal mineralization, but also to regulate phosphate homeostasis. Both DMP1-null mice and individuals with the newly identified disorder, autosomal recessive hypophosphatemic rickets (ARHR), manifest rickets and osteomalacia with isolated renal phosphate wasting associated with elevated FGF23 levels and normocalciuria. Mutational analyses in patients showed that ARHR in one family carried a mutation affecting the DMP1 start codon, and in a second family, carried a 7 bp deletion disrupting the highly conserved DMP1 C terminus. Mechanistic studies using DMP1-null mice demonstrated that absence of DMP1 results in defective osteocyte maturation and increased FGF23 expression, leading to pathological changes in bone mineralization. These findings suggest that DMP1 is another important member in the bone–kidney axis that is central to guiding proper mineral metabolism.13
The role of DMP1 in mineralization was also analysed by comparing bone mineral and matrix properties in DMP1-null female mice to heterozygous (HET) and wild-type (WT) controls by Fourier transform infrared (FTIR) imaging spectroscopy.14 The observed decreased mineral content in DMP1-null mice indicates a key role for DMP1 in bone mineralization. Indirect effects of DMP1 on other systems also determine the knockout (KO) phenotype.
To clarify the biological function of DMP1 protein on in vivo mineralization, this study analysed bone properties of DMP1-KO mice compared with HET and WT controls. Tibias from DMP1-KO and age-, sex-, and background-matched HET and WT mice were examined by FTIR imaging spectroscopy, histology and geometry performed by microcomputed tomography (muCT). The mineral–matrix ratios (spectroscopic parameter of relative mineral content) were significantly lower in DMP1-KO mice tibias compared with WT and HET controls at 4 and 16 weeks. The mineral crystallinity (crystal size/perfection) was significantly increased in DMP1-KO and HET mice relative to WT controls. Collagen cross-link ratios (a spectroscopic parameter related to the relative amounts of non-reducible/reducible collagen cross-links) in DMP1-KO mice were not significantly different from WT and HET controls. Cortical bone cross-sectional areas at 16 weeks were significantly reduced in the KO compared with controls. Maximum, minimum and polar cross-sectional moments of inertia were significantly lower in DMP1-KO than in HET mice also at 16 weeks. The histological analysis and muCT 3-D images suggested that DMP1-KO mice had osteomalacia. DMP1-KO mice had significantly lower ionic calcium and phosphate concentrations relative to WT controls, whereas in the HET, values for phosphate were equivalent, and calcium values were decreased relative to WT values. These results suggest that DMP1 has multiple roles in the regulation of postnatal mineralization through direct effects on mineral formation and crystal growth, and indirect effects on Ca × P concentrations and bone matrix turnover.14
SODIUM-DEPENDENT PHOSPHATE TRANSPORTER IIc (NaPi-IIc)
Members of the SLC34 gene family of solute carriers encode for three Na+-dependent phosphate (Pi) cotransporter proteins, two of which (NaPi-IIa/SLC34A1 and NaPi-IIc/SLC34A3) control renal reabsorption of Pi in the proximal tubule of mammals, whereas NaPi-IIb/SCLC34A2 mediates Pi transport in organs other than the kidney.15 The Pi transport mechanism has been extensively studied in heterologous expression systems, and structure-function studies have begun to reveal the intricacies of the transport cycle at the molecular level. Moreover, sequence differences between the three types of cotransporters have been exploited to obtain information about the molecular determinants of hormonal sensitivity and electrogenicity. Renal handling of Pi is regulated by hormonal and non-hormonal factors, and the changes in urinary excretion of Pi are almost invariably mirrored by changes in the apical expression of NaPi-IIa and NaPi-IIc in proximal tubules.15
The type IIa sodium/inorganic phosphate cotransporter is the key player in the renal handling of renal phosphate reabsorption: in mice, targeted disruption of Npt2 gene leads to phosphaturia and 80% loss of the Na/Pi cotransport rate.16
Homozygous mutants (Npt2(–/–)) exhibit increased urinary Pi excretion, hypophosphatemia, an appropriate elevation in the serum concentration of 1,25-dihydroxyvitamin D with attendant hypercalcemia, hypercalciuria and decreased serum PTH levels, and increased serum alkaline phosphatase activity. These biochemical features are typical of patients with HHRH. However, unlike HHRH patients, Npt2(–/–) mice do not have rickets or osteomalacia. At weaning, Npt2(–/–) mice have poorly developed trabecular bone and retarded secondary ossification, but, with increasing age, there is a dramatic reversal and eventual overcompensation of the skeletal phenotype.16 Associated with the increased urinary Pi excretion, disruption of type IIa sodium/inorganic phosphate cotransporter leads to an absence of response to the two most important regulatory mechanisms of proximal tubule phosphate reabsorption: inhibition induced by PTH, and stimulation produced by a low-phosphate diet.17
Sodium-dependent phosphate cotransport in renal proximal tubules is heterogeneous with respect to proximal tubular segmentation (S1 vs S3) and nephron generation (superficial vs juxtamedullary). Madjdpour et al.18 determined mRNA and protein expression of the Na/Pi-cotransporters NaPi-IIa and NaPi-IIc in laser-microdissected S1 and S3 segments of superficial and juxtamedullary nephrons. Expression of NaPi-IIa mRNA decreased axially in juxtamedullary nephrons. There was no effect of dietary Pi content on NaPi-lla mRNA expression in any proximal tubular segment. The abundance of the NaPi-IIa cotransporter in the brush-border membrane showed inter- and intranephron heterogeneity and increased in response to a low-Pi diet (5 days), suggesting that up-regulation of NaPi-lla occurs via post-transcriptional mechanisms. In contrast, NaPi-IIc mRNA and protein was up-regulated by the low-Pi diet in all nephron generations analysed.
