An update on clinical presentation and responses to therapy of patients with hereditary hypophosphatemic rickets with hypercalciuria (HHRH).

Pathogenic variants in solute carrier family 34, member 3 (SLC34A3), the gene encoding the sodium-dependent phosphate cotransporter 2c (NPT2c), cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH). Here, we report a pooled analysis of clinical and laboratory records of 304 individuals from 145 kindreds, including 20 previously unreported HHRH kindreds, in which two novel SLC34A3 pathogenic variants were identified. Compound heterozygous/homozygous carriers show above 90% penetrance for kidney and bone phenotypes. The biochemical phenotype for heterozygous carriers is intermediate with decreased serum phosphate, tubular reabsorption of phosphate (TRP (%)), fibroblast growth factor 23, and intact parathyroid hormone, but increased serum 1,25-dihydroxy vitamin D, and urine calcium excretion causing idiopathic hypercalciuria in 38%, with bone phenotypes still observed in 23% of patients. Oral phosphate supplementation is the current standard of care, which typically normalizes serum phosphate. However, although in more than half of individuals this therapy achieves correction of hypophosphatemia it fails to resolve the other outcomes. The American College of Medical Genetics and Genomics score correlated with functional analysis of frequent SLC34A3 pathogenic variants in vitro and baseline disease severity. The number of mutant alleles and baseline TRP (%) were identified as predictors for kidney and bone phenotypes, baseline TRP (%) furthermore predicted response to therapy. Certain SLC34A3/NPT2c pathogenic variants can be identified with partial responses to therapy, whereas with some overlap, others present only with kidney phenotypes and a third group present only with bone phenotypes. Thus, our report highlights important novel clinical aspects of HHRH and heterozygous carriers, raises awareness to this rare group of disorders and can be a foundation for future studies urgently needed to guide therapy of HHRH.

Hereditary Hypophosphatemic Rickets with Hypercalciuria Presenting with Enthesopathy, Renal Cysts, and High Serum c-Terminal FGF23: Single-Center Experience and Systematic Review.

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare disorder of phosphate homeostasis. We describe a single-center experience of genetically proven HHRH families and perform systematic review phenotype-genotype correlation in reported biallelic probands and their monoallelic relatives. Detailed clinical, biochemical, radiological, and genetic data were retrieved from our center and a systematic review of Pub-Med and Embase databases for patients and relatives who were genetically proven. Total of nine subjects (probands:5) carrying biallelic SLC34A3 mutations (novel:2) from our center had a spectrum from rickets/osteomalacia to normal BMD, with hypophosphatemia and hypercalciuria in all. We describe the first case of genetically proven HHRH with enthesopathy. Elevated FGF23 in another patient with hypophosphatemia, iron deficiency anemia, and noncirrhotic periportal fibrosis led to initial misdiagnosis as tumoral osteomalacia. On systematic review of 58 probands (with biallelic SLC34A3 mutations; 35 males), early-onset HHRH and renal calcification were present in ~ 70% and late-onset HHRH in 10%. c.575C > T p.(Ser192Leu) variant occurred in 53% of probands without skeletal involvement. Among 110 relatives harboring monoallelic SLC34A3 mutation at median age 38 years, renal calcification, hypophosphatemia, high 1,25(OH)2D, and hypercalciuria were observed in ~30%, 22.3%, 40%, and 38.8%, respectively. Renal calcifications correlated with age but were similar across truncating and non-truncating variants. Although most relatives were asymptomatic for bone involvement, 6/12(50%) had low bone mineral density. We describe the first monocentric HHRH case series from India with varied phenotypes. In a systematic review, frequent renal calcifications and low BMD in relatives with monoallelic variants (HHRH trait) merit identification.

Multifocal heterotopic ossification in a man with germline variants of LIM Mineralization Protein-1 (LMP-1).

