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Hereditary hypophosphatemic rickets with hypercalciuria is an autosomal recessive phosphate-wasting disorder, associated with kidney and skeletal pathologies, which is caused by pathogenic variants of SLC34A3. In this issue, Zhu et al. describe a pooled analysis of 304 individuals carrying SLC34A3 variants. Their study underscores the complexity of hereditary hypophosphatemic rickets with hypercalciuria, as kidney and bone phenotypes generally do not coexist, heterozygous carriers of SLC34A3 variants also can be affected, and the response to oral phosphate supplementation is dependent on the genetic status., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

The kidney controls systemic inorganic phosphate (Pi) levels by adapting reabsorption to Pi intake. Renal Pi reabsorption is mostly mediated by sodium-phosphate cotransporters NaPi-IIa (SLC34A1) and NaPi-IIc (SLC34A3) that are tightly controlled by various hormones including parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23). PTH and FGF23 rise in response to Pi intake and decrease NaPi-IIa and NaPi-IIc brush border membrane abundance enhancing phosphaturia. Phosphaturia and transporter regulation occurs even in the absence of PTH and FGF23 signaling. The calcium-sensing receptor (CaSR) regulates PTH and FGF23 secretion, and may also directly affect renal Pi handling. Here, we combined pharmacological and genetic approaches to examine the role of the CaSR in the acute phosphaturic response to Pi loading. Animals pretreated with the calcimimetic cinacalcet were hyperphosphatemic, had blunted PTH levels upon Pi administration, a reduced Pi-induced phosphaturia, and no Pi-induced NaPi-IIa downregulation. The calcilytic NPS-2143 exaggerated the PTH response to Pi loading but did not abolish Pi-induced downregulation of NaPi-IIa. In mice with a dominant inactivating mutation in the Casr ( Casr BCH002 ), baseline NaPi-IIa expression was higher, whereas downregulation of transporter expression was blunted in double Casr BCH002 /PTH knockout (KO) transgenic animals. Thus, in response to an acute Pi load, acute modulation of the CaSR affects the endocrine and renal response, whereas chronic genetic inactivation, displays only subtle differences in the downregulation of NaPi-IIa and NaPi-IIc renal expression. We did not find evidence that the CaSR impacts on the acute renal response to oral Pi loading beyond its role in regulating PTH secretion. NEW & NOTEWORTHY Consumption of phosphate-rich diets causes an adaptive response of the body leading to the urinary excretion of phosphate. The underlying mechanisms are still poorly understood. Here, we examined the role of the calcium-sensing receptor (CaSR) that senses both calcium and phosphate. We confirmed that the receptor increases the secretion of parathyroid hormone involved in stimulating urinary phosphate excretion. However, we did not find any evidence for a role of the receptor beyond this function.

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., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Urinary stone disease (USD) is a major health burden affecting over 10% of the United Kingdom population. While stone disease is associated with lifestyle, genetic factors also strongly contribute. Common genetic variants at multiple loci from genome-wide association studies account for 5% of the estimated 45% heritability of the disorder. Here, we investigated the extent to which rare genetic variation contributes to the unexplained heritability of USD. Among participants of the United Kingdom 100,000-genome project, 374 unrelated individuals were identified and assigned diagnostic codes indicative of USD. Whole genome gene-based rare variant testing and polygenic risk scoring against a control population of 24,930 ancestry-matched controls was performed. We observed (and replicated in an independent dataset) exome-wide significant enrichment of monoallelic rare, predicted damaging variants in the SLC34A3 gene for a sodium-dependent phosphate transporter that were present in 5% cases compared with 1.6% of controls. This gene was previously associated with autosomal recessive disease. The effect on USD risk of having a qualifying SLC34A3 variant was greater than that of a standard deviation increase in polygenic risk derived from GWAS. Addition of the rare qualifying variants in SLC34A3 to a linear model including polygenic score increased the liability-adjusted heritability from 5.1% to 14.2% in the discovery cohort. We conclude that rare variants in SLC34A3 represent an important genetic risk factor for USD, with effect size intermediate between the fully penetrant rare variants linked with Mendelian disorders and common variants associated with USD. Thus, our findings explain some of the heritability unexplained by prior common variant genome-wide association studies., (Copyright © 2023 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Studies addressing homeostasis of inorganic phosphate (Pi) are mostly restricted to murine models. Data provided by genetically modified mice suggest that renal Pi reabsorption is primarily mediated by the Na + /Pi cotransporter NaPi-IIa/Slc34a1, whereas the contribution of NaPi-IIc/Slc34a3 in adult animals seems negligible. However, mutations in both cotransporters associate with hypophosphatemic syndromes in humans, suggesting major inter-species heterogeneity. Urinary extracellular vesicles (UEV) have been proposed as an alternative source to analyse the intrinsic expression of renal proteins in vivo. Here, we analyse in rats whether the protein abundance of renal Pi transporters in UEV correlates with their renal content. For that, we compared the abundance of NaPi-IIa and NaPi-IIc in paired samples from kidneys and UEV from rats fed acutely and chronically on diets with low or high Pi. In renal brush border membranes (BBM) NaPi-IIa was detected as two fragments corresponding to the full-length protein and to a proteolytic product, whereas NaPi-IIc migrated as a single full-length band. The expression of NaPi-IIa (both fragments) in BBM adapted to acute as well to chronic changes of dietary Pi, whereas adaptation of NaPi-IIc was only detected in response to chronic administration. Both transporters were detected in UEV as well. UEV reflected the renal adaptation of the NaPi-IIa proteolytic fragment (but not the full-length protein) upon chronic but not acute dietary changes, while also reproducing the chronic regulation of NaPi-IIc. Thus, the composition of UEV reflects only partially changes in the expression of NaPi-IIa and NaPi-IIc at the BBM triggered by dietary Pi., (© 2022. The Author(s).)

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) represents an FGF23-independent disease caused by biallelic variants in the solute carrier family 34-member 3 (SLC34A3) gene. HHRH is characterized by chronic hypophosphatemia and an increased risk for nephrocalcinosis and rickets/osteomalacia, muscular weakness, and secondary limb deformity. Biochemical changes, but no relevant skeletal changes, have been reported for heterozygous SLC34A3 carriers. Therefore, we assessed the characteristics of individuals with biallelic and monoallelic SLC34A3 variants. In 8 index patients and 5 family members, genetic analysis was performed using a custom gene panel. The skeletal assessment comprised biochemical parameters, areal bone mineral density (aBMD), and bone microarchitecture. Pathogenic SLC34A3 variants were revealed in 7 of 13 individuals (2 homozygous, 5 heterozygous), whereas 3 of 13 carried monoallelic variants of unknown significance. Whereas both homozygous individuals had nephrocalcinosis, only one displayed a skeletal phenotype consistent with HHRH. Reduced to low-normal phosphate levels, decreased tubular reabsorption of phosphate (TRP), and high-normal to elevated values of 1,25-OH 2 -D 3 accompanied by normal cFGF23 levels were revealed independently of mutational status. Interestingly, individuals with nephrocalcinosis showed significantly increased calcium excretion and 1,25-OH 2 -D 3 levels but normal phosphate reabsorption. Furthermore, aBMD Z-score <-2.0 was revealed in 4 of 8 heterozygous carriers, and HR-pQCT analysis showed a moderate decrease in structural parameters. Our findings highlight the clinical relevance also of monoallelic SLC34A3 variants, including their potential skeletal impairment. Calcium excretion and 1,25-OH 2 -D 3 levels, but not TRP, were associated with nephrocalcinosis. Future studies should investigate the effects of distinct SLC34A3 variants and optimize treatment and monitoring regimens to prevent nephrocalcinosis and skeletal deterioration. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)., (© 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).)

