RESUMO
X-linked hypophosphatemia (XLH), the most common form of hereditary hypophosphatemia, is caused by disrupting variants in the PHEX gene, located on the X chromosome. XLH is inherited in an X-linked pattern with complete penetrance observed for both males and females. Patients experience lifelong symptoms resulting from chronic hypophosphatemia, including impaired bone mineralization, skeletal deformities, growth retardation, and diminished quality of life. This chronic condition requires life-long management with disease-specific therapies, which can improve patient outcomes especially when initiated early in life. To centralize and disseminate PHEX variant information, we have established a new PHEX gene locus-specific database, PHEX LSDB. As of April 30, 2021, 870 unique PHEX variants, compiled from an older database of PHEX variants, a comprehensive literature search, a sponsored genetic testing program, and XLH clinical trials, are represented in the PHEX LSDB. This resource is publicly available on an interactive, searchable website (https://www.rarediseasegenes.com/), which includes a table of variants and associated data, graphical/tabular outputs of genotype-phenotype analyses, and an online submission form for reporting new PHEX variants. The database will be updated regularly with new variants submitted on the website, identified in the published literature, or shared from genetic testing programs.
Assuntos
Bases de Dados Genéticas , Raquitismo Hipofosfatêmico Familiar , Doenças Genéticas Ligadas ao Cromossomo X , Hipofosfatemia , Endopeptidase Neutra Reguladora de Fosfato PHEX , Raquitismo Hipofosfatêmico Familiar/genética , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/genética , Humanos , Hipofosfatemia/genética , Masculino , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Qualidade de VidaRESUMO
Pseudoxanthoma elasticum (PXE), a heritable multisystem ectopic calcification disorder, is predominantly caused by inactivating mutations in ABCC6. The encoded protein, ABCC6, is a hepatic efflux transporter and a key regulator of extracellular inorganic pyrophosphate (PPi). Recent studies demonstrated that deficiency of plasma PPi, a potent endogenous calcification inhibitor, is the underlying cause of PXE. This study examined whether restoring plasma PPi levels by INZ-701, a recombinant human ENPP1 protein, the principal PPi-generating enzyme, prevents ectopic calcification in an Abcc6-/- mouse model of PXE. Abcc6-/- mice, at 6 weeks of age, the time of earliest stages of ectopic calcification, were injected subcutaneously with INZ-701 at 2 or 10 mg/kg for 2 or 8 weeks. INZ-701 at both doses increased steady-state plasma ENPP1 activity and PPi levels. In the 8-week treatment study, histopathologic examination and quantification of the calcium content in INZ-701-treated Abcc6-/- mice revealed significantly reduced calcification in the muzzle skin containing vibrissae, a biomarker of the calcification process in these mice. The extent of calcification corresponds to the local expression of two calcification inhibitors, osteopontin and fetuin-A. These results suggest that INZ-701 might provide a therapeutic approach for PXE, a disease with high unmet needs and no approved treatment.
Assuntos
Calcinose , Diester Fosfórico Hidrolases , Pseudoxantoma Elástico , Pirofosfatases , Animais , Calcinose/tratamento farmacológico , Calcinose/prevenção & controle , Modelos Animais de Doenças , Humanos , Fígado , Camundongos , Camundongos Knockout , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Diester Fosfórico Hidrolases/uso terapêutico , Pseudoxantoma Elástico/genética , Pseudoxantoma Elástico/terapia , Pirofosfatases/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Pele/metabolismoRESUMO
Vascular calcification increases the risk of cardiovascular disease and death in patients with chronic kidney disease (CKD). Increased activity of mammalian target of rapamycin complex 1 (mTORC1) and endoplasmic reticulum (ER) stress-unfolded protein response (UPR) are independently reported to partake in the pathogenesis of vascular calcification in CKD. However, the association between mTORC1 activity and ER stress-UPR remains unknown. We report here that components of the uremic state [activation of the receptor for advanced glycation end products (RAGE) and hyperphosphatemia] potentiate vascular smooth muscle cell (VSMC) calcification by inducing persistent and exaggerated activity of mTORC1. This gives rise to prolonged and excessive ER stress-UPR as well as attenuated levels of sestrin 1 ( Sesn1) and Sesn3 feeding back to inhibit mTORC1 activity. Activating transcription factor 4 arising from the UPR mediates cell death via expression of CCAAT/enhancer-binding protein (c/EBP) homologous protein (CHOP), impairs the generation of pyrophosphate, a potent inhibitor of mineralization, and potentiates VSMC transdifferentiation to the osteochondrocytic phenotype. Short-term treatment of CKD mice with rapamycin, an inhibitor of mTORC1, or tauroursodeoxycholic acid, a bile acid that restores ER homeostasis, normalized mTORC1 activity, molecular markers of UPR, and calcium content of aortas. Collectively, these data highlight that increased and/or protracted mTORC1 activity arising from the uremic state leads to dysregulated ER stress-UPR and VSMC calcification. Manipulation of the mTORC1-ER stress-UPR pathway opens up new therapeutic strategies for the prevention and treatment of vascular calcification in CKD.
