RESUMEN
The sodium phosphate co-transporters Npt2a and Npt2c play important roles in the regulation of phosphate homeostasis. Slc34a1, the gene encoding Npt2a, resides downstream of the gene encoding coagulation factor XII (f12) and was inadvertently modified while generating f12(-/-) mice. In this report, the renal consequences of this modification are described. The combined single allelic mutant Slc34a1m contains two point mutations in exon 13: A499V is located in intracellular loop 5, and V528M is located in transmembrane domain 11. In addition to the expected coagulopathy of the f12(-/-) phenotype, mice homozygous for the double allelic modification (f12(-/-)/slc34a1(m/m)) displayed hypophosphatemia, hypercalcemia, elevated levels of alkaline phosphatase, urolithiasis, and hydronephrosis. Strategic cross-breedings demonstrated that the kidney-related pathology was associated only with autosomal recessive transmission of the slc34a1(m) gene and was not influenced by the simultaneous inactivation of f12. Npt2a[V528M] could be properly expressed in opossum kidney cells, but Npt2a[A499V] could not. These results suggest that a single amino acid substitution in Npt2a can lead to improper translocation of the protein to the cell membrane, disturbance of phosphate homeostasis, and renal calcification. Whether point mutations in the SLC34A1 gene can lead to hypophosphatemia and nephrolithiasis in humans remains unknown.
Asunto(s)
Factor XII/genética , Enfermedades Renales/genética , Fosfatos/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genética , Secuencia de Aminoácidos , Animales , Peso Corporal , Cruzamiento , Calcio/sangre , Calcio/orina , Línea Celular , Análisis Mutacional de ADN , Factor XII/metabolismo , Femenino , Silenciador del Gen , Homeostasis , Hidronefrosis/genética , Hidronefrosis/metabolismo , Riñón/patología , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Mutación Missense , Zarigüeyas , Fosfatos/sangre , Fosfatos/orina , Profilinas/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , Urolitiasis/genética , Urolitiasis/metabolismoRESUMEN
Inorganic phosphate (Pi) is fundamental to cellular metabolism and skeletal mineralization. Ingested Pi is absorbed by the small intestine, deposited in bone, and filtered by the kidney where it is reabsorbed and excreted in amounts determined by the specific needs of the organism. Two distinct renal Na-dependent Pi transporters, type IIa (NPT2a, SLC34A1) and type IIc (NPT2c, SLC34A3), are expressed in brush border membrane of proximal tubular cells where the bulk of filtered Pi is reabsorbed. Both are regulated by dietary Pi intake and parathyroid hormone. Regulation is achieved by changes in transporter protein abundance in the brush border membrane and requires the interaction of the transporter with scaffolding and signaling proteins. The demonstration of hypophosphatemia secondary to decreased renal Pi reabsorption in mice homozygous for the disrupted type IIa gene underscores its crucial role in the maintenance of Pi homeostasis. Moreover, the recent identification of mutations in the type IIc gene in patients with hereditary hypophosphatemic rickets with hypercalciuria attests to the importance of this transporter in Pi conservation and subsequent skeletal mineralization. Two novel Pi regulating genes, PHEX and FGF23, play a role in the pathophysiology of inherited and acquired hypophosphatemic skeletal disorders and studies are underway to define their mechanism of action on renal Pi handling in health and disease.
Asunto(s)
Fosfatos/metabolismo , Animales , Transporte Biológico , Raquitismo Hipofosfatémico Familiar/complicaciones , Raquitismo Hipofosfatémico Familiar/metabolismo , Raquitismo Hipofosfatémico Familiar/fisiopatología , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/biosíntesis , Humanos , Hipercalciuria/complicaciones , Hipercalciuria/metabolismo , Hipercalciuria/fisiopatología , Ratones , Ratones Noqueados , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genéticaRESUMEN
Fibroblast growth factor-23 (FGF-23) is a novel circulating peptide that regulates phosphorus (Pi) and vitamin D metabolism, but the mechanisms by which circulating FGF-23 itself is regulated are unknown. To determine whether the serum FGF-23 concentration is regulated by dietary intake of Pi, we fed wild-type (WT), Npt2a gene-ablated (Npt2a(-/-)), and Hyp mice diets containing varying Pi contents (0.02-1.65%). In WT mice, increases in dietary Pi intake from 0.02-1.65% induced a 7-fold increase in serum FGF-23 and a 3-fold increase in serum Pi concentrations. Across the range of dietary Pi, serum FGF-23 concentrations varied directly with serum Pi concentrations (r(2) = 0.72; P < 0.001). In Npt2a(-/-) mice, serum FGF-23 concentrations were significantly lower than in WT mice, and these differences could be accounted for by the lower serum Pi levels in Npt2a(-/-) mice. The serum concentrations of FGF-23 in Hyp mice were 5- to 25-fold higher than values in WT mice, and the values varied with dietary Pi intake. Fgf-23 mRNA abundance in calvaria was significantly higher in Hyp mice than in WT mice on the 1% Pi diet; in both groups of mice, fgf-23 mRNA abundance in calvarial bone was suppressed by 85% on the low (0.02%) Pi diet. In WT mice fed the low (0.02%) Pi diet, renal mitochondrial 1alpha-hydroxylase activity and renal 1alpha-hydroxylase (P450c1alpha) mRNA abundance were significantly higher than in mice fed the higher Pi diets and varied inversely with serum FGF-23 concentrations (r(2) = 0.86 and r(2) = 0.64; P < 0.001, respectively). The present data demonstrate that dietary Pi regulates the serum FGF-23 concentration in mice, and such regulation is independent of phex function. The data suggest that genotype-dependent and dietary Pi-induced changes in the serum FGF-23 concentration reflect changes in fgf-23 gene expression in bone.