Mutations in CYP24A1 (vitamin D 24-hydroxylase) and SLC34A1 (renal phosphate transporter NPT2a) cause autosomal recessive Infantile Hypercalcemia type 1 and 2, illustrating links between vitamin D and phosphate metabolism. Patients may present with hypercalciuria and alternate between chronic phases with normal serum calcium but inappropriately high 1,25-(OH) 2 D and appropriately low PTH, and acute phases with hypercalcemia with suppressed PTH. Mutations in SLC34A3 and SLC9A3R1 have been associated with phosphate wasting without hypercalcemia. The aims of this study were to evaluate the frequency of mutations in these genes in patients with a medical history suggestive of CYP24A1 mutation to search for a specific pattern. Using next generation sequencing, we screened for mutations in 185 patients with PTH levels < 20 pg/mL, hypercalcemia and/or hypercalciuria, and relatives. Twenty-eight (15%) patients harbored biallelic mutations in CYP24A1 (25) and SLC34A3 (3), mostly associated with renal disease (lithiasis, nephrocalcinosis) (86%). Hypophosphatemia was found in 7 patients with biallelic mutations in CYP24A1 and a normal phosphatemia was reported in 2 patients with biallelic mutations in SLC34A3 . Rare variations in SLC34A1 and SLC34A3 were mostly of uncertain significance. Fifteen patients (8%) carried only one heterozygous mutation. Heterozygous relatives carrying SLC34A1 or SLC34A3 variation may present with biochemical changes in mineral metabolism. Two patients' genotype may suggest digenism (heterozygous variations in different genes). No variation was found in SLC9A3R1. As no specific pattern can be found, patients with medical history suggestive of CYP24A1 mutation should benefit from SLC34A1 and SLC34A3 analysis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Molin, Lemoine, Kaufmann, Breton, Nowoczyn, Ballandonne, Coudray, Mittre, Richard, Ryckwaert, Lavillaureix, Jones, Bacchetta and Kottler.)

Context: Idiopathic infantile hypercalcemia (IIH), an uncommon disorder characterized by elevated serum concentrations of 1,25 dihydroxyvitamin D (1,25(OH)2D) and low parathyroid hormone (PTH) levels, may present with mild to severe hypercalcemia during the first months of life. Biallelic variants in the CYP24A1 or SLC34A1 genes are associated with severe IIH. Little is known about milder forms., Objective: This work aims to characterize the genetic associations and biochemical profile of mild IIH., Methods: This is a cross-sectional study including children between age 6 months and 17 years with IIH who were followed in the Calcium Clinic at the Hospital for Sick Children (SickKids), Toronto, Canada. Twenty children with mild IIH on calcium-restricted diets were evaluated. We performed a dietary assessment and analyzed biochemical measures including vitamin D metabolites and performed a stepwise molecular genetic analysis. Complementary biochemical assessments and renal ultrasounds were offered to first-degree family members of positive probands., Results: The median age was 16 months. Median serum levels of calcium (2.69 mmol/L), urinary calcium:creatinine ratio (0.72 mmol/mmol), and 1,25(OH)2D (209 pmol/L) were elevated, whereas intact PTH was low normal (22.5 ng/L). Mean 1,25(OH)2D/PTH and 1,25(OH)2D/25(OH)D ratios were increased by comparison to healthy controls. Eleven individuals (55%) had renal calcification. Genetic variants were common (65%), with the majority being heterozygous variants in SLC34A1 and SLC34A3, while a minority showed variants of CYP24A1 and other genes related to hypercalciuria., Conclusion: The milder form of IIH has a distinctive vitamin D metabolite profile and is primarily associated with heterozygous SLC34A1 and SLC34A3 variants., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Purpose: Tumor-induced osteomalacia (TIO) is an acquired form of hypophosphatemia caused by tumors with excess production of fibroblast growth factor 23 (FGF23). Some reports showed that TIO patients had renal Fanconi syndrome (FS) with unidentified mechanism. In this study, we investigated the association between genetic polymorphisms of phosphate transporters in renal proximal tubules and TIO with FS., Methods: We recruited 30 TIO patients with FS (TIO-FS) as well as 30 TIO patients (TIO-nonFS) without any urine abnormalities matched by age and gender. We collected clinical manifestations and conducted targeted sequencing of SLC34A1, SLC34A3 and XPR1 genes and the association analysis between variants in TIO with FS and phenotypes., Results: TIO-FS group had lower levels of serum phosphate (0.44 ± 0.12 vs. 0.51 ± 0.07 mmol/L, p < 0.05) than TIO-nonFS group. Among the 16 SNPs in SLC34A1, SLC34A3 and XPR1 genes, GG/GC genotypes of rs148196667 in XPR1 and AA/TA genotypes of rs35535797 in SLC34A3 were associated with a reduced susceptibility to have FS. The G allele of rs148196667 in XPR1 decreased the risk of FS. The GGAA haplotype in SLC34A3 and GCT haplotype in XPR1 were associated with a decreased risk for FS., Conclusions: The polymorphisms of XPR1 and SCL34A3 are associated with TIO patients with Fanconi syndrome. It provides novel insight to the relationship of phosphate transportation and general functions of renal proximal tubules.

Context: Hypophosphatemia and metabolic bone disease are associated with hereditary hypophosphatemic rickets with hypercalciuria (HHRH) due to biallelic mutations of SLC34A3 encoding the NPT2C sodium-phosphate cotransporter and nephrolithiasis/osteoporosis, hypophosphatemic 1 (NPHLOP1) due to monoallelic mutations in SLC34A1 encoding the NPT2A sodium-phosphate cotransporter., Objective: To identify a genetic cause of apparent dominant transmission of HHRH., Design and Setting: Retrospective and prospective analysis of clinical and molecular characteristics of patients studied in 2 academic medical centers., Methods: We recruited 4 affected and 3 unaffected members of a 4-generation family in which the proband presented with apparent HHRH. We performed clinical examinations, biochemical and radiological analyses, and molecular studies of genomic DNA., Results: The proband and her affected sister and mother carried pathogenic heterozygous mutations in 2 related genes, SLC34A1 (exon 13, c.1535G>A; p.R512H) and SLC34A3 (exon 13, c.1561dupC; L521Pfs*72). The proband and her affected sister inherited both gene mutations from their mother, while their clinically less affected brother, father, and paternal grandmother carried only the SLC34A3 mutation. Renal phosphate-wasting exhibited both a gene dosage-effect and an age-dependent attenuation of severity., Conclusions: We describe a kindred with autosomal dominant hypophosphatemic rickets in which whole exome analysis identified digenic heterozygous mutations in SLC34A1 and SLC34A3. Subjects with both mutations were more severely affected than subjects carrying only one mutation. These findings highlight the challenges of assigning causality to plausible genetic variants in the next generation sequencing era., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Barakat syndrome is an autosomal dominant disorder characterized by the triad of hypoparathyroidism, sensorineural deafness, and renal anomalies and is caused by mutations in GATA3 gene. SLC34A3 is the cause gene of hypophosphatemic rickets with hypercalciuria, and heterozygous carriers may have milder clinical symptoms. The aim of this study was to identify the underlying genetic cause of a patient who initially presented with renal failure, hypercalciuria, kidney stone, and bilateral sensorineural deafness., Methods: A 6-year-old boy with complex clinical presentations was investigated. Comprehensive medical evaluations were performed including auditory function tests, endocrine function tests, metabolic studies, and imaging examinations. Molecular diagnoses were analyzed by trio whole-exome sequencing., Results: One novel de novo deleterious variant (c. 324del) of the GATA3 gene was identified in the patient. The patient can be diagnosed with Barakat syndrome. In addition, one novel variant (c. 589A>G) of the SLC34A3 gene was detected, which was inherited from the father. This heterozygous variant can explain the hypercalciuria and kidney stone that occurred in both the patient and his father., Conclusion: This study provides a special case which is phenotype-driven dual diagnoses, and the two novel variants can parsimoniously explain the complex clinical presentations of this patient., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

