RESUMO
Osteocytes are embedded in lacunae and connected by canaliculi (lacuno-canalicular network, LCN). Bones from mice with X-linked hypophosphatemia (Hyp), which have impaired production of 1,25 dihydroxyvitamin D (1,25D) and hypophosphatemia, have abnormal LCN structure that is improved by treatment with 1,25D or an anti-FGF23 targeting antibody, supporting roles for 1,25D and phosphate in regulating LCN remodeling. Bones from mice lacking the vitamin D receptor (VDR) in osteocytes (Vdrf/f;Dmp1Cre+) and mice lacking the sodium phosphate transporter 2a (Npt2aKO), which have low serum phosphate with high serum 1,25D, have impaired LCN organization, demonstrating that osteocyte-specific actions of 1,25D and hypophosphatemia regulate LCN remodeling. In osteoclasts, nuclear factor of activated T cells cytoplasmic 1 (NFATc1) is critical for stimulating bone resorption. Since osteocytes also resorb matrix, we hypothesize that NFATc1 plays a role in 1,25D and phosphate-mediated LCN remodeling. Consistent with this, 1,25D and phosphate suppress Nfatc1 mRNA expression in IDG-SW3 osteocytes, and knockdown of Nfatc1 expression in IDG-SW3 cells blocks 1,25D- and phosphate-mediated suppression of matrix resorption gene expression and 1,25D- and phosphate-mediated suppression of RANKL-induced acidification of the osteocyte microenvironment. To determine the role of NFATc1 in 1,25D- and phosphate-mediated LCN remodeling in vivo, histomorphometric analyses of tibiae from mice lacking osteocyte-specific Nfatc1 in Vdrf/f;Dmp1Cre+ and Npt2aKO mice were performed, demonstrating that bones from these mice have decreased lacunar size and expression of matrix resorption genes, and improved canalicular structure compared to Vdrf/f;Dmp1Cre+ and Npt2aKO control. This study demonstrates that NFATc1 is necessary for 1,25D- and phosphate-mediated regulation of LCN remodeling.
Assuntos
Remodelação Óssea , Fator de Crescimento de Fibroblastos 23 , Fatores de Transcrição NFATC , Osteócitos , Fosfatos , Vitamina D , Animais , Masculino , Camundongos , Remodelação Óssea/efeitos dos fármacos , Raquitismo Hipofosfatêmico Familiar/metabolismo , Raquitismo Hipofosfatêmico Familiar/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição NFATC/metabolismo , Fatores de Transcrição NFATC/genética , Osteócitos/metabolismo , Osteócitos/efeitos dos fármacos , Fosfatos/metabolismo , Receptores de Calcitriol/metabolismo , Receptores de Calcitriol/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismo , Vitamina D/farmacologia , Vitamina D/análogos & derivados , FemininoRESUMO
Vitamin D3 is clinically used for the treatment of vitamin D3 deficiency or osteoporosis, partially because of its role in regulating phosphate (Pi) and calcium (Ca2+) homeostasis. The renal sodium-phosphate cotransporter 2a (Npt2a) plays an important role in Pi homeostasis; however, the role of vitamin D3 in hypophosphatemia has never been investigated. We administered vehicle or vitamin D3 to wild-type (WT) mice or hypophosphatemic Npt2a-/- mice. In contrast to WT mice, vitamin D3 treatment increased plasma Pi levels in Npt2a-/- mice, despite similar levels of reduced parathyroid hormone and increased fibroblast growth factor 23. Plasma Ca2+ was increased ~ twofold in both genotypes. Whereas WT mice were able to increase urinary Pi and Ca2+/creatinine ratios, in Npt2a-/- mice, Pi/creatinine was unchanged and Ca2+/creatinine drastically decreased, coinciding with the highest kidney Ca2+ content, highest plasma creatinine, and greatest amount of nephrocalcinosis. In Npt2a-/- mice, vitamin D3 treatment completely diminished Npt2c abundance, so that mice resembled Npt2a/c double knockout mice. Abundance of intestinal Npt2b and claudin-3 (tight junctions protein) were reduced in Npt2a-/- only, the latter might facilitate the increase in plasma Pi in Npt2a-/- mice. Npt2a might function as regulator between renal Ca2+ excretion and reabsorption in response to vitamin D3.