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Fósforo Dietético/farmacología , Fósforo/sangre , Vitamina D/análogos & derivados , Animales , Huesos/metabolismo , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/sangre , Factores de Crecimiento de Fibroblastos/genética , Hipofosfatemia Familiar/sangre , Riñón/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Concentración Osmolar , ARN Mensajero/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/deficiencia , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismo , Vitamina D/metabolismo , Vitamina D3 24-HidroxilasaRESUMEN
Mice homozygous for the disrupted renal type IIa sodium/phosphate (Na/Pi) cotransporter gene (Npt2-/-) exhibit renal Pi wasting, hypophosphatemia, and an adaptive increase in the serum concentration of 1,25-dihydroxyvitamin D with associated hypercalcemia and hypercalciuria. Because hypercalciuria is a risk factor for nephrocalcinosis, we determined whether Npt2-/- mice form renal stones. Analysis of renal sections by von Kossa staining and intact kidneys by microcomputed tomography revealed renal calcification in adult Npt2-/- mice but not in Npt2+/+ littermates. Energy-dispersive spectroscopy and selected-area electron diffraction indicated that the calcifications are comprised of calcium and Pi with an apatitic mineral phase. To determine the age of onset of nephrocalcinosis, we examined renal sections of newborn and weanling mice. At both ages, mutant but not wild-type mice display renal calcification, which is associated with renal Pi wasting and hypercalciuria. Immunohistochemistry revealed that osteopontin co-localizes with the calcifications. Furthermore, renal osteopontin messenger RNA abundance is significantly elevated in Npt2-/- mice compared with Npt2+/+ mice. The onset of renal stones correlated developmentally with the absence of Npt2 expression and the expression of the genes responsible for the renal production (1alpha-hydroxylase) and catabolism (24-hydroxylase) of 1,25-dihydroxyvitamin D. In summary, we show that Npt2 gene ablation is associated with renal calcification and suggest that mutations in the NPT2 gene may contribute to nephrocalcinosis in a subset of patients with familial hypercalciuria.
Asunto(s)
Cálculos Renales/genética , Simportadores/deficiencia , Simportadores/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , Animales , Secuencia de Bases , Calcio/orina , Sistema Enzimático del Citocromo P-450/genética , ADN/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Hipercalcemia/genética , Hipercalcemia/metabolismo , Riñón/metabolismo , Cálculos Renales/metabolismo , Cálculos Renales/patología , Ratones , Ratones Noqueados , Mutación , Fosfatos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo I , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa , Esteroide Hidroxilasas/genética , Simportadores/metabolismo , Vitamina D3 24-HidroxilasaRESUMEN
X-linked hypophosphatemic (Hyp) mice exhibit hypophosphatemia, impaired renal phosphate reabsorption, defective skeletal mineralization, and disordered regulation of vitamin D metabolism: In Hyp mice, restriction of dietary phosphorus induces a decrease in serum concentration of 1,25-dihydroxyvitamin D and renal activity of 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase), and induces an increase in renal activity of 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase). In contrast, in wild-type mice, phosphorus restriction stimulates renal 1alpha-hydroxylase gene expression and suppresses that of 24-hydroxylase. To determine the molecular basis for the disordered regulation of vitamin D metabolism in Hyp mice, we determined renal mitochondrial 1alpha-hydroxylase activity and the renal abundance of p450c1alpha and p450c24 mRNA in wild-type and Hyp mice fed either control, low-, or high-phosphorus diets for 5 d. In wild-type mice, phosphorus restriction increased 1alpha-hydroxylase activity and p450c1alpha mRNA expression by 6-fold and 3-fold, respectively, whereas in the Hyp strain the same diet induced changes of similar magnitude but opposite in direction. Phosphorus supplementation was without effect in wild-type mice, whereas in Hyp mice the same diet induced 3-fold and 2-fold increases, respectively, in enzyme activity and p450c1alpha mRNA abundance. In wild-type mice, both renal 1alpha-hydroxylase activity and p450c1alpha mRNA abundance varied inversely and significantly with serum phosphorus concentrations, whereas in Hyp mice the relationship between both renal parameters and serum phosphorus concentration was direct. In Hyp mice, phosphorus restriction induced a significant increase in renal p450c24 mRNA abundance, in contrast to the lack of effect observed in wild-type mice. The present findings demonstrate that regulation of both the p450c1alpha and p45024 genes by phosphorus is disordered in Hyp mice at the level of renal 1alpha-hydroxylase activity and renal p450c1alpha and p450c24 mRNA expression.