SLC34A3/NPT2c/NaPi-2c/Npt2c is a growth-related NaPi cotransporter that mediates the uptake of renal sodium-dependent phosphate (Pi). Mutation of human NPT2c causes hereditary hypophosphatemic rickets with hypercalciuria. Mice with Npt2c knockout, however, exhibit normal Pi metabolism. To investigate the role of Npt2c in Pi homeostasis, we generated α-klotho -/- /Npt2c -/- (KL2cDKO) mice and analyzed Pi homeostasis. α-Klotho -/- (KLKO) mice exhibit hyperphosphatemia and markedly increased kidney Npt2c protein levels. Genetic disruption of Npt2c extended the lifespan of KLKO mice similar to that of α-Klotho -/- /Npt2a -/- mice. Adult KL2cDKO mice had hyperphosphatemia, but analysis of Pi metabolism revealed significantly decreased intestinal and renal Pi (re)absorption compared with KLKO mice. The 1,25-dihydroxy vitamin D3 concentration was not reduced in KL2cDKO mice compared with that in KLKO mice. The KL2cDKO mice had less severe soft tissue and vascular calcification compared with KLKO mice. Juvenile KL2cDKO mice had significantly reduced plasma Pi levels, but Pi metabolism was not changed. In Npt2cKO mice, plasma Pi levels began to decrease around the age of 15 days and significant hypophosphatemia developed within 21 days. The findings of the present study suggest that Npt2c contributes to regulating plasma Pi levels in the juvenile stage and affects Pi retention in the soft and vascular tissues in KLKO mice., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Loss-of-function mutations of SLC34A3 represent an established cause of a distinct renal phosphate wasting disorder termed hereditary hypophosphatemic rickets with hypercalciuria (HHRH). SLC34A3 encodes the renal phosphate transporter NaPi2c expressed at the apical brush border of proximal renal tubules. Substitution of p.Ser192Leu is one of the most frequent genetic changes among HHRH patients in Europe, but has never been systematically evaluated, clinically or on a cellular level. Identification of a 32-year-old female with a homozgyous c.575C>T, p.Ser192Leu substitution enabled a more comprehensive assessment of the impact of this missense variant. Clinically, the patient showed renal phosphate wasting and nephrocalcinosis without any bone abnormalities. Heterozygous carriers of deleterious SLC34A3 variants were previously described to harbor an increased risk of kidney stone formation and renal calcification. We hence examined the frequency of p.Ser192Leu variants in our adult kidney stone cohort and compared the results to clinical findings of previously published cases of both mono- and biallelic p.Ser192Leu changes. On a cellular level, p.Ser192Leu-mutated transporters localize to the plasma membrane in different cellular systems, but lead to significantly reduced transport activity of inorganic phosphate upon overexpression in Xenopus oocytes. Despite the reduced function in ectopic cellular systems, the clinical consequences of p.Ser192Leu may appear relatively mild, at least in our index patient, and can potentially be missed in clinical practice.

Background: Injection of parathyroid hormone (PTH) rapidly stimulates renal Pi excretion, in part by downregulating NaPi-IIa (Npt2a/SLC34A1) and NaPi-IIc (Npt2c/SLC34A3) transporters. The mechanisms underlying the effects of PTH on NaPi-IIc are not fully elucidated., Methods: We analyzed the effect of PTH on inorganic phosphate (Pi) reabsorption in Npt2a -/- mice to eliminate the influence of Npt2a on renal Pi reabsorption. In opossum kidney (OK) cells and Xenopus oocytes, we investigated the effect of NaPi-IIc transporter phosphorylation. Studies of mice with mutations of NaPi-IIc protein in which serine and threonine were replaced with either alanine (A), which prevents phosphorylation, or aspartic acid (D), which mimics the charged state of phosphorylated NaPi-IIc, were also performed to evaluate the involvement of phosphorylation in the regulation of transport function., Results: The Npt2a -/- experiments showed that PTH administration rapidly inactivated NaPi-IIc function in the apical membrane of proximal tubular cells. Analysis of mutant proteins (S71, S138, T151, S174, T583) at putative protein kinase C sites, revealed that S138 markedly suppressed the function and cellular expression of mouse NaPi-IIc in Xenopus oocytes and OK cells. In addition, 138D had a short half-life compared with wild-type protein., Conclusions: The present study suggests that acute regulation of NaPi-IIc protein by PTH is involved in the inactivation of Na + -dependent Pi cotransporter activity and that phosphorylation of the transporter is involved in the rapid modification.

Nucleotide sugar transporters (NSTs) regulate the flux of activated sugars from the cytosol into the lumen of the Golgi apparatus where glycosyltransferases use them for the modification of proteins, lipids, and proteoglycans. It has been well-established that NSTs are antiporters that exchange nucleotide sugars with the respective nucleoside monophosphate. Nevertheless, information about the molecular basis of ligand recognition and transport is scarce. Here, using topology predictors, cysteine-scanning mutagenesis, expression of GFP-tagged protein variants, and phenotypic complementation of the yeast strain Kl3, we identified residues involved in the activity of a mouse UDP-GlcNAc transporter, murine solute carrier family 35 member A3 (mSlc35a3). We specifically focused on the putative transmembrane helix 2 (TMH2) and observed that cells expressing E47C or K50C mSlc35a3 variants had lower levels of GlcNAc-containing glycoconjugates than WT cells, indicating impaired UDP-GlcNAc transport activity of these two variants. A conservative substitution analysis revealed that single or double substitutions of Glu-47 and Lys-50 do not restore GlcNAc glycoconjugates. Analysis of mSlc35a3 and its genetic variants reconstituted into proteoliposomes disclosed the following: (i) all variants act as UDP-GlcNAc/UMP antiporters; (ii) conservative substitutions (E47D, E47Q, K50R, or K50H) impair UDP-GlcNAc uptake; and (iii) substitutions of Glu-47 and Lys-50 dramatically alter kinetic parameters, consistent with a critical role of these two residues in mSlc35a3 function. A bioinformatics analysis revealed that an E XX K motif in TMH2 is highly conserved across SLC35 A subfamily members, and a 3D-homology model predicted that Glu-47 and Lys-50 are facing the central cavity of the protein., (© 2019 Toscanini et al.)

Mutations in the SLC34A3 gene, encoding the sodium/phosphate cotransporter 2C (NPTIIc), induce decreased renal phosphate reabsorption, hypophosphatemia, decreased fibroblast growth factor 23 and parathyroid hormone, and increased 1,25-dihydroxyvitamin D (1,25[OH] 2 D) levels. The complete phenotype is characterized by hypophosphatemia, hypercalciuria, and nephrolithiasis/nephrocalcinosis, leading to chronic kidney disease and osteoporosis in adults. We report a 15-year-old boy referred for nephrocalcinosis. The patient demonstrated hypercalcemia, hypercalciuria, normal serum phosphate level, normal tubular phosphate reabsorption, and increased serum 1,25(OH) 2 D level with suppressed serum parathyroid hormone. Compound heterozygous mutations in SLC34A3 were found. Hydrochlorothiazide failed to decrease calciuria. Fluconazole, an inhibitor of 1α-hydroxylase, was effective in normalizing calciuria without decreasing glomerular filtration rate. We conclude that children with SLC334A3 mutations can present with a less-typical phenotype, having normal serum phosphate levels and normal renal phosphate reabsorption. Genetic abnormalities of NPTIIc should be considered in cases of increased 1,25(OH) 2 D levels without mutations in CYP24A1. The utility of fluconazole to decrease 1,25(OH) 2 D levels requires confirmation in larger studies., (Copyright © 2019 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