Assuntos
Cálcio , Colecalciferol , Homeostase , Camundongos Knockout , Fosfatos , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa , Animais , Fosfatos/metabolismo , Cálcio/metabolismo , Homeostase/efeitos dos fármacos , Camundongos , Colecalciferol/farmacologia , Colecalciferol/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Rim/metabolismo , Rim/efeitos dos fármacos , Fator de Crescimento de Fibroblastos 23 , Hormônio Paratireóideo/metabolismo , Masculino , Hipofosfatemia/metabolismo , Hipofosfatemia/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIbRESUMO
Infantile hypercalcemia (IH) is a rare genetic disorder characterized by hypercalcemia, hypercalciuria, low parathyroid hormone, and nephrocalcinosis during the first months of life. Biallelic variants in the genes CYP24A1 and SCL34A1 cause IH1 and 2, respectively. We present the case of a newborn with an antenatal diagnosis of IH2 due to the identification of echogenic, yet normal-sized kidneys at 23 weeks gestation. Trio whole-exome sequencing initially identified only a heterozygous pathogenic variant in SLC34A1. Re-analysis of the exome data because of the clinical suspicion of IH2 revealed a 21-basepair deletion in trans that had initially been filtered out because of its high allele frequency. The diagnosis of IH2 enabled postnatal screening for hypercalcemia, present already at week 1, resulting in early treatment with phosphate supplementation and vitamin D avoidance. In the subsequent course, biochemical parameters were normalized, and the patient showed no obvious clinical complications of IH2, apart from the nephrocalcinosis.
Assuntos
Hipercalcemia , Humanos , Hipercalcemia/genética , Hipercalcemia/diagnóstico , Recém-Nascido , Feminino , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Gravidez , Sequenciamento do Exoma , Vitamina D3 24-Hidroxilase/genética , Nefrocalcinose/genética , Nefrocalcinose/diagnóstico , Masculino , Vitamina D/sangue , Vitamina D/uso terapêutico , Fosfatos/sangue , Diagnóstico Pré-Natal/métodos , Ultrassonografia Pré-NatalRESUMO
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 (CasrBCH002), baseline NaPi-IIa expression was higher, whereas downregulation of transporter expression was blunted in double CasrBCH002/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.
Assuntos
Fator de Crescimento de Fibroblastos 23 , Rim , Camundongos Knockout , Hormônio Paratireóideo , Fosfatos , Receptores de Detecção de Cálcio , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIc , Receptores de Detecção de Cálcio/metabolismo , Receptores de Detecção de Cálcio/genética , Animais , Hormônio Paratireóideo/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Fosfatos/metabolismo , Rim/metabolismo , Rim/efeitos dos fármacos , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIc/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIc/genética , Camundongos , Reabsorção Renal/efeitos dos fármacos , Masculino , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Camundongos Endogâmicos C57BLRESUMO
OBJECTIVE: Idiopathic infantile hypercalcemia (IIH) is a rare disorder of PTH-independent hypercalcemia. CYP24A1 and SLC34A1 gene mutations cause two forms of hereditary IIH. In this study, the clinical manifestations and molecular aspects of six new Chinese patients were investigated. METHODS: The clinical manifestations and laboratory study of six patients with idiopathic infantile hypercalcemia were analyzed retrospectively. RESULTS: Five of the patients were diagnosed with hypercalcemia, hypercalciuria, and bilateral medullary nephrocalcinosis. Their clinical symptoms and biochemical abnormalities improved after treatment. One patient presented at age 11 years old with arterial hypertension, hypercalciuria and nephrocalcinosis, but normal serum calcium. Gene analysis showed that two patients had compound heterozygous mutations of CYP24A1, one patient had a monoallelic CYP24A1 variant, and three patients had a monoallelic SLC34A1 variant. Four novel CYP24A1 variants (c.116G > C, c.287T > A, c.476G > A and c.1349T > C) and three novel SLC34A1 variants (c.1322 A > G, c.1697_1698insT and c.1726T > C) were found in these patients. CONCLUSIONS: A monoallelic variant of CYP24A1 or SLC34A1 gene contributes to symptomatic hypercalcemia, hypercalciuria and nephrocalcinosis. Manifestations of IIH vary with onset age. Hypercalcemia may not necessarily present after infancy and IIH should be considered in patients with nephrolithiasis either in older children or adults.