Asunto(s)
25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hipofosfatemia Familiar/enzimología , Riñón/enzimología , Fósforo/farmacología , Animales , Sistema Enzimático del Citocromo P-450/genética , Femenino , Ligamiento Genético , Masculino , Ratones , Ratones Endogámicos C57BL , Fósforo/sangre , Fósforo Dietético/administración & dosificación , ARN Mensajero/análisis , Esteroide Hidroxilasas/genética , Vitamina D3 24-Hidroxilasa , Cromosoma XRESUMEN
Mutations in the fibroblast growth factor 23 gene, FGF23, cause autosomal dominant hypophosphatemic rickets (ADHR). The gene product, FGF-23, is produced by tumors from patients with oncogenic osteomalacia (OOM), circulates at increased levels in most patients with X-linked hypophosphatemia (XLH) and is phosphaturic when injected into rats or mice, suggesting involvement in the regulation of phosphate (Pi) homeostasis. To better define the precise role of FGF-23 in maintaining Pi balance and bone mineralization, we generated transgenic mice that express wild-type human FGF-23, under the control of the alpha1(I) collagen promoter, in cells of the osteoblastic lineage. At 8 wk of age, transgenic mice were smaller (body weight = 17.5 +/- 0.57 vs. 24.3 +/- 0.37 g), exhibited decreased serum Pi concentrations (1.91 +/- 0.27 vs. 2.75 +/- 0.22 mmol/liter) and increased urinary Pi excretion when compared with wild-type littermates. The serum concentrations of human FGF-23 (undetectable in wild-type mice) was markedly elevated in transgenic mice (>7800 reference units/ml). Serum PTH levels were increased in transgenic mice (231 +/- 62 vs. 139 +/- 44 pg/ml), whereas differences in calcium and 1,25-dihydroxyvitamin D were not apparent. Expression of Npt2a, the major renal Na(+)/Pi cotransporter, as well as Npt1 and Npt2c mRNAs, was significantly decreased in the kidneys of transgenic mice. Histology of tibiae displayed a disorganized and widened growth plate and peripheral quantitative computerized tomography analysis revealed reduced bone mineral density in transgenic mice. The data indicate that FGF-23 induces phenotypic changes in mice resembling those of patients with ADHR, OOM, and XLH and that FGF-23 is an important determinant of Pi homeostasis and bone mineralization.
Asunto(s)
Colágeno Tipo I/genética , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Trastornos del Crecimiento/metabolismo , Osteomalacia/metabolismo , Fosfatos/sangre , Animales , Huesos/patología , Huesos/fisiología , Calcificación Fisiológica , Calcio/sangre , Calcio/orina , Factor-23 de Crecimiento de Fibroblastos , Trastornos del Crecimiento/patología , Trastornos del Crecimiento/fisiopatología , Homeostasis/fisiología , Humanos , Riñón/citología , Riñón/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Osteomalacia/patología , Osteomalacia/fisiopatología , Glándulas Paratiroides/patología , Glándulas Paratiroides/fisiología , Fenotipo , Fosfatos/orina , Regiones Promotoras Genéticas , Vitamina D/metabolismoRESUMEN
Synthesis of the hormone 1,25-dihydroxyvitamin D, the biologically active form of vitamin D, occurs in the kidney and is catalyzed by the mitochondrial cytochrome P450 enzyme, 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase). We sought to characterize the effects of changes in dietary phosphorus on the kinetics of renal mitochondrial 1alpha-hydroxylase activity and the renal expression of P450c1alpha and P450c24 mRNA, to localize the nephron segments involved in such regulation, and to determine whether transcriptional mechanisms are involved. In intact mice, restriction of dietary phosphorus induced rapid, sustained, approximately 6- to 8-fold increases in renal mitochondrial 1alpha-hydroxylase activity and renal P450c1alpha mRNA abundance. Immunohistochemical analysis of renal sections from mice fed the control diet revealed the expression of 1alpha-hydroxylase protein in the proximal convoluted and straight tubules, epithelial cells of Bowman's capsule, thick ascending limb of Henle's loop, distal tubule, and collecting duct. In mice fed a phosphorus-restricted diet, immunoreactivity was significantly increased in the proximal convoluted and proximal straight tubules and epithelial cells of Bowman's capsule, but not in the distal nephron. Dietary phosphorus restriction induced a 2-fold increase in P450c1alpha gene transcription, as shown by nuclear run-on assays. Thus, the increase in renal synthesis of 1,25-dihydroxyvitamin D induced in normal mice by restricting dietary phosphorus can be attributed to an increase in the renal abundance of P450c1alpha mRNA and protein. The increase in P450c1alpha gene expression, which occurs exclusively in the proximal renal tubule, is due at least in part to increased transcription of the P450c1alpha gene.