Renal phosphate handling critically determines plasma phosphate and whole body phosphate levels. Filtered phosphate is mostly reabsorbed by Na + -dependent phosphate transporters located in the brush border membrane of the proximal tubule: NaPi-IIa (SLC34A1), NaPi-IIc (SLC34A3), and Pit-2 (SLC20A2). Here we review new evidence for the role and relevance of these transporters in inherited disorders of renal phosphate handling. The importance of NaPi-IIa and NaPi-IIc for renal phosphate reabsorption and mineral homeostasis has been highlighted by the identification of mutations in these transporters in a subset of patients with infantile idiopathic hypercalcemia and patients with hereditary hypophosphatemic rickets with hypercalciuria. Both diseases are characterized by disturbed calcium homeostasis secondary to elevated 1,25-(OH) 2 vitamin D 3 as a consequence of hypophosphatemia. In vitro analysis of mutated NaPi-IIa or NaPi-IIc transporters suggests defective trafficking underlying disease in most cases. Monoallelic pathogenic mutations in both SLC34A1 and SLC34A3 appear to be very frequent in the general population and have been associated with kidney stones. Consistent with these findings, results from genome-wide association studies indicate that variants in SLC34A1 are associated with a higher risk to develop kidney stones and chronic kidney disease, but underlying mechanisms have not been addressed to date.

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) 2 D levels, which sets this fibroblast growth factor 23 (FGF23)-independent disorder apart from the more common X-linked hypophosphatemia. The elevated 1,25(OH) 2 D 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) 2 D 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.

Objective: To describe clinical findings, biochemical profile and genetic analysis in an Iranian kindred with hereditary hypophosphatemic rickets with hypercalciuria (HHRH)., Methods: Clinical examination and biochemical profile results and gene analysis of 12 members of a family of a patient previously diagnosed with HHRH due to SLC34A3 mutation. Ten healthy controls were also evaluated., Results: Of the twelve family members three were homozygote and seven heterozygote for the same SLC34A3 variant found in the proband while two others were unaffected. All patients had significantly increased risk of kidney stone formation, bone deformities and short stature compared with unrelated healthy controls. The heterozygous patients displayed milder clinical symptoms compared with homozygous patients. In particular they had mild or no hypophosphatemia and they did not develop skeletal deformities. Recurrent renal stones and hypercalciuria were the main presentations of the heterozygous patients which may be confused with familial hypercalciuria. In addition, biochemical analysis showed significantly low serum sodium and elevated alkaline phosphatase levels in these patients., Conclusion: Genetic counseling and screening for SLC34A3 mutations can be helpful in adult onset phenotype with unexplained osteoporosis, bone deformities and especial recurrent renal stones. In subjects with vitamin D deficiency the results should be interpreted cautiously.

Objective: To detect potential mutations of PHEX gene in four pedigrees affected with hypophosphatemic rickets (HR) and provide prenatal diagnosis for a fetus at 13th gestational week., Methods: The coding regions and exon/intron boundaries of PHEX, FGF23, DMP1, ENPP1, CLCN5 and SLC34A3 genes of the probands were analyzed by targeted next-generation sequencing (NGS). Suspected mutations were verified by Sanger sequencing among unaffected relatives and 200 unrelated healthy individuals. Deletions were confirmed by multiplex ligation-dependent probe amplification (MLPA) detection of probands, unaffected relatives and 20 unrelated healthy individuals. Prenatal diagnosis for a fetus with high risk was carried out through MLPA analysis., Results: Four PHEX mutations were respectively detected in the pedigrees, which included c.850-3C>G, exon 11 deletion, exon 13 deletion and c.1753G>A (p.G585R). Among these, exon 11 deletion, exon 13 deletion and c.1753G>A (p.G585R) were novel mutations and not found among unaffected relatives and healthy controls. In pedigree 3, the same mutation was not found in the fetus., Conclusion: Mutations of the PHEX gene probably underlies the disease among the four pedigrees. NGS combined with Sanger sequencing and/or MLPA detection can ensure accurate diagnosis for this disease.

Circulating inorganic phosphate exhibits a remarkable daily oscillation based on food intake. In humans and rodents, the daily oscillation in response to food intake may be coordinated to control the intestinal absorption, renal excretion, cellular shifts, and extracellular concentration of inorganic phosphate. However, mechanisms regulating the resulting oscillation are unknown. Here we investigated the roles of the sodium phosphate cotransporter SLC34 (Npt2) family and nicotinamide phosphoribosyltransferase (Nampt) in the daily oscillation of plasma inorganic phosphate levels. First, it is roughly linked to urinary inorganic phosphate excretion. Second, expression of renal Npt2a and Npt2c, and intestinal Npt2b proteins also exhibit a dynamic daily oscillation. Analyses of Npt2a, Npt2b, and Npt2c knockout mice revealed the importance of renal inorganic phosphate reabsorption and cellular inorganic phosphate shifts in the daily oscillation. Third, experiments in which nicotinamide and a specific Nampt inhibitor (FK866) were administered in the active and rest phases revealed that the Nampt/NAD + system is involved in renal inorganic phosphate excretion. Additionally, for cellular shifts, liver-specific Nampt deletion disturbed the daily oscillation of plasma phosphate during the rest but not the active phase. In systemic Nampt +/- mice, NAD levels were significantly reduced in the liver, kidney, and intestine, and the daily oscillation (active and rest phases) of the plasma phosphate concentration was attenuated. Thus, the Nampt/NAD + system for Npt2 regulation and cellular shifts to tissues such as the liver play an important role in generating daily oscillation of plasma inorganic phosphate levels., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: Hereditary hypophosphatemia is a group of rare renal phosphate wasting disorders. The diagnosis is based on clinical, radiological, and biochemical features, and may require genetic testing to be confirmed., Methodology: Clinical features and mutation spectrum were investigated in patients with hereditary hypophosphatemia. Genomic DNA of 23 patients from 15 unrelated families were screened sequentially by PCR-sequencing analysis for mutations in the following genes: PHEX, FGF23, DMP1, ENPP1, CLCN5, SLC34A3 and SLC34A1. CytoScan HD Array was used to identify large deletions., Results: Genetic evaluation resulted in the identification of an additional asymptomatic but intermittent hypophosphatemic subject. Mutations were detected in 21 patients and an asymptomatic sibling from 13 families (86.6%, 13/15). PHEX mutations were identified in 20 patients from 12 families. Six of them were novel mutations present in 9 patients: c.983&#95;987dupCTACC, c.1586+2T>G, c.1206delA, c.436+1G>T, c.1217G>T, and g.22,215,887-22,395,767del (179880 bp deletion including exon 16-22 and ZNF645). Six previously reported mutations were found in 11 patients. Among 12 different PHEX mutations, 6 were de novo mutations. Patients with de novo PHEX mutations often had delayed diagnosis and significantly shorter in height than those who had inherited PHEX mutations. Novel compound heterozygous mutations in SLC34A3 were found in one patient and his asymptomatic sister: c.1335+2T>A and c.1639&#95;1652del14. No mutation was detected in two families., Conclusions: This is the largest familial study on Turkish patients with hereditary hypophosphatemia. PHEX mutations, including various novel and de novo variants, are the most common genetic defect. More attention should be paid to hypophosphatemia by clinicians since some cases remain undiagnosed both during childhood and adulthood.