A 54-year-old man with a history of unimelic, post-traumatic multifocal heterotopic ossification (HO) and normal genetic analysis of ACVR1 and GNAS had variants of unknown significance (VUS) in PDLIM-7 (PDZ and LIM Domain Protein 7), the gene encoding LMP-1 (LIM Mineralization Protein-1), an intracellular protein involved in the bone morphogenetic protein (BMP) pathway signaling and ossification. In order to determine if the LMP-1 variants were plausibly responsible for the phenotype observed, a series of in vitro experiments were conducted. C2C12 cells were co-transfected with a BMP-responsive reporter as well as the LMP-1 wildtype (wt) construct or the LMP-1T161I or the LMP-1D181G constructs (herein designated as LMP-161 or LMP-181) corresponding to the coding variants detected in the patient. A significantly increased BMP-reporter activity was observed in LMP-161 or LMP-181 transfected cells compared to the wt cells. The LMP-181 variant exhibited BMP-reporter activity with a four-fold increase over the LMP-1 wt protein. Similarly, mouse pre-osteoblastic MC3T3 cells transfected with the patient's LMP-1 variants expressed higher levels of osteoblast markers both at mRNA and protein levels and preferentially mineralized when stimulated with recombinant BMP-2 compared to control cells. Presently, there are no pathogenic variants of LMP-1 known to induce HO in humans. Our findings suggest that the germline variants in LMP-1 detected in our patient are plausibly related to his multifocal HO (LMP1-related multifocal HO). Further observations will be required to firmly establish this gene-disease relationship.

Phosphorus bioaccessibility measured in four amino acid-based formulas using in-vitro batch digestion translates well into phosphorus bioavailability in mice.

OBJECTIVE: The aim of this study was to quantify the bioaccessibility of phosphorus from amino acid-based formulas (AAFs) under different digestive conditions. METHODS: We developed in-vitro batch digestion models with stomach digestion at different pH mimicking the normal digestive condition and conditions representing use of acid-suppressive medication. To validate bioaccessibility findings, we devised a low phosphorus murine model to test phosphorus bioavailability under compromised digestive conditions using proton pump inhibitors (PPIs) to neutralize stomach pH. RESULTS: In vitro phosphorus bioaccessibility of AAFs Neocate® Infant and Neocate Junior ranged between 57% and 65% under normal digestive conditions for infants (stomach pH 3.5) and between 38% and 46% under conditions that simulated bypass of stomach acidification, which is comparable to control diet and two EleCare® AAFs. In vivo bioavailability analysis showed that both Neocate formulas were able to normalize plasma phosphorus levels when administered to low phosphorus mice along with PPIs (control diet + PPI 8 ± 0.4; Neocate Infant 10.1 ± 0.9; Neocate Junior 9.2 ± 0.6; EleCare Infant 8.6 ± 0.4; EleCare Junior 8.7 ± 0.5; n = 8-10; P < 0.0001 versus baseline 3.4 ± 0.2 mg/dL). In comparison, plasma phosphorus levels remained lower on the low phosphorus diet (5.7 ± 0.2 mg/dL). Furthermore, urinary phosphorus/creatinine and intact fibroblast growth factor 23 were significantly lowered by low phosphorus diet. In contrast, intact parathyroid hormone and 1,25-dihydroxy vitamin D decreased and increased, respectively, and these parameters likewise normalized in mice administered AAFs. CONCLUSION: The present findings indicated that phosphorus bioaccessibility in the in-vitro batch digestion model translates well into phosphorus bioavailability in mice even under compromised digestive conditions that bypass gastric acidification.

Different elemental infant formulas show equivalent phosphorus and calcium bioavailability in healthy volunteers.