Asunto(s)
25-Hidroxivitamina D3 1-alfa-Hidroxilasa/biosíntesis , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Túbulos Renales Proximales/enzimología , Fósforo Dietético/administración & dosificación , Animales , Western Blotting , Núcleo Celular/metabolismo , Regulación Enzimológica de la Expresión Génica/genética , Inmunohistoquímica , Túbulos Renales Proximales/efectos de los fármacos , Cinética , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/enzimología , Nefronas/metabolismo , ARN Mensajero/biosíntesis , Activación Transcripcional/fisiologíaRESUMEN
The PHEX gene that is mutated in patients with X-linked hypophosphatemia (XLH) encodes a protein homologous to the M13 family of zinc metallopeptidases. The present study was undertaken to assess the impact of nine PHEX missense mutations on cellular trafficking, endopeptidase activity, and protein conformation. Secreted forms of wild-type and mutant PHEX proteins were generated by PCR mutagenesis; these included C85R, D237G, Y317F, G579R, G579V, S711R, A720T, and F731Y identified in XLH patients, and E581V, which in neutral endopeptidase 24.11 abolishes catalytic activity but not plasma membrane localization. The wild-type and D237G, Y317F, E581V, and F731Y proteins were terminally glycosylated and secreted into the medium, whereas the C85R, G579R, G579V, S711R, and A720T proteins were trapped inside the transfected cells. Growing the cells at 26 C permitted the secretion of G579V, S711R, and A720T proteins, although the yield of rescued G579V was insufficient for further analysis. Endopeptidase activity of secreted and rescued PHEX proteins, assessed using a novel internally quenched fluorogenic peptide substrate, revealed that E581V and S711R are completely inactive; D237G and Y317F exhibit 50-60% of wild-type activity; and A720T and F731Y retain full catalytic activity. Conformational analysis by limited proteolysis demonstrated that F731Y is more sensitive to trypsin and D237G is more resistant to endoproteinase Glu-c than the wild-type protein. Thus, defects in protein trafficking, endopeptidase activity, and protein conformation account for loss of PHEX function in XLH patients harboring these missense mutations.
Asunto(s)
Hipofosfatemia Familiar/genética , Mutación Missense , Proteínas/genética , Proteínas/fisiología , Western Blotting , Línea Celular , Cromosomas Humanos X , Endopeptidasas/metabolismo , Estabilidad de Enzimas , Expresión Génica , Ligamiento Genético , Calor , Humanos , Hipofosfatemia Familiar/enzimología , Mutagénesis , Endopeptidasa Neutra Reguladora de Fosfato PHEX , Reacción en Cadena de la Polimerasa , Conformación Proteica , Proteínas/química , Serina Endopeptidasas/metabolismo , Relación Estructura-Actividad , Transfección , Tripsina/metabolismoRESUMEN
During metabolic acidosis (MA), urinary phosphate excretion increases and contributes to acid removal. Two Na(+)-dependent phosphate transporters, NaPi-IIa (Slc34a1) and NaPi-IIc (Slc34a3), are located in the brush border membrane (BBM) of the proximal tubule and mediate renal phosphate reabsorption. Transcriptome analysis of kidneys from acid-loaded mice revealed a large decrease in NaPi-IIc messenger RNA (mRNA) and a smaller reduction in NaPi-IIa mRNA abundance. To investigate the contribution of transporter regulation to phosphaturia during MA, we examined renal phosphate transporters in normal and Slc34a1-gene ablated (NaPi-IIa KO) mice acid-loaded for 2 and 7 days. In normal mice, urinary phosphate excretion was transiently increased after 2 days of acid loading, whereas no change was found in Slc34a1-/- mice. BBM Na/Pi cotransport activity was progressively and significantly decreased in acid-loaded KO mice, whereas in WT animals, a small increase after 2 days of treatment was seen. Acidosis increased BBM NaPi-IIa abundance in WT mice and NaPi-IIc abundance in WT and KO animals. mRNA abundance of NaPi-IIa and NaPi-IIc decreased during MA. Immunohistochemistry did not indicate any change in the localization of NaPi-IIa and NaPi-IIc along the nephron. Interestingly, mRNA abundance of both Slc20 phosphate transporters, Pit1 and Pit2, was elevated after 7 days of MA in normal and KO mice. These data demonstrate that phosphaturia during acidosis is not caused by reduced protein expression of the major Na/Pi cotransporters NaPi-IIa and NaPi-IIc and suggest a direct inhibitory effect of low pH mainly on NaPi-IIa. Our data also suggest that Pit1 and Pit2 transporters may play a compensatory role.