Objective: To identify a genetic basis for markedly reduced bone density and multiple fractures in an adult patient with hypophosphatemia and hypercalciuria., Subjects: A 54-year-old Vietnamese man, his unaffected two daughters and wife., Methods: We performed biochemical studies and sequenced the SLC34A3 gene using genomic DNA from peripheral blood mononuclear cells., Results: Biochemical evaluation of the proband revealed hypophosphatemia with increased renal phosphate wasting, hypercalciuria, low serum parathyroid hormone (PTH) and an elevated serum 1,25(OH) 2 D level. Mutation analysis of SLC34A3 gene revealed that the patient was a compound heterozygote for two nonsynonymous nucleotide substitutions: a novel c.571G>A (p.G191S) damaging mutation and the previously reported c.200G>A (p.R67H) polymorphism, consistent with the clinical diagnosis of late-onset hereditary hypophosphatemic rickets with hypercalciuria (HHRH). His wife and older daughter both carried the p.R67H polymorphism, while his younger daughter was compound heterozygous for p.R67H and p.G191S., Conclusions: HHRH is an uncommon autosomal recessive disease that generally manifests in childhood as rickets or nephrolithiasis, but an adult onset phenotype may occur in heterozygous carriers of SLC34A3 mutations. The severe presentation of this proband in adulthood with marked nephrolithiasis, multiple fractures and low bone density emphasizes the importance of measuring the serum phosphorus level in patients with suspected but unexplained osteoporosis and/or recurrent renal stones. The recognition of late-onset HHRH facilitates timely institution of appropriate therapy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 the American Physiological Society.)

Massively parallel sequencing of whole genomes and exomes has facilitated a direct assessment of causative genetic variation, now enabling the identification of genetic factors involved in rare diseases (RD) with Mendelian inheritance patterns on an almost routine basis. Here, we describe the illustrative case of a single consanguineous family where this strategy suffered from the difficulty to distinguish between two etiologically distinct disorders, namely the co-occurrence of hereditary hypophosphatemic rickets (HRR) and congenital myopathies (CM), by their phenotypic manifestation alone. We used parametric linkage analysis, homozygosity mapping and whole exome-sequencing to identify mutations underlying HRR and CM. We also present an approximate approach for assessing the probability of co-occurrence of two unlinked recessive RD in a single family as a function of the degree of consanguinity and the frequency of the disease-causing alleles. Linkage analysis and homozygosity mapping yielded elusive results when assuming a single RD, but whole-exome sequencing helped to identify two mutations in two genes, namely SLC34A3 and SEPN1, that segregated independently in this family and that have previously been linked to two etiologically different diseases. We assess the increase in chance co-occurrence of rare diseases due to consanguinity, i.e. under circumstances that generally favor linkage mapping of recessive disease, and show that this probability can increase by several orders of magnitudes. We conclude that such potential co-occurrence represents an underestimated risk when analyzing rare or undefined diseases in consanguineous families and should be given more consideration in the clinical and genetic evaluation.

NaPi-IIc/SLC34A3 is a sodium-dependent inorganic phosphate (Pi) transporter in the renal proximal tubules and its mutations cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH). In the present study, we created a specific antibody for opossum SLC34A3, NaPi-IIc (oNaPi-IIc), and analyzed its localization and regulation in opossum kidney cells (a tissue culture model of proximal tubular cells). Immunoreactive oNaPi-IIc protein levels increased during the proliferative phase and decreased during differentiation. Moreover, stimulating cell growth upregulated oNaPi-IIc protein levels, whereas suppressing cell proliferation downregulated oNaPi-IIc protein levels. Immunocytochemistry revealed that endogenous and exogenous oNaPi-IIc proteins localized at the protrusion of the plasma membrane, which is a phosphatidylinositol 4,5-bisphosphate (PIP2) rich-membrane, and at the intracellular vacuolar membrane. Exogenous NaPi-IIc also induced cellular vacuoles and localized in the plasma membrane. The ability to form vacuoles is specific to electroneutral NaPi-IIc, and not electrogenic NaPi-IIa or NaPi-IIb. In addition, mutations of NaPi-IIc (S138F and R468W) in HHRH did not cause cellular PIP2-rich vacuoles. In conclusion, our data anticipate that NaPi-IIc may regulate PIP2 production at the plasma membrane and cellular vesicle formation.

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., (Copyright © 2014 by the American Society of Nephrology.)

The proximal renal epithelia express three different Na-dependent inorganic phosphate (Pi) cotransporters: NaPi-IIa/SLC34A1, NaPi-IIc/SLC34A3, and PiT2/SLC20A2. Constitutive mouse knockout models of NaPi-IIa and NaPi-IIc suggested that NaPi-IIa mediates the bulk of renal reabsorption of Pi whereas the contribution of NaPi-IIc to this process is minor and probably restricted to young mice. However, many reports indicate that mutations of NaPi-IIc in humans lead to hereditary hypophosphatemic rickets with hypercalciuria (HHRH). Here, we report the generation of a kidney-specific and inducible NaPi-IIc-deficient mouse model based on the loxP-Cre system. We found that the specific removal of the cotransporter from the kidneys of young mice does not impair the capacity of the renal epithelia to transport Pi. Moreover, the levels of Pi in plasma and urine as well as the circulating levels of parathyroid hormone, FGF-23, and vitamin D3 remained unchanged. These findings are in agreement with the data obtained with the constitutive knockout model and suggest that, under steady-state conditions of normal dietary Pi, NaPi-IIc is not an essential Na-Pi cotransporter in murine kidneys. However, and unlike the constitutive mutants, the kidney-specific depletion of NaPi-IIc does not result in alteration of the homeostasis of calcium. This suggests that the calcium-related phenotype observed in constitutive knockout mice may not be related to inactivation of the cotransporter in kidney.

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare metabolic disorder inherited in an autosomal recessive fashion and characterized by hypophosphatemia, short stature, rickets and/or osteomalacia, and secondary absorptive hypercalciuria. HHRH was recently mapped to chromosome 9q34, which contains the gene SLC34A3 which encodes the renal proximal tubular sodium-phosphate cotransporter NaPi-IIc. Here we describe a 29-year-old man with a history of childhood rickets who presented with increased renal phosphate clearance leading to hypophosphatemia, hypercalciuria, low serum parathyroid hormone (PTH), elevated serum 1,25-dihydroxyvitamin D (1,25(OH)2D) and recurrent nephrolithiasis. We performed a mutation analysis of SLC34A3 (exons and adjacent introns) of the proband and his parents to determine if there was a genetic contribution. The proband proved to be compound heterozygous for two missense mutations in SLC34A3: one novel mutation in exon 7 c.571G>C (p.G191R) and one previously identified mutation in exon 13 c.1402C>T (p.R468W). His parents were both asymptomatic heterozygous carriers of one of these two mutations. We also performed an oral phosphate loading test and compared serum phosphate, intact PTH, and intact fibroblast growth factor 23 (iFGF23) in this patient versus patients with other forms of hypophosphatemic rickets, the results of which further revealed that the mechanism of hypophosphatemia in HHRH is independent of FGF23. This is the first report of HHRH in the Chinese population. Our findings of the novel mutation in exon 7 add to the list of more than 20 reported mutations of SLC34A3., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare metabolic disorder, characterized by hypophosphatemia, variable degrees of rickets/osteomalacia, and hypercalciuria secondary to increased serum 1,25-dihydroxyvitamin D [1,25(OH)2D] levels. HHRH is caused by mutations in the SLC34A3 gene, which encodes sodium-phosphate co-transporter type IIc. A 6-1/2-year-old female presented with a history of nephrolithiasis. Her metabolic evaluation revealed increased 24-hour urine calcium excretion with high serum calcium, low intact parathyroid hormone (PTH), and elevated 1,25(OH)2D. In addition, the patient had low to low-normal serum phosphorus with high urine phosphorus. The patient had normal stature; without rachitic or boney deformities or a history of fractures. Genetic analysis of SLC34A3 revealed the patient to be a compound heterozygote for a novel single base pair deletion in exon 12 (c.1304delG) and 30-base pair deletion in intron 6 (g.1440-1469del). The single-base pair mutation causes a frameshift, which results in premature stop codon. The intronic deletion is likely caused by misalignment of the 4-basepair homologous repeats and results in the truncation of an already small intron to 63bp, which would impair proper RNA splicing of the intron. This is the fourth unique intronic deletion identified in patients with HHRH, suggesting the frequent occurrence of sequence misalignments in SLC34A3 and the importance of screening introns in patients with HHRH., (© 2013.)