Retrospective chart reviews have reported hypophosphatemia associated with elemental formula use in infants and children with systemic disease involving multiple diagnoses. The present study aims to evaluate the bioavailability of phosphorus from 2 commercial elemental formulas and to test our hypothesis of bioequivalence of the 2 products in healthy volunteers receiving gastric acid-suppressive medication. A single-center, double-blind, randomized, cross-over study was conducted in healthy volunteers with esomeprazole-induced hypochlorhydria. After a standardized low phosphorus meal followed by overnight fasting, subjects consumed 1 gram of phosphorus in a single oral dose of 1217 kcal of Product A (Neocate) or Product B (Elecare). The alternate product was given following a 1-week washout period. Blood and urine were collected at baseline and different time-points for up to 6 hours after product consumption. Area-under-the-curve (AUC) and peak values (Cpeak) for serum phosphate and calcium and urinary creatinine-corrected phosphate and calcium were assessed for bioequivalence of Products A and B. Results show that the geometric mean ratio (GMR) and 90% CI for serum phosphate were 1.041 (0.998-1.086) and 1.020 (0.963-1.080) for AUC0-360 and Cpeak, respectively, meeting the predetermined criteria for bioequivalence. Urinary creatinine-corrected phosphate followed a similar pattern after intake of Product A and B, but did not reach bioequivalence criteria (GMR: AUC70-370 = 1.105 (0.918-1.330); Cpeak = 1.182 (1.040-1.343)). Serum calcium concentrations (GMR: AUC0-360 = 1.002 (0.996-1.009); Cpeak = 0.991 (0.983-0.999)) and urinary creatinine-corrected calcium excretion (GMR: AUC70-370 = 1.117 (1.023-1.219); Cpeak = 1.157 (1.073-1.247)) demonstrated bioequivalence of the products. In conclusion, both elemental infant formulas showed equivalent serum phosphorus and calcium bioavailability in healthy volunteers even if combined with treatment with acid-suppressive medication.

FGF23 signalling and physiology.

Fibroblast growth factor 23 (FGF23) is a phosphotropic hormone that belongs to a subfamily of endocrine FGFs with evolutionarily conserved functions in worms and fruit flies. FAM20C phosphorylates FGF23 post-translationally, targeting it to proteolysis through subtilisin-like proprotein convertase FURIN, resulting in secretion of FGF23 fragments. O-glycosylation of FGF23 through GALNT3 appears to prevent proteolysis, resulting in secretion of biologically active intact FGF23. In the circulation, FGF23 may undergo further processing by plasminogen activators. Crystal structures show that the ectodomain of the cognate FGF23 receptor FGFR1c binds with the ectodomain of the co-receptor alpha-KLOTHO. The KLOTHO-FGFR1c double heterodimer creates a high-affinity binding site for the FGF23 C-terminus. The topology of FGF23 deviates from that of paracrine FGFs, resulting in poor affinity for heparan sulphate, which may explain why FGF23 diffuses freely in the bone matrix to enter the bloodstream following its secretion by cells of osteoblastic lineage. Intact FGF23 signalling by this canonical pathway activates FRS2/RAS/RAF/MEK/ERK1/2. It reduces serum phosphate by inhibiting 1,25-dihydroxyvitamin D synthesis, suppressing intestinal phosphate absorption, and by downregulating the transporters NPT2a and NPT2c, suppressing phosphate reabsorption in the proximal tubules. The physiological role of FGF23 fragments, which may be inhibitory, remains unclear. Pharmacological and genetic activation of canonical FGF23 signalling causes hypophosphatemic disorders, while its inhibition results in hyperphosphatemic disorders. Non-canonical FGF23 signalling through binding and activation of FGFR3/FGFR4/calcineurin/NFAT in an alpha-KLOTHO-independent fashion mainly occurs at extremely elevated circulating FGF23 levels and may contribute to mortality due to cardiovascular disease and left ventricular hypertrophy in chronic kidney disease.

Importance of Dietary Phosphorus for Bone Metabolism and Healthy Aging.

Inorganic phosphate (Pi) plays a critical function in many tissues of the body: for example, as part of the hydroxyapatite in the skeleton and as a substrate for ATP synthesis. Pi is the main source of dietary phosphorus. Reduced bioavailability of Pi or excessive losses in the urine causes rickets and osteomalacia. While critical for health in normal amounts, dietary phosphorus is plentiful in the Western diet and is often added to foods as a preservative. This abundance of phosphorus may reduce longevity due to metabolic changes and tissue calcifications. In this review, we examine how dietary phosphorus is absorbed in the gut, current knowledge about Pi sensing, and endocrine regulation of Pi levels. Moreover, we also examine the roles of Pi in different tissues, the consequences of low and high dietary phosphorus in these tissues, and the implications for healthy aging.

Description of a novel SLC34A3.c.671delT mutation causing hereditary hypophosphatemic rickets with hypercalciuria in two adolescent boys and response to recombinant human growth hormone.