Asunto(s)
Acidosis/complicaciones , Hipofosfatemia Familiar/etiología , Túbulos Renales Proximales/metabolismo , Fosfatos/orina , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , Acidosis/orina , Animales , Modelos Animales de Enfermedad , Concentración de Iones de Hidrógeno , Hipofosfatemia Familiar/orina , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microvellosidades/metabolismo , ARN Mensajero/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/deficiencia , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/metabolismo , Factores de TiempoRESUMEN
Fibroblast growth factor-23 (FGF-23) is critical to the pathogenesis of a distinct group of renal phosphate wasting disorders: tumor-induced osteomalacia, X-linked hypophosphatemia, and autosomal dominant and autosomal recessive hypophosphatemic rickets. Excess circulating FGF-23 is responsible for their major phenotypic features which include hypophosphatemia due to renal phosphate wasting and inappropriately low serum 1,25(OH)2D concentrations. To characterize the effects of FGF-23 on renal sodium-phosphate (Na/P(i)) cotransport and vitamin D metabolism, we administered FGF-23(R176Q) to normal mice. A single injection (0.33 microg/g body wt) induced significant hypophosphatemia, 20 and 29% decreases (P < 0.001) in brush-border membrane (BBM) Na/Pi cotransport at 5 and 17 h after injection, respectively, and comparable decreases in the abundance of type IIa Na/P(i) cotransporter protein in BBM. Multiple injections (6, 12, and 24 mug/day for 4 days) induced dose-dependent decreases (38, 63, and 75%, respectively) in renal abundance of 1alpha-hydroxylase mRNA (P < 0.05). To determine whether FGF-23(R176Q) exerts a direct action on 1alpha-hydroxylase gene expression, we examined its effects in cultured human (HKC-8) and mouse (MCT) renal proximal tubule cells. FGF-23(R176Q) (1 to 10 ng/ml) induced a dose-dependent decrease in 1alpha-hydroxylase mRNA with a maximum suppression of 37% (P < 0.05). Suppression was detectable after 6 h of exposure and maximal after 21 h. In MCT cells, FGF-23(R176Q) suppressed 1alpha-hydroxylase mRNA and activated the ERK1/2 signaling pathway. The MAPK inhibitor PD98059 effectively abolished FGF-23-induced suppression of 1alpha-hydroxylase mRNA by blocking signal transduction via ERK1/2. These novel findings provide evidence that FGF-23 directly regulates renal 1alpha-hydroxylase gene expression via activation of the ERK1/2 signaling pathway.
Asunto(s)
25-Hidroxivitamina D3 1-alfa-Hidroxilasa/antagonistas & inhibidores , Factores de Crecimiento de Fibroblastos/farmacología , Riñón/metabolismo , Fósforo/metabolismo , Vitamina D/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , Animales , Arginina , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/genética , Glucuronidasa/genética , Glutamina , Humanos , Proteínas Klotho , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Microvellosidades/metabolismo , Fósforo/sangre , Isoformas de Proteínas/genética , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Esteroide Hidroxilasas/genética , Vitamina D3 24-HidroxilasaRESUMEN
Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare disorder of autosomal recessive inheritance that was first described in a large consanguineous Bedouin kindred. HHRH is characterized by the presence of hypophosphatemia secondary to renal phosphate wasting, radiographic and/or histological evidence of rickets, limb deformities, muscle weakness, and bone pain. HHRH is distinct from other forms of hypophosphatemic rickets in that affected individuals present with hypercalciuria due to increased serum 1,25-dihydroxyvitamin D levels and increased intestinal calcium absorption. We performed a genomewide linkage scan combined with homozygosity mapping, using genomic DNA from a large consanguineous Bedouin kindred that included 10 patients who received the diagnosis of HHRH. The disease mapped to a 1.6-Mbp region on chromosome 9q34, which contains SLC34A3, the gene encoding the renal sodium-phosphate cotransporter NaP(i)-IIc. Nucleotide sequence analysis revealed a homozygous single-nucleotide deletion (c.228delC) in this candidate gene in all individuals affected by HHRH. This mutation is predicted to truncate the NaP(i)-IIc protein in the first membrane-spanning domain and thus likely results in a complete loss of function of this protein in individuals homozygous for c.228delC. In addition, compound heterozygous missense and deletion mutations were found in three additional unrelated HHRH kindreds, which supports the conclusion that this disease is caused by SLC34A3 mutations affecting both alleles. Individuals of the investigated kindreds who were heterozygous for a SLC34A3 mutation frequently showed hypercalciuria, often in association with mild hypophosphatemia and/or elevations in 1,25-dihydroxyvitamin D levels. We conclude that NaP(i)-IIc has a key role in the regulation of phosphate homeostasis.