Assuntos
Hipercalcemia , Doenças do Recém-Nascido , Erros Inatos do Metabolismo , Nefrocalcinose , Criança , Humanos , Hipercalcemia/genética , Hipercalciúria/genética , Mutação/genética , Nefrocalcinose/genética , Estudos Retrospectivos , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Vitamina D3 24-Hidroxilase/genética , Vitamina D3 24-Hidroxilase/metabolismoRESUMO
SIGNIFICANCE STATEMENT: Kidneys are gatekeepers of systemic inorganic phosphate balance because they control urinary phosphate excretion. In yeast and plants, inositol hexakisphosphate kinases (IP6Ks) are central to regulate phosphate metabolism, whereas their role in mammalian phosphate homeostasis is mostly unknown. We demonstrate in a renal cell line and in mice that Ip6k1 and Ip6k2 are critical for normal expression and function of the major renal Na + /Pi transporters NaPi-IIa and NaPi-IIc. Moreover, Ip6k1/2-/- mice also show symptoms of more generalized kidney dysfunction. Thus, our results suggest that IP6Ks are essential for phosphate metabolism and proper kidney function in mammals. BACKGROUND: Inorganic phosphate is an essential mineral, and its plasma levels are tightly regulated. In mammals, kidneys are critical for maintaining phosphate homeostasis through mechanisms that ultimately regulate the expression of the Na + /Pi cotransporters NaPi-IIa and NaPi-IIc in proximal tubules. Inositol pyrophosphate 5-IP 7 , generated by IP6Ks, is a main regulator of phosphate metabolism in yeast and plants. IP6Ks are conserved in mammals, but their role in phosphate metabolism in vivo remains unexplored. METHODS: We used in vitro (opossum kidney cells) and in vivo (renal tubular-specific Ip6k1/2-/- mice) models to analyze the role of IP6K1/2 in phosphate homeostasis in mammals. RESULTS: In both systems, Ip6k1 and Ip6k2 are responsible for synthesis of 5-IP 7 . Depletion of Ip6k1/2 in vitro reduced phosphate transport and mRNA expression of Na + /Pi cotransporters, and it blunts phosphate transport adaptation to changes in ambient phosphate. Renal ablation of both kinases in mice also downregulates the expression of NaPi-IIa and NaPi-IIc and lowered the uptake of phosphate into proximal renal brush border membranes. In addition, the absence of Ip6k1 and Ip6k2 reduced the plasma concentration of fibroblast growth factor 23 and increased bone resorption, despite of which homozygous males develop hypophosphatemia. Ip6k1/2-/- mice also show increased diuresis, albuminuria, and hypercalciuria, although the morphology of glomeruli and proximal brush border membrane seemed unaffected. CONCLUSIONS: Depletion of renal Ip6k1/2 in mice not only altered phosphate homeostasis but also dysregulated other kidney functions.