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is an autosomal recessive disorder characterized by hypophosphatemia, rickets, hyperphosphaturia, elevated 1,25(OH)2D, and hypercalciuria. Mutations in SLC34A3, the gene encoding the sodium-dependent cotransporter NPT2c, have previously been described as a cause of HHRH. Here, we describe two male siblings with rickets and hypercalciuric nephrolithiasis born to unrelated parents, and their response to oral phosphate supplementation and growth hormone therapy. Whole exome sequencing of the oldest brother, and polymerase chain reaction and Sanger sequence analysis of the identified SLC34A3 mutations, was performed for confirmation and to evaluate his siblings and parents. Serum and urine biochemical parameters of mineral homeostasis before and after therapy were evaluated. Whole exome sequencing analysis identified a previously reported heterozygous deletion SLC34A3.g.2259-2359del101bp on the maternal allele, and a novel heterozygous single nucleotide deletion SLC34A3.c.671delT on the paternal allele of the two affected brothers. The parents and the unaffected brother are heterozygous carriers. Recombinant human growth hormone (rHGH) plus oral phosphate in one affected brother improved the renal phosphate leak and resulted in accelerated linear growth superior to that seen with oral phosphate supplementation alone in the other affected brother. Our case study is the first to demonstrate that rHGH can be considered in addition to oral supplementation with phosphorus to improve linear growth in patients with this disorder, and suggests that renal phosphate reabsorption in response to rHGH is NPT2c-independent.

Slc20a1/Pit1 and Slc20a2/Pit2 are essential for normal skeletal myofiber function and survival.

Low blood phosphate (Pi) reduces muscle function in hypophosphatemic disorders. Which Pi transporters are required and whether hormonal changes due to hypophosphatemia contribute to muscle function is unknown. To address these questions we generated a series of conditional knockout mice lacking one or both house-keeping Pi transporters Pit1 and Pit2 in skeletal muscle (sm), using the postnatally expressed human skeletal actin-cre. Simultaneous conditional deletion of both transporters caused skeletal muscle atrophy, resulting in death by postnatal day P13. smPit1-/-, smPit2-/- and three allele mutants are fertile and have normal body weights, suggesting a high degree of redundance for the two transporters in skeletal muscle. However, these mice show a gene-dose dependent reduction in running activity also seen in another hypophosphatemic model (Hyp mice). In contrast to Hyp mice, grip strength is preserved. Further evaluation of the mechanism shows reduced ERK1/2 activation and stimulation of AMP kinase in skeletal muscle from smPit1-/-; smPit2-/- mice consistent with energy-stress. Similarly, C2C12 myoblasts show a reduced oxygen consumption rate mediated by Pi transport-dependent and ERK1/2-dependent metabolic Pi sensing pathways. In conclusion, we here show that Pit1 and Pit2 are essential for normal myofiber function and survival, insights which may improve management of hypophosphatemic myopathy.

Transgenic mouse model for conditional expression of influenza hemagglutinin-tagged human SLC20A1/PIT1.

To further investigate the role of the phosphate (Pi) transporter PIT1 in Pi homeostasis and tissue mineralization, we developed a transgenic mouse expressing the C-terminal influenza hemagglutinin (HA) epitope-tagged human PIT1 transporter under control of the cytomegalovirus/chicken beta actin/rabbit beta-globin gene (CAG) promotor and a loxP-stop-loxP (LSL) cassette permitting conditional activation of transgene expression (LSL-HA-hPITtg/+). For an initial characterization of this conditional mouse model, germline excision of the LSL cassette was performed to induce expression of the transgene in all mouse tissues (HA-hPIT1tg/+). Recombination was confirmed using genomic DNA obtained from blood samples of these mice. Furthermore, expression of HA-hPIT1 was found to be at least 10-fold above endogenous mouse Pit1 in total RNA isolated from multiple tissues and from cultured primary calvaria osteoblasts (PCOB) estimated by semi-quantitative RT-PCR. Robust expression of the HA-hPIT1 protein was also observed upon immunoblot analysis in most tissues and permits HA-mediated immunoprecipitation of the transporter. Characterization of the phenotype of HA-hPIT1tg/+ mice at 80 days of age when fed a standard chow (0.7% Pi and 1% calcium) showed elevated plasma Pi, but normal plasma iPTH, iFGF23, serum calcium, BUN, 1,25-dihydroxy vitamin D levels and urine Pi, calcium and protein excretion when compared to WT littermates. Likewise, no change in bone mineral density was observed upon uCT analysis of the distal femur obtained from these mice. In conclusion, heterozygous overexpression of HA-hPIT1 is compatible with life and causes hyperphosphatemia while bone and mineral metabolism of these mice are otherwise normal.