Asunto(s)
Raquitismo Hipofosfatémico Familiar/genética , Ligamiento Genético , Hipercalciuria/genética , Fosfatos/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/fisiología , Adolescente , Adulto , Secuencia de Aminoácidos , Árabes/genética , Niño , Mapeo Cromosómico , Femenino , Heterocigoto , Homeostasis , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , LinajeRESUMEN
The type IIa Na/phosphate (Pi) cotransporter (Npt2a) is expressed in the brush border membrane (BBM) of renal proximal tubular cells where the bulk of filtered Pi is reabsorbed. Disruption of the Npt2a gene in mice elicits hypophosphatemia, renal Pi wasting, and an 80% decrease in renal BBM Na/Pi cotransport, and led to the demonstration that Npt2a is the target for hormonal and dietary regulation of renal Pi reabsorption. Regulation is achieved by changes in BBM abundance of Npt2a protein and requires the interaction of Npt2a with various scaffolding and regulatory proteins. Molecular studies in patients with renal Pi wasting resulted in the identification of novel regulators of Pi homeostasis: fibroblast growth factor-23 (FGF-23) and a phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX). In mouse models, increased FGF-23 production or loss of Phex function causes hypophosphatemia and decreased renal Pi reabsorption, secondary to decreased BBM Npt2a protein abundance. Thus, Npt2a plays a major role in the maintenance of Pi homeostasis in both health and disease.
Asunto(s)
Homeostasis , Fósforo/metabolismo , Simportadores/fisiología , Absorción , Animales , Calcio/metabolismo , Dieta , Factor-23 de Crecimiento de Fibroblastos , Humanos , Riñón/metabolismo , Ratones , Ratones Noqueados , Hormona Paratiroidea/fisiología , Fosfatos/administración & dosificación , Fosfatos/deficiencia , Proteínas Cotransportadoras de Sodio-Fosfato , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa , Simportadores/deficiencia , Simportadores/genéticaRESUMEN
Over the past decade, three classes of Na/Pi cotransporters have been identified in mammalian kidney. The type IIa Na/Pi cotransporter, Npt2, is the most abundant and is expressed in the brush-border membrane of renal proximal tubular cells where the bulk of filtered inorganic phosphate (Pi) is reabsorbed. Disruption of the Npt2 gene in mice underscored the importance of Npt2 in the overall maintenance of Pi homeostasis and demonstrated that Npt2 is the target for regulation of proximal tubular Pi reabsorption by parathyroid hormone and dietary Pi. The regulation is post-transcriptional and largely occurs by brush-border membrane retrieval and insertion of Npt2 protein. Of great interest is the recent identification of novel Pi regulating genes, PHEX and FGF23, that play a role in the pathophysiology of inherited (X-linked hypophosphatemia and autosomal dominant hypophosphatemic rickets) and acquired (oncogenic hypophosphatemic rickets) disorders characterized by renal Pi wasting and associated skeletal abnormalities. Studies are currently underway to elucidate the molecular basis for impaired renal Pi reabsorption in these disorders and to determine the precise physiological role of PHEX and FGF-23 in the regulation of Pi homeostasis.
Asunto(s)
Hipofosfatemia Familiar/etiología , Hipofosfatemia/etiología , Enfermedades Renales/etiología , Simportadores/genética , Animales , Factor-23 de Crecimiento de Fibroblastos , Humanos , Hipofosfatemia/genética , Hipofosfatemia Familiar/genética , Enfermedades Renales/genética , Proteínas Cotransportadoras de Sodio-Fosfato , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo I , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa , Simportadores/metabolismoRESUMEN
Disruption of the major renal Na-phosphate (Pi) cotransporter gene Npt2a in mice leads to a substantial decrease in renal brush-border membrane Na-Pi cotransport, hypophosphatemia, and appropriate adaptive increases in renal 25-hydroxyvitamin D3-1alpha-hydroxylase (1alphaOHase) activity and the serum concentration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D]. The latter is associated with increased intestinal Ca absorption, hypercalcemia, hypercalciuria, and renal calcification in Npt2-/- mice. To determine the contribution of elevated serum 1,25(OH)2D levels to the development of hypercalciuria and nephrocalcinosis in Npt2-/- mice, we examined the effects of 1alphaOHase gene ablation and long-term Pi supplementation on urinary Ca excretion and renal calcification by microcomputed tomography. We show that the urinary Ca/creatinine ratio is significantly decreased in Npt2-/-/1alphaOHase-/- mice compared with Npt2-/- mice. In addition, renal calcification, determined by estimating the calcified volume to total renal volume (CV/TV), is reduced by 80% in Npt2-/-/1alphaOHase-/- mice compared with that in Npt2-/- mice. In Npt2-/- mice derived from dams fed a 1% Pi diet and maintained on the same diet, we observed a significant decrease in urinary Ca/creatinine that was also associated with 80% reduction in CV/TV when compared with counterparts fed a 0.6% diet. Taken together, the present data demonstrate that both 1alphaOHase gene ablation and Pi supplementation inhibit renal calcification in Npt2-/- mice and that 1,25(OH)2D is essential for the development of hypercalciuria and nephrocalcinosis in the mutant strain.