Assuntos
Túbulos Renais , Fosfotransferases (Aceptor do Grupo Fosfato) , Animais , Masculino , Camundongos , Rim/metabolismo , Fosfatos/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Túbulos Renais/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismoRESUMO
BACKGROUND: The proximal tubules play a critical role in phosphate (Pi) homeostasis by reabsorbing Pi via sodium-dependent Pi cotransporters. NPT2A is a major proximal-specific Pi cotransporter, whose expression is regulated by circulating hormones, such as parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23). In this study, we aimed to find a novel regulator in Pi homeostasis. METHODS: Using RNA-seq and RT-qPCR analysis, we identified proximal tubule cell-enriched genes. We next used RNAi screening of the identified proximal tubular cell-enriched genes to identify a novel proximal tubule-specific gene that contributes to FGF23- and PTH-mediated inhibition of Pi uptake and NPT2 reduction. We created mice lacking this novel regulator of Pi homeostasis to examine whether the novel regulator contributes to Pi homeostasis in vivo. RESULTS: We identified 54 kidney-enriched genes, 19 of which are expressed in renal primary proximal tubule cells. One of the proximal tubule-specific genes, TMEM174, interacted with NPT2A, and its knockdown blocked the reduction of NPT2A protein by FGF23 and PTH treatments in human and opossum proximal tubule cells. TMEM174 KO mice had significantly increased levels of serum Pi, FGF23, and PTH, resulting in vascular calcification. CONCLUSIONS: TMEM174 is a novel regulator of Pi homeostasis that interacts with NPT2A.
Assuntos
Hiperfosfatemia , Proteínas de Membrana , Calcificação Vascular , Animais , Fatores de Crescimento de Fibroblastos , Humanos , Hiperfosfatemia/genética , Túbulos Renais Proximais/metabolismo , Proteínas de Membrana/genética , Camundongos , Hormônio Paratireóideo , Fosfatos , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Calcificação Vascular/genéticaRESUMO
Kidneys are key regulators of phosphate homeostasis. Biallelic mutations of the renal Na+/phosphate cotransporter SLC34A1/NaPi-IIa cause idiopathic infantile hypercalcemia, whereas monoallelic mutations were frequently noted in adults with kidney stones. Genome-wide-association studies identified SLC34A1 as a risk locus for chronic kidney disease. Pathogenic mutations in SLC34A1 are present in 4% of the general population. Here, we characterize a mouse model carrying the 91del7 in-frame deletion, a frequent mutation whose significance remains unclear. Under normal dietary conditions, 12 weeks old heterozygous and homozygous males have similar plasma and urinary levels of phosphate as their wild type (WT) littermates, and comparable concentrations of parathyroid hormone, fibroblast growth factor 23 (FGF-23) and 1,25(OH)2 vitamin D3. Renal phosphate transport, and expression of NaPi-IIa and NaPi-IIc cotransporters, was indistinguishable in the three genotypes. Challenging mice with low dietary phosphate did not result in differences between genotypes with regard to urinary and plasma phosphate. Urinary and plasma phosphate, plasma FGF-23 and expression of cotransporters were similar in all genotypes after weaning. Urinary phosphate and bone mineral density were also comparable in 300 days old WT and mutant mice. In conclusion, mice carrying the 91del7 truncation do not show signs of impaired phosphate homeostasis.
Assuntos
Fosfatos , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa , Animais , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Homeostase , Humanos , Masculino , Camundongos , Minerais/metabolismo , Mutação , Fosfatos/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismoRESUMO
AIMS: Dietary inorganic phosphate (Pi) modulates renal Pi reabsorption by regulating the expression of the NaPi-IIa and NaPi-IIc Pi transporters. Here, we aimed to clarify the role of several Pi-regulatory mechanisms including parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23) and inositol hexakisphosphate kinases (IP6-kinases) in the acute regulation of NaPi-IIa and NaPi-IIc. METHODS: Wildtype (WT) and PTH-deficient mice (PTH-KO) with/without inhibition of FGF23 signalling were gavaged with Pi/saline and examined at 1, 4 and 12 h. RESULTS: Pi-gavage elevated plasma Pi and decreased plasma Ca2+ in both genotypes after 1 h Within 1 h, Pi-gavage decreased NaPi-IIa abundance in WT and PTH-KO mice. NaPi-IIc was downregulated 1 h post-administration in WT and after 4 h in PTH-KO. PTH increased after 1 h in WT animals. After 4 h Pi-gavage, FGF23 increased in both genotypes being higher in the KO group. PTHrp and dopamine were not altered by Pi-gavage. Blocking FGF23 signalling blunted PTH upregulation in WT mice and reduced NaPi-IIa downregulation in PTH-KO mice 4 h after Pi-gavage. Inhibition of IP6-kinases had no effect. CONCLUSIONS: (1) Acute downregulation of renal Pi transporters in response to Pi intake occurs also in the absence of PTH and FGF23 signalling, (2) when FGF23 signalling is blocked, a partial contribution of PTH is revealed, (3) IP6 kinases, intracellular Pi-sensors in yeast and bacteria, are not involved, and (4) Acute Pi does not alter PTHrp and dopamine. Thus, signals other than PTH, PTHrp, FGF23 and dopamine contribute to renal adaption.