Background: Calcium nephrolithiasis is a multifactorial disease with a polygenic milieu. Association studies identified genetic polymorphisms potentially implicated in the pathogenesis of calcium nephrolithiasis. The present article reviews the mechanisms of calcium stone formation and the potential contribution of gene polymorphisms to lithogenic mechanisms., Summary: Endoscopy observations suggested that precipitation of calcium-oxalate on the Randall's plaque at the papilla surface may cause idiopathic calcium-oxalate stones. The Randall's plaque is a hydroxyapatite deposit in the interstitium of the kidney medulla, which resembles a soft tissue calcification. Conversely, calcium-phosphate stones may develop from crystalline deposits located at the tip of the Bellini duct. Polymorphisms of eleven genes have been associated with stones in genome-wide association studies and replicated candidate-gene association studies: VDR, SLC34A1, SLC34A4, CLDN14, and CaSR genes coding for proteins regulating tubular phosphate and calcium reabsorption; CaSR, MGP, OPN, PLAU, and UMOD genes coding for proteins preventing calcium salt precipitation; AQP1 gene coding for a water channel in the proximal tubule. The renal activity of the last gene, DGKH, is unknown. Polymorphisms in these genes may predispose to calcium-oxalate and -phosphate stones by increasing the risk of calcium-phosphate precipitation in the tubular fluid. Key Messages: Genetic findings suggest that tubular fluid supersaturation with respect to calcium and phosphate predisposes to calcium-oxalate stones by triggering cellular mechanisms that lead to the Randall's plaque formation., (© 2014 S. Karger AG, Basel.)

Background: Mutations in SLC34A3 have been shown to cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH). Patients with compound heterozygous or homozygous mutations develop skeletal lesions in addition to hypercalciuria, hypophosphatemia and/or elevated 1,25-dihydroxy vitamin D [1,25-(OH)2D] levels. Here, we report a case of hypercalciuria without skeletal lesions in a patient with compound heterozygous mutations of SLC34A3., Case Presentation: A 3-year-old girl presented with microscopic hematuria. Laboratory data revealed elevated 1,25-(OH)2D levels and serum calcium, reduced serum inorganic phosphorus and hypercalciuria. In addition, the ratio of maximal rate of renal tubular reabsorption of phosphate to glomerular filtration rate was reduced. Abdominal ultrasound revealed bilateral nephrocalcinosis. These data were consistent with HHRH, but the patient had no clinical features of rickets or any family history of skeletal disease. Genetic analysis revealed compound heterozygous mutations of c.175+1 G>A and c.1234 C>T in SLC34A3., Conclusions: This is the report of a patient with compound heterozygous mutations of SLC34A3 and normal skeletal features. Biallelic mutations in SLC34A3 can thus be associated with hypercalciuria not accompanied by rickets. Orally administered inorganic phosphate is predicted to improve symptoms in these patients, hence screening for SLC34A3 mutations should be considered in patients with hypercalciuria of unknown etiology.

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) , an autosomal recessive disorder first identified in a large Bedouin tribe, is characterized by hypophosphatemia secondary to renal inorganic phosphate (Pi) wasting, resulting in increased serum1,25-dihydroxyvitamin D3 concentrations with associated intestinal calcium hyperabsorption, hypercalciuria, rickets, and osteomalacia. Recent studies identified several mutations in the NaPi-2c/NPT2c transporter gene (SLC34A3) as the cause of HHRH. The fact that HHRH is caused by NaPi-2c loss-of-function mutations is compatible with the HHRH phenotype and the prevailing view of renal Pi regulation. The NaPi-2c mutants in HHRH show defective processing and stability.

The SLC34 family of Na(+)-dependent inorganic phosphate cotransporters comprises two electrogenic isoforms (NaPi-IIa, NaPi-IIb) and an electroneutral isoform (NaPi-IIc). Both fulfill essential physiological roles in mammalian phosphate homeostasis. By substitution of three conserved amino acids, found in all electrogenic isoforms, at corresponding sites in NaPi-IIc, electrogenicity was re-established and the Na(+)/P i stoichiometry increased from 2:1 to 3:1. However, this engineered electrogenic construct (AAD-IIc) had a reduced apparent P i affinity and different presteady-state kinetics from the wild-type NaPi-IIa/b. We investigated AAD-IIc using electrophysiology and voltage clamp fluorometry to elucidate the compromised behavior. The activation energy for cotransport was threefold higher than for NaPi-IIc and 1.5-fold higher than for NaPi-IIa and the temperature dependence of presteady-state charge displacements suggested that the large activation energy was associated with the empty carrier reorientation. AAD-IIc shows a weak interaction of external Na(+) ions with the electric field, and thus retains the electroneutral cooperative interaction of two Na(+) ions preceding external P i binding of NaPi-IIc. Most of the presteady-state charge movement was accounted for by the empty carrier (in the absence of external P i ), and the cytosolic release of one Na(+) ion (in the presence of P i ). Simulations using a kinetic model recapitulated the presteady-state and steady-state behavior and allowed identification of two critical partial reactions: the final release of Na(+) to the cytosol and external P i binding. Fluorometric recordings from AAD-IIc mutants with Cys substituted at functionally important sites established that AAD-IIc undergoes substrate- and voltage-dependent conformational changes that correlated qualitatively with its presteady-state kinetics.

PTH increases urinary Pi excretion by reducing expression of two renal cotransporters [NaPi-IIa (Npt2a) and NaPi-IIc (Npt2c)]. In contrast to acute transporter regulation that is cAMP/protein kinase A dependent, long-term effects require phospholipase C (PLC) signaling by the PTH/PTHrP receptor (PPR). To determine whether the latter pathway regulates Pi through Npt2a and/or Npt2c, wild-type mice (Wt) and animals expressing a mutant PPR incapable of PLC activation (DD) were tested in the absence of one (Npt2a(-/-) or Npt2c(-/-)) or both phosphate transporters (2a/2c-dko). PTH infusion for 8 days caused a rapid and persistent decrease in serum Pi in Wt mice, whereas serum Pi in DD mice fell only transiently for the first 2 days. Consistent with these findings, fractional Pi excretion index was increased initially in both animals, but this increase persisted only when the PPR Wt was present. The hypophosphatemic response to PTH infusion was impaired only slightly in PPR Wt/Npt2c(-/-) or DD/Npt2c(-/-) mice. Despite lower baselines, PTH infusion in PPR Wt/Npt2a(-/-) mice decreased serum Pi further, an effect that was attenuated in DD/Npt2a(-/-) mice. Continuous PTH had no effect on serum Pi in 2a/2c-dko mice. PTH administration increased serum 1,25 dihydroxyvitamin D3 levels in Wt and DD mice and increased levels above the elevated baseline with ablation of either but not of both transporters. Continuous PTH elevated serum fibroblast growth factor 23 and blood Ca(2+) equivalently in all groups of mice. Our data indicate that PLC signaling at the PPR contributes to the long-term effect of PTH on Pi homeostasis but not to the regulation of 1,25 dihydroxyvitamin D3, fibroblast growth factor 23, or blood Ca(2+).