Asunto(s)
25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , Nefrocalcinosis/fisiopatología , Simportadores/genética , Animales , Calcinosis/fisiopatología , Calcitriol/sangre , Calcio/sangre , Calcio/orina , Femenino , Homocigoto , Riñón/patología , Riñón/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Nefrocalcinosis/patología , Fósforo Dietético/farmacología , Proteínas Cotransportadoras de Sodio-Fosfato , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo I , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIIRESUMEN
Mutations in the PHEX gene are responsible for X-linked hypophosphatemia, a renal phosphate-wasting disorder associated with defective skeletal mineralization. PHEX is predominantly expressed in bones and teeth and in the parathyroid gland of patients with chronic renal failure and tertiary hyperparathyroidism. The purpose of the present study was to examine the effects of renal insufficiency and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the regulation of PHEX expression in rat tibia and parathyroid gland. In rats fed a high-phosphate (Pi) diet, nephrectomy elicited a significant increase in the serum parathyroid hormone (PTH) concentration that was associated with a significant increase in the abundance of PHEX mRNA and protein in the tibia and a significant increase in PHEX mRNA in the parathyroid gland. In contrast, 1,25(OH)2D3 administration to intact rats fed a control diet elicited a significant decrease in the serum PTH concentration that was accompanied by a significant decrease in PHEX mRNA and protein abundance in the tibia and a significant decrease in PHEX mRNA in the parathyroid gland. In addition, the increases in serum PTH levels and PHEX mRNA in the tibia and parathyroid gland in nephrectomized rats fed a high-Pi diet were blunted by 1,25(OH)2D3. Serum PTH concentration was positively and significantly correlated with tibial PHEX mRNA and protein abundance. In summary, we demonstrate that PHEX expression in the tibia and parathyroid gland is increased by chronic renal insufficiency and decreased by 1,25(OH)2D3 administration and suggest that PTH status may play an important role in mediating these changes in PHEX expression.
Asunto(s)
Calcitriol/análogos & derivados , Calcitriol/farmacología , Fallo Renal Crónico/fisiopatología , Glándulas Paratiroides/fisiología , Proteínas/genética , Tibia/fisiología , Animales , Calcio/sangre , Calcio/orina , Creatinina/sangre , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Masculino , Nefrectomía , Endopeptidasa Neutra Reguladora de Fosfato PHEX , Hormona Paratiroidea/sangre , Fósforo Dietético/sangre , Fósforo Dietético/farmacología , Fósforo Dietético/orina , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Urea/sangreRESUMEN
The present study was undertaken to define the mechanisms governing the regulation of the novel renal brush-border membrane (BBM) Na-phosphate (Pi) cotransporter designated type IIc (Npt2c). To address this issue, the renal expression of Npt2c was compared in two hypophosphatemic mouse models with impaired renal BBM Na-Pi cotransport. In mice homozygous for the disrupted Npt2a gene (Npt2-/-), BBM Npt2c protein abundance, relative to actin, was increased 2.8-fold compared with Npt2+/+ littermates, whereas a corresponding increase in renal Npt2c mRNA abundance, relative to beta-actin, was not evident. In contrast, in X-linked Hyp mice, which harbor a large deletion in the Phex gene, the renal abundance of both Npt2c protein and mRNA was significantly decreased by 80 and 50%, respectively, relative to normal littermates. Pi deprivation elicited a 2.5-fold increase in BBM Npt2c protein abundance in Npt2+/+ mice but failed to elicit a further increase in Npt2c protein in Npt2-/- mice. Pi restriction led to an increase in BBM Npt2c protein abundance in both normal and Hyp mice without correcting its renal expression in the mutants. In summary, we report that BBM Npt2c protein expression is differentially regulated in Npt2-/- mice and Hyp mice and that the Npt2c response to low-Pi challenge differs in both hypophosphatemic mouse strains. We demonstrate that Npt2c protein is maximally upregulated in Npt2-/- mice and suggest that Npt2c likely accounts for residual BBM Na-Pi cotransport in the knockout model. Finally, our data indicate that loss of Phex function abrogates renal Npt2c protein expression.