Assuntos
Fosfatos , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa , Animais , Dopamina/metabolismo , Fatores de Crescimento de Fibroblastos , Rim/metabolismo , Camundongos , Hormônio Paratireóideo/metabolismo , Hormônio Paratireóideo/farmacologia , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/farmacologia , Proteínas de Transporte de Fosfato/metabolismo , Fosfatos/metabolismo , Fosfatos/farmacologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismoRESUMO
Phosphate homeostasis, mediated by dietary intake, renal absorption, and bone deposition, is incompletely understood because of the uncharacterized roles of numerous implicated protein factors. Here, we identified a novel role for one such element, regulator of G protein signaling 14 (RGS14), suggested by genome-wide association studies to associate with dysregulated Pi levels. We show that human RGS14 possesses a carboxy-terminal PDZ ligand required for sodium phosphate cotransporter 2a (NPT2A) and sodium hydrogen exchanger regulatory factor-1 (NHERF1)-mediated renal Pi transport. In addition, we found using isotope uptake measurements combined with bioluminescence resonance energy transfer assays, siRNA knockdown, pull-down and overlay assays, and molecular modeling that secreted proteins parathyroid hormone (PTH) and fibroblast growth factor 23 inhibited Pi uptake by inducing dissociation of the NPT2A-NHERF1 complex. PTH failed to affect Pi transport in cells expressing RGS14, suggesting that it suppresses hormone-sensitive but not basal Pi uptake. Interestingly, RGS14 did not affect PTH-directed G protein activation or cAMP formation, implying a postreceptor site of action. Further pull-down experiments and direct binding assays indicated that NPT2A and RGS14 bind distinct PDZ domains on NHERF1. We showed that RGS14 expression in human renal proximal tubule epithelial cells blocked the effects of PTH and fibroblast growth factor 23 and stabilized the NPT2A-NHERF1 complex. In contrast, RGS14 genetic variants bearing mutations in the PDZ ligand disrupted RGS14 binding to NHERF1 and subsequent PTH-sensitive Pi transport. In conclusion, these findings identify RGS14 as a novel regulator of hormone-sensitive Pi transport. The results suggest that changes in RGS14 function or abundance may contribute to the hormone resistance and hyperphosphatemia observed in kidney diseases.
Assuntos
Fosfoproteínas/metabolismo , Proteínas RGS , Trocadores de Sódio-Hidrogênio/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Ligantes , Hormônio Paratireóideo/metabolismo , Fosfatos/metabolismo , Proteínas RGS/genética , Proteínas RGS/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismoRESUMO
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)2D 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.
Assuntos
Hipercalcemia/genética , Mutação , Fenótipo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIc/genética , Vitamina D3 24-Hidroxilase/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
OBJECTIVES: Biallelic pathogenic variants in CYPA24A1 and SLC34A1 are causes of idiopathic infantile hypercalcemia. Pathogenic variants in both may also give rise to hypercalciuria with nephrocalcinosis or nephrolithiasis without previous hypercalcemia (renal group). Our objective was to examine the frequency of CYP24A1 or SLC34A1 variants in children with early hypercalcemia or late-onset hypercalciuria. METHOD: Forty-one children from 7 centers across Canada were recruited. Local investigations were undertaken. The serum was evaluated by liquid chromatography tandem-mass spectrometry for the ratio of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3, (25-OH-D3:24,25-(OH)2D3), an elevation pathognomonic for the loss of function of the CYP24A1 enzyme. Mutational analyses were undertaken. Family cascade screening was performed if pathogenic variants were detected in probands. RESULTS: Twenty-nine children had early-onset hypercalcemia; none had elevated 25-OH-D3:24,25-(OH)2D3 or variants. Interestingly, 2 of 12 in the renal group had elevated 25-OH-D3:24,25-(OH)2D3 and presented as preadolescents. In case 1, cascade testing revealed a sibling and parent with asymptomatic pathogenic variants in CYP24A1. Four CYP24A1 pathogenic variants were identified in these 2 probands: 3 have been described in European populations, and 1 is a rare variant in exon 7 (c931delC) that is likely pathogenic. No SLC34A1 pathogenic variants were detected. CONCLUSION: In Canada, pathogenic variants in CYP24A1 appear to manifest with late-onset hypercalciuria and its sequelae. The 25-OH-D3:24,25-(OH)2D3 ratio is an excellent tool for screening for biallelic pathogenic variants in CYP24A1. We confirm that cascade testing is important for these variants.