Hereditary hypophosphatemic rickets with hypercalciuria is a rare disorder of autosomal recessive inheritance that was first described in a large consanguineous Bedouin kindred. HHRH is characterized by the presence of hypophosphatemia secondary to renal phosphate wasting, radiographic and/or histological evidence of rickets, limb deformities, muscle weakness and bone pain. HHRH is distinct from other forms of hypophosphatemic rickets in that affected individuals present with hypercalciuria due to increased serum 1,25-dihydroxyvitamin D levels and increased intestinal calcium absorption.
Bergwitz et al. have recently performed a genome-wide linkage scan combined with homozygosity mapping, using genomic DNA from a large consanguineous Bedouin kindred that included 10 patients who received the diagnosis of HHRH.19 The disease was mapped to a 1.6 Mbp region on chromosome 9q34, which contains SLC34A3, the gene encoding the renal sodium-phosphate cotransporter NaPi-IIc. Nucleotide sequence analysis revealed a homozygous single-nucleotide deletion (c.228delC) in this candidate gene in all individuals affected by HHRH. This mutation is predicted to truncate the NaPi-IIc protein in the first membrane-spanning domain, and thus likely results in a complete loss of function of this protein in individuals homozygous for c.228delC. In addition, compound HET missense and deletion mutations were found in three additional unrelated HHRH kindreds, which supports the conclusion that this disease is caused by SLC34A3 mutations affecting both alleles. This was the first demonstration that NaPi-IIc has a key role in the regulation of phosphate homeostasis.19 Individuals of the investigated kindreds who were HET for a SLC34A3 mutation frequently showed hypercalciuria, often in association with mild hypophosphatemia and/or elevations in 1,25-dihydroxyvitamin D levels. ‘Idiopathic’ hypercalciuria, which is present in 50% of patients with calcium nephrolithiasis, is characterized by hypercalciuria, normal serum calcium levels in the absence of other hypercalciuric conditions. The most probable mechanism of the hypercalciuria in this condition is increased intestinal calcium absorption, and controversy exists as to the cause of this high absorption rate. Calcitriol levels have been found elevated in many of these hypercalciuric patients, although this has not been a universal finding. So HET mutations in the NaPi-IIc transporter could be one of the causes of this frequently found disorder. In a study of 20 patients with urolithiasis or bone demineralization and persistent idiopathic hypophosphatemia associated with a decrease in maximal renal phosphate reabsorption, the coding region of the NaPi-IIa gene was sequenced.20 Only two patients, one with recurrent urolithiasis and the other with bone demineralization, were found to be HET for two distinct mutations in that transporter.
Using another approach (single nucleotide polymorphism array genotyping), Lorenz-Depiereux et al. also mapped the HHRH locus in two consanguineous families to the end of the long arm of chromosome 9.21 The candidate region has been shown to contain the sodium-phosphate cotransporter gene, SLC34A3, which is expressed in proximal tubular cells. Sequencing of this gene revealed disease-associated mutations in five families, including two frame-shift and one splice-site mutation. Loss of function of the SLC34A3 protein presumably results in a primary renal tubular defect and is compatible with the HHRH phenotype. These investigators also showed that the phosphaturic factor FGF23, which is increased in XLH and carries activating mutations in ADHR, is at normal or low-normal serum levels in the patients with HHRH, further supporting a primary renal defect as the cause of this disease. Identification of the gene mutated in a further form of hypophosphatemia adds to the understanding of phosphate homeostasis and may help elucidate the interaction of the proteins involved in this pathway.21
Another mutation analysis of exons and adjacent introns in the SLC34A3 gene was conducted in members of two unrelated families with HHRH.22 Two affected siblings in one family were homozygous for a 101 bp deletion in intron 9. Haplotype analysis of the SLC34A3 locus in the family showed that the two deletions are on different haplotypes. An unrelated individual with HHRH was a compound heterozygote for an 85 bp deletion in intron 10 and a G-to-A substitution at the last nucleotide in exon 7. The intron-9 deletion (and likely the other two mutations) identified in this study causes aberrant RNA splicing. Sequence analysis of the deleted regions revealed the presence of direct repeats of homologous sequences. This shows again that HHRH is caused by biallelic mutations in the SLC34A3 gene. Haplotype analysis suggests that the two intron-9 deletions arose independently. The identification of three independent deletions in introns 9 and 10 suggests that the SLC34A3 gene may be susceptible to unequal crossing over because of sequence misalignment during meiosis.22
Dentin matrix protein 1 (DMP1) and NaPi-IIc add two new members to the bone–kidney axis that provide a mechanism for the skeleton to communicate with the kidney to coordinate the mineralization of extracelular matrix and the renal handling of phosphate. Osteoblasts/osteocytes are well suited for coordinating systemic phosphate homeostasis and mineralization, because they express all of the implicated components of a possible bone–kidney axis, including PHEX, FGF23, MEPE and DMP1, as well as frizzled, FGF and PTH receptors. In addition, autocrine effects of phosphatonins on osteoblasts could regulate the production of extracellular matrix proteins that regulate mineralization. The kidney provides one of the effectors of the axis that regulates phosphate balance through the apical expression of NaPi-IIa and NaPi-IIc in proximal tubules. Central in this axis is FGF23, produced by osteoblasts, which has phosphaturic actions on the kidney and autocrine effects on osteoblasts to modulate the mineralization of bone, which is regulated in some way by PHEX, MEPE and DMP1. Proving the existence of this bone–kidney axis and defining its physiological role will require additional investigations.