A magnesium (Mg) deficiency induces changes in calcium (Ca) and phosphorus (P) metabolism; however, the mechanisms responsible for these effects remain unclear. Since 1,25-dihydroxyvitamin D3 and type II sodium-phosphate (Na/Pi) cotransporters are essential regulators of Ca and P metabolism, this study examined the effects of Mg deficiency on the mRNA expression of vitamin D metabolizing enzymes (25-hydroxyvitamin D-1α-hydroxylase (1α(OH)ase) and 25-hydroxyvitamin D-24-hydroxylase (24(OH)ase)), and Na/Pi cotransporters (type IIa and IIc) in the rat kidney. Rats were divided into two groups and fed a control diet (Mg concentration: 0.05%) or a Mg-deficient diet (Mg concentration: Mg-free) for 21 days. 1α(OH)ase mRNA levels were significantly decreased in rats fed the Mg-deficient diet, while 24(OH)ase mRNA levels were significantly increased, compared to rats fed the control diet. Type IIa and IIc Na/Pi cotransporter mRNA levels in rats fed the Mg-deficient diet were significantly decreased compared to rats fed the control diet. These results suggest that Mg deficiency induces downregulation of 1α(OH)ase and type IIa and IIc Na/Pi cotransporters, and upregulation of 24(OH)ase in the kidney.

Three siblings, aged 12, 4 and 2 years, presented at a Gambian clinic with bone deformities. Radiographs of knees and wrists confirmed the presence of florid rickets. The family (including 2 unaffected siblings and the mother) were investigated for hereditary rickets. The three affected siblings had biochemical features of hereditary hypophosphataemic rickets with hypercalciuria (HHRH) with normal plasma calcium and 25-hydroxyvitamin D concentrations, elevated 1,25-dihydroxyvitamin D, hypophosphataemia, hyperphosphaturia and hypercalciuria. At presentation, two of the three affected siblings had an elevated fibroblast growth factor-23 (FGF23) concentration. The mother and clinically unaffected siblings had largely normal biochemistry. Genetic analysis of the SLC34A3 gene, encoding the type IIc sodium-phosphate cotransporter, in DNA samples from the siblings and their mother was conducted. Three single nucleotide polymorphisms (SNPs) S168F, E513V and L599L were identified. E513V and L599L had been previously identified as benign polymorphisms. S168F however, is a previously unreported variant. In silico mutation evaluation predicted that the S168F mutation causes changes in the protein product which are damaging to its function. In addition, the three clinically affected siblings were homozygous in the S168F variant whereas the unaffected family members were carriers. This study describes a biochemical profile and complementary gene data consistent with a rare genetic hypophosphataemic rickets disease in a family from rural Gambia. To our knowledge, this study reports the first cases of HHRH in Africa and describes a novel causal mutation within the SLC34A3 gene., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Context: Many inherited disorders of calcium and phosphate homeostasis are unexplained at the molecular level., Objective: The objective of the study was to identify the molecular basis of phosphate and calcium abnormalities in two unrelated, consanguineous families., Patients: The affected members in family 1 presented with rickets due to profound urinary phosphate-wasting and hypophosphatemic rickets. In the previously reported family 2, patients presented with proximal renal tubulopathy and hypercalciuria yet normal or only mildly increased urinary phosphate excretion., Methods: Genome-wide linkage scans and direct nucleotide sequence analyses of candidate genes were performed. Transport of glucose and phosphate by glucose transporter 2 (GLUT2) was assessed using Xenopus oocytes. Renal sodium-phosphate cotransporter 2a and 2c (Npt2a and Npt2c) expressions were evaluated in transgenically rescued Glut2-null mice (tgGlut2-/-)., Results: In both families, genetic mapping and sequence analysis of candidate genes led to the identification of two novel homozygous mutations (IVS4-2A>G and R124S, respectively) in GLUT2, the gene mutated in Fanconi-Bickel syndrome, a rare disease usually characterized by renal tubulopathy, impaired glucose homeostasis, and hepatomegaly. Xenopus oocytes expressing the [R124S]GLUT2 mutant showed a significant reduction in glucose transport, but neither wild-type nor mutant GLUT2 facilitated phosphate import or export; tgGlut2-/- mice demonstrated a profound reduction of Npt2c expression in the proximal renal tubules., Conclusions: Homozygous mutations in the facilitative glucose transporter GLUT2, which cause Fanconi-Bickel syndrome, can lead to very different clinical and biochemical findings that are not limited to mild proximal renal tubulopathy but can include significant hypercalciuria and highly variable degrees of urinary phosphate-wasting and hypophosphatemia, possibly because of the impaired proximal tubular expression of Npt2c.

We report a case of a male aged 50 years who consulted for renal disease recurrent lithiasis and nephrocalcinosis. The clinical examination showed external signs of rickets/osteomalacia and biochemical data as well as a severe loss of renal phosphate with hypophosphatemia, normal 25 OH vitamin D, high 1,25 OH vitamin D and hypercalciuria. Parathyroid hormone was low and renal ultrasound confirmed the existence of severe bilateral medullary nephrocalcinosis. They also found incipient chronic renal failure and incomplete renal tubular acidosis, both secondary to nephrocalcinosis and unrelated to the underlying disease. The molecular study found a change in homozygosity in intron 5 of gene SLC34A3 (NM&#95;080877.2:c[ 448 +5G&gt;A] + [ 448 +5G&gt;A] ). His three children were carriers of the same variant in heterozygosis and although they were clinically asymptomatic two of them had hypercalciuria. All these data suggest that the patient had hereditary hypophosphataemic rickets with hypercalciuria (HHRH) secondary to an alteration in the sodium dependent phosphate cotransporter located in proximal tubule (NaPi-IIc). The HHRH is transmitted by autosomal recessive inheritance and is an extremely rare form of hypophosphatemic rickets. The diagnosis and treatment are essential to prevent bone sequelae of rickets and nephrocalcinosis. A correct differential diagnosis with other forms of hypophosphatemic rickets has implications on the treatment, as the management based only on phosphorus supplementation usually corrects all clinical and biochemical abnormalities, except for the loss of phosphorus in the urine. The exogenous supply of calcitriol, as advised in other hypophosphatemic rickets, may induce renal calcium deposits and nephrocalcinosis and worsens the prognosis.

This study aimed to identify the underlying genetic mutation in patients with hypophosphatemic rickets (HR). Genomic DNA was analysed for mutations in PHEX, FGF23 and CLCN5 by polymerase chain reaction (PCR) followed by denaturing high-performance liquid chromatography (dHPLC). Bi-directional sequencing was performed in samples with deviating chromatographic profiles. DMP1 and SLC34A3 were sequenced, only. In addition, a multiplex ligation-dependent probe amplification (MLPA) analysis was performed to detect larger deletions/duplications in PHEX or FGF23. Familial cases accounted for 12 probands while 12 cases were sporadic. In 20 probands, mutations were detected in PHEX of which 12 were novel, and one novel frameshift mutation was found in DMP1. Three PHEX mutations were identified by the MLPA analysis only; that is, two large deletions and one duplication. No mutations were identified in FGF23, SLC34A3 or CLCN5. By the methods used, a disease causing mutation was identified in 83% of the familial and 92% of the sporadic cases, thereby in 88% of the tested probands. Genetic analysis performed in HR patients by PCR, dHPLC, sequencing and in addition by MLPA analysis revealed a high identification rate of gene mutations causing HR, including 12 novel PHEX and one novel DMP1 mutation.