Assuntos
Sequência de Bases , Éxons , Hipercalcemia/genética , Hipercalciúria/genética , Deleção de Sequência , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Vitamina D3 24-Hidroxilase/genética , Canadá , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Estudos RetrospectivosRESUMO
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.
Assuntos
Hipercalcemia/genética , Hormônio Paratireóideo/sangue , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIc/genética , Vitamina D/análogos & derivados , Adolescente , Cálcio/sangue , Cálcio/urina , Criança , Pré-Escolar , Creatinina/urina , Estudos Transversais , Feminino , Variação Genética , Heterozigoto , Humanos , Hipercalcemia/sangue , Hipercalcemia/urina , Lactente , Masculino , Vitamina D/sangue , Vitamina D3 24-Hidroxilase/genéticaRESUMO
The type II sodium-dependent phosphate cotransporter (NPT2A) mediates renal phosphate uptake. The NPT2A is regulated by parathyroid hormone (PTH) and fibroblast growth factor 23, which requires Na+/H+ exchange regulatory factor-1 (NHERF1), a multidomain PDZ-containing phosphoprotein. Phosphocycling controls the association between NHERF1 and the NPT2A. Here, we characterize the critical involvement of G protein-coupled receptor kinase 6A (GRK6A) in mediating PTH-sensitive phosphate transport by targeted phosphorylation coupled with NHERF1 conformational rearrangement, which in turn allows phosphorylation at a secondary site. GRK6A, through its carboxy-terminal PDZ recognition motif, binds NHERF1 PDZ1 with greater affinity than PDZ2. However, the association between NHERF1 PDZ2 and GRK6A is necessary for PTH action. Ser162, a PKCα phosphorylation site in PDZ2, regulates the binding affinity between PDZ2 and GRK6A. Substitution of Ser162 with alanine (S162A) blocks the PTH action but does not disrupt the interaction between NHERF1 and the NPT2A. Replacement of Ser162 with aspartic acid (S162D) abrogates the interaction between NHERF1 and the NPT2A and concurrently PTH action. We used amber codon suppression to generate a phosphorylated Ser162(pSer162)-PDZ2 variant. KD values determined by fluorescence anisotropy indicate that incorporation of pSer162 increased the binding affinity to the carboxy terminus of GRK6A 2-fold compared with WT PDZ2. Molecular dynamics simulations predict formation of an electrostatic network between pSer162 and Asp183 of PDZ2 and Arg at position -1 of the GRK6A PDZ-binding motif. Our results suggest that PDZ2 plays a regulatory role in PTH-sensitive NPT2A-mediated phosphate transport and phosphorylation of Ser162 in PDZ2 modulates the interaction with GRK6A.