Asunto(s)
Hipofosfatemia/fisiopatología , Riñón/fisiología , Simportadores/genética , Simportadores/metabolismo , Animales , Membrana Basal/fisiología , Femenino , Expresión Génica , Hipofosfatemia/genética , Hipofosfatemia/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Fosfatos/sangre , Sodio/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo I , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III , Cromosoma XRESUMEN
Mice homozygous for the disrupted type-II Na/P(i) cotransporter gene ( Npt2(-/-)) exhibit hypophosphataemia, increased serum concentration of 1,25-dihydroxyvitamin D (1,25-(OH)(2)D) and calcium (Ca) and elevated urinary Ca excretion. To determine whether the hypercalcaemia and hypercalciuria are secondary to 1,25-(OH)(2)D-stimulated intestinal Ca absorption, we examined the effect of Npt2 gene disruption on serum Ca and urinary Ca excretion after an overnight fast, and on duodenal Ca absorption. We also compared the duodenal expression of the epithelial Ca channels, ECaC1 and ECaC2, and calbindinD(9K) mRNAs, relative to that of beta-actin mRNA, in Npt2(+/+) and Npt2(-/-) mice. Both serum Ca and urine Ca/creatinine were significantly decreased in Npt2(-/-) mice after an overnight fast and were no longer different from that in wild-type mice. Absorption of (45)Ca from isolated duodenal segments in vivo and (45)Ca appearing in the plasma were significantly increased in Npt2(-/-) compared with Npt2(+/+) mice. In addition, the duodenal abundance of ECaC1, ECaC2 and calbindinD(9K) mRNAs was significantly elevated in mutant mice relative to that in wild-type mice. In contrast, both duodenal Ca absorption and ECaC1 and ECaC2 mRNA abundance were lower in mice with X-linked hypophosphataemia ( Hyp) than in normal littermates. In summary, we provide evidence for increased duodenal Ca absorption in Npt2(-/-) mice and suggest a role for ECaC1, ECaC2 and calbindinD(9K) in mediating this response.
Asunto(s)
Canales de Calcio/genética , Calcio/orina , Duodeno/metabolismo , Hipofosfatemia/metabolismo , Simportadores/genética , Simportadores/metabolismo , Animales , Calbindinas , Calcio/sangre , Ayuno/fisiología , Femenino , Expresión Génica/fisiología , Hipofosfatemia/genética , Absorción Intestinal/fisiología , Masculino , Ratones , Ratones Noqueados , Mutagénesis , Mutación , ARN Mensajero/análisis , Proteína G de Unión al Calcio S100/genética , Proteínas Cotransportadoras de Sodio-Fosfato , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo I , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo II , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III , Canales Catiónicos TRPVRESUMEN
We previously demonstrated that inhibition of Na-dependent phosphate (P(i)) transport in osteoclasts led to reduced ATP levels and diminished bone resorption. These findings suggested that Na/P(i) cotransporters in the osteoclast plasma membrane provide P(i) for ATP synthesis and that the osteoclast may utilize part of the P(i) released from bone resorption for this purpose. The present study was undertaken to define the cellular localization of Na/P(i) cotransporters in the mouse osteoclast and to identify the proteins with which they interact. Using glutathione S-transferase (GST) fusion constructs, we demonstrate that the type IIa Na/P(i) cotransporter (Npt2a) in osteoclast lysates interacts with the Na/H exchanger regulatory factor, NHERF-1, a PDZ protein that is essential for the regulation of various membrane transporters. In addition, NHERF-1 in osteoclast lysates interacts with Npt2a in spite of deletion of a putative PDZ-binding domain within the carboxy terminus of Npt2a. In contrast, deletion of the carboxy-terminal TRL amino acid motif of Npt2a significantly reduced its interaction with NHERF-1 in kidney lysates. Studies in osteoclasts transfected with green fluorescent protein-Npt2a constructs indicated that Npt2a colocalizes with NHERF-1 and actin at or near the plasma membrane of the osteoclast and associates with ezrin, a linker protein associated with the actin cytoskeleton, likely via NHERF-1. Furthermore, we demonstrate by RT/PCR of osteoclast RNA and in situ hybridization that the type III Na/P(i) cotransporter, PiT-1, is also expressed in mouse osteoclasts. To examine the cellular distribution of PiT-1, we infected mouse osteoclasts with a retroviral vector encoding PiT-1 fused to an epitope tag. PiT-1 colocalizes with actin and is present on the basolateral membrane of the polarized osteoclast, similar to that previously reported for Npt2a. Taken together, our data suggest that association of Npt2a with NHERF-1, ezrin, and actin, and of PiT-1 with actin, may be responsible for membrane sorting and regulation of these Na/P(i) cotransporters in the osteoclast.