Endocrine regulation of MFS2 by branchless controls phosphate excretion and stone formation in Drosophila renal tubules.

How inorganic phosphate (Pi) homeostasis is regulated in Drosophila is currently unknown. We here identify MFS2 as a key Pi transporter in fly renal (Malpighian) tubules. Consistent with its role in Pi excretion, we found that dietary Pi induces MFS2 expression. This results in the formation of Malpighian calcium-Pi stones, while RNAi-mediated knockdown of MFS2 increases blood (hemolymph) Pi and decreases formation of Malpighian tubule stones in flies cultured on high Pi medium. Conversely, microinjection of adults with the phosphaturic human hormone fibroblast growth factor 23 (FGF23) induces tubule expression of MFS2 and decreases blood Pi. This action of FGF23 is blocked by genetic ablation of MFS2. Furthermore, genetic overexpression of the fly FGF branchless (bnl) in the tubules induces expression of MFS2 and increases Malpighian tubule stones suggesting that bnl is the endogenous phosphaturic hormone in adult flies. Finally, genetic ablation of MFS2 increased fly life span, suggesting that Malpighian tubule stones are a key element whereby high Pi diet reduces fly longevity previously reported by us. In conclusion, MFS2 mediates excretion of Pi in Drosophila, which is as in higher species under the hormonal control of FGF-signaling.

Hereditary hypophosphatemic rickets with hypercalciuria: pathophysiology, clinical presentation, diagnosis and therapy.

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH; OMIM: 241530) is a rare autosomal recessive disorder with an estimated prevalence of 1:250,000 that was originally described by Tieder et al. Individuals with HHRH carry compound-heterozygous or homozygous (comp/hom) loss-of-function mutations in the sodium-phosphate co-transporter NPT2c. These mutations result in the development of urinary phosphate (Pi) wasting and hypophosphatemic rickets, bowing, and short stature, as well as appropriately elevated 1,25(OH)2D levels, which sets this fibroblast growth factor 23 (FGF23)-independent disorder apart from the more common X-linked hypophosphatemia. The elevated 1,25(OH)2D levels in turn result in hypercalciuria due to enhanced intestinal calcium absorption and reduced parathyroid hormone (PTH)-dependent calcium-reabsorption in the distal renal tubules, leading to the development of kidney stones and/or nephrocalcinosis in approximately half of the individuals with HHRH. Even heterozygous NPT2c mutations are frequently associated with isolated hypercalciuria (IH), which increases the risk of kidney stones or nephrocalcinosis threefold in affected individuals compared with the general population. Bone disease is generally absent in individuals with IH, in contrast to those with HHRH. Treatment of HHRH and IH consists of monotherapy with oral Pi supplements, while active vitamin D analogs are contraindicated, mainly because the endogenous 1,25(OH)2D levels are already elevated but also to prevent further worsening of the hypercalciuria. Long-term studies to determine whether oral Pi supplementation alone is sufficient to prevent renal calcifications and bone loss, however, are lacking. It is also unknown how therapy should be monitored, whether secondary hyperparathyroidism can develop, and whether Pi requirements decrease with age, as observed in some FGF23-dependent hypophosphatemic disorders, or whether this can lead to osteoporosis.

Role of phosphate sensing in bone and mineral metabolism.