Assuntos
Quinases de Receptores Acoplados a Proteína G/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Transporte Biológico , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/metabolismo , Quinases de Receptores Acoplados a Proteína G/genética , Humanos , Transporte de Íons , Simulação de Dinâmica Molecular , Domínios PDZ/genética , Hormônio Paratireóideo/metabolismo , Fosfatos/metabolismo , Fosfoproteínas/genética , Fosforilação , Ligação Proteica , Conformação Proteica , Trocadores de Sódio-Hidrogênio/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismoRESUMO
Infantile hypercalcemia (IH), is a rare disorder caused by CYP24A1 or SLC34A1 variants which lead to disturbed catabolism of 25(OH)D3 and 125(OH)2D3 or increased generation of 125(OH)2D3. AIM OF STUDY: To assess the status of 2425(OH)2D3 and other markers of vitamin D in IH survivors, in whom variants of CYP24A1 or SLC34A1 gene were found and to compare these unique biochemical features with those obtained from subjects who were diagnosed in the first year of life with hypercalcemia, elevated 25(OH)D3 and low PTH but in whom neither CYP24A1 nor SLC34A1 variant was found. PATIENTS AND METHODS: 16 IH survivors in whom CYP24A1 (n = 13) or SLC34A1 (n = 3) variants were found and 41 subjects in whom hypercalcemia was diagnosed in the first year of life but in whom CYP24A1 or SLC34A1 variants were not found were included in the study. 25(OH)D3, 3-epi-25(OH)D3, 25(OH)D2, 2425(OH)2D3 were assessed by liquid chromatography coupled with tandem mass spectrometry. 125(OH)2D3 concentrations were assessed by chemiluminescence. RESULTS: Subjects with CYP24A1 variants, despite normal 25(OH)D3 levels, had higher 25(OH)D3/2425(OH)2D3 ratio values (487; 265-1073 ng/mL) when compared to subjects with SLC34A1 variants (16; 16-23 ng/mL) and with subjects in whom CYP24A1 or SLC34A1 were not found (56; 9-56 ng/mL) (p = 0.00003). Separation of interfering metabolite further increased differences between subjects with and without CYP24A1 mutation. CONCLUSIONS: Survivors of IH with CYP24A1 variant, despite being normocalcemic, still presented extremely high 25(OH)D3/2425(OH)2D3 ratio values. Separation of interfering compound further increased differences between subjects with CYP24A1 mutation and without this mutation.
Assuntos
Colecalciferol/metabolismo , Hipercalcemia/tratamento farmacológico , Hipercalcemia/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Vitamina D3 24-Hidroxilase/genética , Colecalciferol/administração & dosagem , Colecalciferol/genética , Cromatografia Líquida , Feminino , Humanos , Hipercalcemia/metabolismo , Hipercalcemia/patologia , Lactente , Recém-Nascido , Masculino , Mutação , Espectrometria de Massas em Tandem , Vitamina D/genética , Vitamina D/metabolismo , Vitamina D3 24-Hidroxilase/sangueRESUMO
BACKGROUND: Infantile hypercalcaemia (IH) is a vitamin D3 metabolism disorder. The molecular basis for IH is biallelic mutations in the CYP24A1 or SLC34A1 gene. These changes lead to catabolism disorders (CYP24A1 mutations) or excessive generation of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] (SLC34A1 mutations). The incidence rate of IH in children and the risk level for developing end-stage renal disease (ESRD) are still unknown. The aim of this study was to analyse the long-term outcome of adolescents and young adults who suffered from IH in infancy. DESIGN: Forty-two children (23 girls; average age 10.7 ± 6.3 years) and 26 adults (14 women; average age 24.2 ± 4.4 years) with a personal history of hypercalcaemia with elevated 1,25(OH)2D3 levels were included in the analysis. In all patients, a genetic analysis of possible IH mutations was conducted, as well as laboratory tests and renal ultrasonography. RESULTS: IH was confirmed in 20 studied patients (10 females). CYP24A1 mutations were found in 16 patients (8 females) and SLC34A1 in 4 patients (2 females). The long-term outcome was assessed in 18 patients with an average age of 23.8 years (age range 2-34). The average glomerular filtration rate (GFR) was 72 mL/min/1.73 m2 (range 15-105). Two patients with a CYP24A1 mutation developed ESRD and underwent renal transplantation. A GFR <90 mL/min/1.73 m2 was found in 14 patients (77%), whereas a GFR <60 mL/min/1.73 m2 was seen in 5 patients (28%), including 2 adults after renal transplantation. Three of 18 patients still had serum calcium levels >2.6 mmol/L. A renal ultrasound revealed nephrocalcinosis in 16 of 18 (88%) patients, however, mild hypercalciuria was detected in only one subject. CONCLUSIONS: Subjects who suffered from IH have a greater risk of progressive chronic kidney disease and nephrocalcinosis. This indicates that all survivors of IH should be closely monitored, with early implementation of preventive measures, e.g. inhibition of active metabolites of vitamin D3 synthesis.