Objective: Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is an autosomal recessive form of hypophosphatemia with hyperphosphaturia, hypercalciuria, and hypercalcemia. In two reports on six affected kindreds with HHRH, the disease was mapped to chromosome 9q34, which contains the SLC34A3 gene that encodes the renal type 2c sodium-phosphate cotransporter. Our objective was to define the clinical course of these cases in a family with HHRH and to screen for SLC34A3 gene in order to determine whether these mutations are responsible for HHRH., Methods: After clinical and biochemical evaluations, the entire SLC34A3 gene was screened using PCR amplification followed by direct sequencing technique. In this paper, we describe a new kindred with HHRH and a case of progressive and complicated HHRH presenting at age 27 years., Results: We found 101-bp deletion in intron 9 of the SLC34A3 gene. The index patient was homozygous for this mutation which has been previously reported in a Caucasian population. This is the first report for presence of SLC34A3 intron 9 deletion in an Iranian population., Conclusions: These data showed that HHRH can be easily missed or underdiagnosed. Genetic evaluation of patients with familial hypercalciuria, hypophosphatemia and nephrolithiasis is needed for further information on the prevalence and management of this rare disorder.

Mutations in the apically located Na(+)-dependent phosphate (NaPi) cotransporter, SLC34A3 (NaPi-IIc), are a cause of hereditary hypophosphatemic rickets with hypercalciuria (HHRH). We have characterized the impact of several HHRH mutations on the processing and stability of human NaPi-IIc. Mutations S138F, G196R, R468W, R564C, and c.228delC in human NaPi-IIc significantly decreased the levels of NaPi cotransport activities in Xenopus oocytes. In S138F and R564C mutant proteins, this reduction is a result of a decrease in the V(max) for P(i), but not the K(m). G196R, R468W, and c.228delC mutants were not localized to oocyte membranes. In opossum kidney (OK) cells, cell surface labeling, microscopic confocal imaging, and pulse-chase experiments showed that G196R and R468W mutations resulted in an absence of cell surface expression owing to endoplasmic reticulum (ER) retention. G196R and R468W mutants could be partially stabilized by low temperature. In blue native-polyacrylamide gel electrophoresis analysis, G196R and R468W mutants were either denatured or present in an aggregation complex. In contrast, S138F and R564C mutants were trafficked to the cell surface, but more rapidly degraded than WT protein. The c.228delC mutant did not affect endogenous NaPi uptake in OK cells. Thus, G196R and R468W mutations cause ER retention, while S138F and R564C mutations stimulate degradation of human NaPi-IIc in renal epithelial cells. Together, these data suggest that the NaPi-IIc mutants in HHRH show defective processing and stability.

Homozygous and compound heterozygous mutations in SLC34A3, the gene encoding the sodium-dependent co-transporter NaPi-IIc, cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a disorder characterized by renal phosphate-wasting resulting in hypophosphatemia, elevated 1,25(OH)(2) vitamin D levels, hypercalciuria, rickets/osteomalacia, and frequently kidney stones or nephrocalcinosis. Similar albeit less severe biochemical changes are also observed in heterozygous carriers, which are furthermore indistinguishable from those encountered in idiopathic hypercalciuria (IH). We now searched for SLC34A3 mutations (exons and introns) in two previously not reported HHRH kindreds, which resulted in the identification of three novel mutations. The affected members of kindred A were compound heterozygous for two different mutations, c.1046&#95;47del and the intronic mutation c.560+23&#95;561-42del, while the index case in kindred B was homozygous for the nonsense SLC34A3 mutation c.1764C>G (p.Y588X). The patient in kindred C was diagnosed with IH because of bilateral medullary nephrocalcinosis, suppressed PTH levels, and hypercalciuria; she was found to have a novel heterozygous c.1571&#95;1880del mutation. The HHRH patients in kindred A were treated for up to 7years with oral phosphate, which led to reversal of hypophosphatemia, hypercalciuria, and prevention or healing of the mild bone abnormalities. PTH levels were normal throughout the observation period, while 1,25(OH)(2) vitamin D levels remained elevated and may thus be helpful for assessing treatment efficacy and patient compliance in HHRH., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Mutations in the SLC34A3 gene, a sodium-dependent inorganic phosphate (Pi) cotransporter, also referred to as NaPi IIc, causes hereditary hypophosphatemic rickets with hypercalciuria (HHRH), an autosomal recessive disorder. In human and rodent, NaPi IIc is mainly localized in the apical membrane of renal proximal tubular cells. In this study, we identified mouse NaPi IIc variant (Npt2c-v1) that lacks the part of the exon 3 sequence that includes the assumed translation initiation site of Npt2c. Microinjection of mouse Npt2c-v1 cRNA into Xenopus oocytes demonstrated that Npt2c-v1 showed sodium-dependent Pi cotransport activity. The characterization of pH dependency showed activation at extracellular alkaline-pH. Furthermore, Npt2c-v1 mediated Pi transport activity was significantly higher at any pH value than those of Npt2c. In an in vitro study, the localization of the Npt2c-v1 protein was detected in the apical membrane in opossum kidney cells. The expression of Npt2c-v1 mRNA was detected in the heart, spleen, testis, uterus, placenta, femur, cerebellum, hippocampus, diencephalon and brain stem of mouse. Using mouse bone primary cultured cells, we showed the expression of Npt2c-v1 mRNA. In addition, the Npt2c protein was detected in the spermatozoa head. Thus, Npt2c-v1 was expressed in extra-renal tissues such as epididymal spermatozoa and may function as a sodium-dependent phosphate transporter.

The kidney is a key regulator of phosphate homeostasis. There are two predominant renal sodium phosphate cotransporters, NaPi2a and NaPi2c. Both are regulated by parathyroid hormone (PTH), which decreases the abundance of the NaPi cotransporters in the apical membrane of renal proximal tubule cells. The time course of PTH-induced removal of the two cotransporters from the apical membrane, however, is markedly different for NaPi2a compared with NaPi2c. In animals and in cell culture, PTH treatment results in almost complete removal of NaPi2a from the brush border (BB) within 1 h whereas for NaPi2c this process in not complete until 4 to 8 h after PTH treatment. The reason for this is poorly understood. We have previously shown that the unconventional myosin motor myosin VI is required for PTH-induced removal of NaPi2a from the proximal tubule BB. Here we demonstrate that myosin VI is also necessary for PTH-induced removal of NaPi2c from the apical membrane. In addition, we show that, while at baseline the two cotransporters have similar diffusion coefficients within the membrane, after PTH addition the diffusion coefficient for NaPi2a initially exceeds that for NaPi2c. Thus NaPi2c appears to remain "tethered" in the apical membrane for longer periods of time after PTH treatment, accounting, at least in part, for the difference in response times to PTH of NaPi2a versus NaPi2c.

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is characterized by rickets, hyperphosphaturia, hypophosphatemia, elevated 1,25-dihydroxyvitamin-D, increased gastrointestinal calcium absorption and hypercalciuria. Serum calcium, 25-hydroxyvitamin-D and PTH levels are normal. Here we describe a boy with HHRH, nephrolithiasis, and compound heterozygosity for one previously described mutation (g.4225&#95;50del) and a novel splice mutation (g.1226G>A) in SLC34A3, the gene encoding the renal sodium-phosphate co-transporter NaPi-IIc. The patient's mother and grandmother are carriers of g.4225&#95;50del, and both have a history of nephrolithiasis associated with hypercalciuria and elevated 1,25-dihydroxyvitamin-D. His three siblings (2-6 years old), who are also carriers of g.4225&#95;50del, have hypercalciuria but so far their renal ultrasounds are normal. Thus, SLC34A3/NaPi-IIc mutations appear to be associated with variable phenotypic changes at presentation, which can include recurrent nephrolithiasis., (Copyright © 2011 Wiley-Liss, Inc.)