Inorganic phosphate (Pi) is essential for signal transduction and cell metabolism, and is also an essential structural component of the extracellular matrix of the skeleton. Pi is sensed in bacteria and yeast at the plasma membrane, which activates intracellular signal transduction to control the expression of Pi transporters and other genes that control intracellular Pi levels. In multicellular organisms, Pi homeostasis must be maintained in the organism and at the cellular level, requiring an endocrine and metabolic Pi-sensing mechanism, about which little is currently known. This Review will discuss the metabolic effects of Pi, which are mediated by Pi transporters, inositol pyrophosphates and SYG1-Pho81-XPR1 (SPX)-domain proteins to maintain cellular phosphate homeostasis in the musculoskeletal system. In addition, we will discuss how Pi is sensed by the human body to regulate the production of fibroblast growth factor 23 (FGF23), parathyroid hormone and calcitriol to maintain serum levels of Pi in a narrow range. New findings on the crosstalk between iron and Pi homeostasis in the regulation of FGF23 expression will also be outlined. Mutations in components of these metabolic and endocrine phosphate sensors result in genetic disorders of phosphate homeostasis, cardiomyopathy and familial basal ganglial calcifications, highlighting the importance of this newly emerging area of research.

Intraperitoneal pyrophosphate treatment reduces renal calcifications in Npt2a null mice.

Mutations in the proximal tubular sodium-dependent phosphate co-transporters NPT2a and NPT2c have been reported in patients with renal stone disease and nephrocalcinosis, however the relative contribution of genotype, dietary calcium and phosphate, and modifiers of mineralization such as pyrophosphate (PPi) to the formation of renal mineral deposits is unclear. In the present study, we used Npt2a-/- mice to model the renal calcifications observed in these disorders. We observed elevated urinary excretion of PPi in Npt2a-/- mice when compared to WT mice. Presence of two hypomorphic Extracellular nucleotide pyrophosphatase phosphodiesterase 1 (Enpp1asj/asj) alleles decreased urine PPi and worsened renal calcifications in Npt2a-/- mice. These studies suggest that PPi is a thus far unrecognized factor protecting Npt2a-/- mice from the development of renal mineral deposits. Consistent with this conclusion, we next showed that renal calcifications in these mice can be reduced by intraperitoneal administration of sodium pyrophosphate. If confirmed in humans, urine PPi could therefore be of interest for developing new strategies to prevent the nephrocalcinosis and nephrolithiasis seen in phosphaturic disorders.

Response of Npt2a knockout mice to dietary calcium and phosphorus.

Mutations in the renal sodium-dependent phosphate co-transporters NPT2a and NPT2c have been reported in patients with renal stone disease and nephrocalcinosis, but the relative contribution of genotype, dietary calcium and phosphate to the formation of renal mineral deposits is unclear. We previously reported that renal calcium phosphate deposits persist and/or reappear in older Npt2a-/- mice supplemented with phosphate despite resolution of hypercalciuria while no deposits are seen in wild-type (WT) mice on the same diet. Addition of calcium to their diets further increased calcium phosphate deposits in Npt2a-/-, but not WT mice. The response of PTH to dietary phosphate of Npt2a-/- was blunted when compared to WT mice and the response of the urinary calcium x phosphorus product to the addition of calcium and phosphate to the diet of Npt2a-/- was increased. These finding suggests that Npt2a-/- mice respond differently to dietary phosphate when compared to WT mice. Further evaluation in the Npt2a-/- cohort on different diets suggests that urinary calcium excretion, plasma phosphate and FGF23 levels appear to be positively correlated to renal mineral deposit formation while urine phosphate levels and the urine anion gap, an indirect measure of ammonia excretion, appear to be inversely correlated. Our observations in Npt2a-/- mice, if confirmed in humans, may be relevant for the optimization of existing and the development of novel therapies to prevent nephrolithiasis and nephrocalcinosis in human carriers of NPT2a and NPT2c mutations.

Impaired urinary osteopontin excretion in Npt2a-/- mice.

Mutations in the renal sodium-dependent phosphate cotransporters NPT2a and NPT2c have been reported in patients with renal stone disease and nephrocalcinosis. Oral phosphate supplementation is currently thought to reduce risk by reversing the hypercalciuria, but the exact mechanism remains unclear and the relative contribution of modifiers of mineralization such as osteopontin (Opn) to the formation of renal mineral deposits in renal phosphate wasting disorders has not been studied. We observed a marked decrease of renal gene expression and urinary excretion of Opn in Npt2a-/- mice, a mouse model of these disorders, at baseline. Following supplementation with phosphate Opn gene expression was restored to wild-type levels in Npt2a-/- mice; however, urine excretion of the protein remained low. To further investigate the role of Opn, we used a double-knockout strategy, which provides evidence that loss of Opn worsens the nephrocalcinosis and nephrolithiasis observed in these mice on a high-phosphate diet. These studies suggest that impaired Opn gene expression and urinary excretion in Npt2a-/- mice may be an additional risk factor for nephrolithiasis, and normalizing urine Opn levels may improve the therapy of phosphaturic disorders.