Assuntos
Hipercalcemia , Nefrocalcinose , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa , Vitamina D3 24-Hidroxilase , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Hipercalcemia/genética , Masculino , Mutação , Nefrocalcinose/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Sobreviventes , Vitamina D3 24-Hidroxilase/genética , Adulto JovemRESUMO
Objectives Both CYP24A1 and SLC34A1 gene mutations are responsible for idiopathic infantile hypercalcemia, whereas loss-of-function mutations in CYP24A1 (25-OH-vitamin D-24-hydroxylase) lead to a defect in the inactivation of active 1.25(OH)2D; mutations in SLC34A1 encoding renal sodium phosphate cotransporter NaPi-IIa lead to primary renal phosphate wasting combined with an inappropriate activation of vitamin D. The presence of mutations in both genes has not been reported in the same patient until today. Case presentation Hypercalcemia was incidentally detected when a 13-month-old boy was being examined for urinary tract infection. After 21 months, hypercalcemia was detected in his six-month-old sister. High dose of vitamin D was not given to both siblings. Both of them also had hypophosphatemia and decreased tubular phosphate reabsorption. Intensive hydration, furosemide and oral phosphorus treatment were given. Bilateral medullary nephrocalcinosis was detected in both siblings and their father. Serum Ca and P levels were within normal limits at follow-up in both siblings. Siblings and their parents all carry a homozygous stop codon mutation (p.R466*) in CYP24A1. Interestingly, both siblings and the father also have a heterozygous splice-site mutation (IVS6(+1)G>A) in SLC34A1. The father has nephrocalcinosis. Conclusions A biallelic loss-of-function mutation in the CYP24A1 gene was identified as responsible for hypercalcemia, hypercalciuria and nephrocalcinosis. In addition, a heterozygous mutation in the SLC34A1 gene, although not being the main pathogenic factor, might contribute to the severe phenotype of both patients.
Assuntos
Hipercalcemia/genética , Hipercalcemia/patologia , Mutação , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Vitamina D3 24-Hidroxilase/genética , Adulto , Criança , Pai , Feminino , Humanos , Lactente , Masculino , IrmãosRESUMO
The sodium-phosphate cotransporter NPT2a plays a key role in the reabsorption of filtered phosphate in proximal renal tubules, thereby critically contributing to phosphate homeostasis. Inadequate urinary phosphate excretion can lead to severe hyperphosphatemia as in tumoral calcinosis and chronic kidney disease (CKD). Pharmacological inhibition of NPT2a may therefore represent an attractive approach for treating hyperphosphatemic conditions. The NPT2a-selective small-molecule inhibitor PF-06869206 was previously shown to reduce phosphate uptake in human proximal tubular cells in vitro. Here, we investigated the acute and chronic effects of the inhibitor in rodents and report that administration of PF-06869206 was well tolerated and elicited a dose-dependent increase in fractional phosphate excretion. This phosphaturic effect lowered plasma phosphate levels in WT mice and in rats with CKD due to subtotal nephrectomy. PF-06869206 had no effect on Npt2a-null mice, but promoted phosphate excretion and reduced phosphate levels in normophophatemic mice lacking Npt2c and in hyperphosphatemic mice lacking Fgf23 or Galnt3. In CKD rats, once-daily administration of PF-06869206 for 8 weeks induced an unabated acute phosphaturic and hypophosphatemic effect, but had no statistically significant effect on FGF23 or PTH levels. Selective pharmacological inhibition of NPT2a thus holds promise as a therapeutic option for genetic and acquired hyperphosphatemic disorders.