Hypophosphatemia promotes lower rates of muscle ATP synthesis.

Hypophosphatemia can lead to muscle weakness and respiratory and heart failure, but the mechanism is unknown. To address this question, we noninvasively assessed rates of muscle ATP synthesis in hypophosphatemic mice by using in vivo saturation transfer [31P]-magnetic resonance spectroscopy. By using this approach, we found that basal and insulin-stimulated rates of muscle ATP synthetic flux (VATP) and plasma inorganic phosphate (Pi) were reduced by 50% in mice with diet-induced hypophosphatemia as well as in sodium-dependent Pi transporter solute carrier family 34, member 1 (NaPi2a)-knockout (NaPi2a-/-) mice compared with their wild-type littermate controls. Rates of VATP normalized in both hypophosphatemic groups after restoring plasma Pi concentrations. Furthermore, VATP was directly related to cellular and mitochondrial Pi uptake in L6 and RC13 rodent myocytes and isolated muscle mitochondria. Similar findings were observed in a patient with chronic hypophosphatemia as a result of a mutation in SLC34A3 who had a 50% reduction in both serum Pi content and muscle VATP After oral Pi repletion and normalization of serum Pi levels, muscle VATP completely normalized in the patient. Taken together, these data support the hypothesis that decreased muscle ATP synthesis, in part, may be caused by low blood Pi concentrations, which may explain some aspects of muscle weakness observed in patients with hypophosphatemia.-Pesta, D. H., Tsirigotis, D. N., Befroy, D. E., Caballero, D., Jurczak, M. J., Rahimi, Y., Cline, G. W., Dufour, S., Birkenfeld, A. L., Rothman, D. L., Carpenter, T. O., Insogna, K., Petersen, K. F., Bergwitz, C., Shulman, G. I. Hypophosphatemia promotes lower rates of muscle ATP synthesis.

Mutations in SLC34A3/NPT2c are associated with kidney stones and nephrocalcinosis.

Compound heterozygous and homozygous (comp/hom) mutations in solute carrier family 34, member 3 (SLC34A3), the gene encoding the sodium (Na(+))-dependent phosphate cotransporter 2c (NPT2c), cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a disorder characterized by renal phosphate wasting resulting in hypophosphatemia, correspondingly elevated 1,25(OH)2 vitamin D levels, hypercalciuria, and rickets/osteomalacia. Similar, albeit less severe, biochemical changes are observed in heterozygous (het) carriers and indistinguishable from those changes encountered in idiopathic hypercalciuria (IH). Here, we report a review of clinical and laboratory records of 133 individuals from 27 kindreds, including 5 previously unreported HHRH kindreds and two cases with IH, in which known and novel SLC34A3 mutations (c.1357delTTC [p.F453del]; c.G1369A [p.G457S]; c.367delC) were identified. Individuals with mutations affecting both SLC34A3 alleles had a significantly increased risk of kidney stone formation or medullary nephrocalcinosis, namely 46% compared with 6% observed in healthy family members carrying only the wild-type SLC34A3 allele (P=0.005) or 5.64% in the general population (P<0.001). Renal calcifications were also more frequent in het carriers (16%; P=0.003 compared with the general population) and were more likely to occur in comp/hom and het individuals with decreased serum phosphate (odds ratio [OR], 0.75, 95% confidence interval [95% CI], 0.59 to 0.96; P=0.02), decreased tubular reabsorption of phosphate (OR, 0.41; 95% CI, 0.23 to 0.72; P=0.002), and increased serum 1,25(OH)2 vitamin D (OR, 1.22; 95% CI, 1.05 to 1.41; P=0.008). Additional studies are needed to determine whether these biochemical parameters are independent of genotype and can guide therapy to prevent nephrocalcinosis, nephrolithiasis, and potentially, CKD.