Assuntos
Doenças da Aorta/enzimologia , Estresse do Retículo Endoplasmático , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Músculo Liso Vascular/enzimologia , Resposta a Proteínas não Dobradas , Uremia/complicações , Calcificação Vascular/enzimologia , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/enzimologia , Aorta/patologia , Doenças da Aorta/tratamento farmacológico , Doenças da Aorta/etiologia , Doenças da Aorta/patologia , Morte Celular , Proliferação de Células , Transdiferenciação Celular , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos Mutantes , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Osteogênese , Fosforilação , Receptor para Produtos Finais de Glicação Avançada/genética , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Proteínas S100/genética , Proteínas S100/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Ácido Tauroquenodesoxicólico/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Calcificação Vascular/tratamento farmacológico , Calcificação Vascular/etiologia , Calcificação Vascular/patologiaRESUMO
Chronic Kidney Disease (CKD)-Mineral and Bone Disorder (CKD-MBD) is a complex disease that is not completely understood. However, some factors secreted by the osteocytes might play an important role in its pathophysiology. Therefore, we evaluated the bone expression of proteins in a group of patients with CKD 2-3, CKD 4, and CKD 5 on dialysis and healthy individuals. We also tested several bone remodeling markers, and correlated these levels with bone biopsy findings. As expected, as serum calcium decreased, serum phosphate, alkaline phosphatase, fibroblast growth factor-23 (FGF-23), parathyroid hormone, and osteoprotegerin increased, as CKD progressed. Additionally, there was a gradual increase in bone resorption associated with a decrease in bone formation and impairment in bone mineralization. Bone expression of sclerostin and parathyroid hormone receptor-1 seemed to be increased in earlier stages of CKD, whereas FGF-23 and phosphorylated ß-catenin had increased expression in the late stages of CKD, although all these proteins were elevated relative to healthy individuals. Immunohistochemical studies showed that FGF-23 and sclerostin did not co-localize, suggesting that distinct osteocytes produce these proteins. Moreover, there was a good correlation between serum levels and bone expression of FGF-23. Thus, our studies help define the complex mechanism of bone and mineral metabolism in patients with CKD. Linkage of serum markers to bone expression of specific proteins may facilitate our understanding and management of this disease.
Assuntos
Remodelação Óssea , Osso e Ossos/metabolismo , Distúrbio Mineral e Ósseo na Doença Renal Crônica/sangue , Osteócitos/metabolismo , Insuficiência Renal Crônica/sangue , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Idoso , Biomarcadores/sangue , Biópsia , Proteínas Morfogenéticas Ósseas/metabolismo , Osso e Ossos/patologia , Cálcio/sangue , Estudos de Casos e Controles , Distúrbio Mineral e Ósseo na Doença Renal Crônica/diagnóstico , Distúrbio Mineral e Ósseo na Doença Renal Crônica/terapia , Feminino , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Marcadores Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Osteócitos/patologia , Osteoprotegerina/sangue , Hormônio Paratireóideo/sangue , Fosforilação , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Diálise Renal , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/terapia , Índice de Gravidade de Doença , beta Catenina/metabolismoRESUMO
PURPOSE OF REVIEW: Maintaining phosphate homeostasis is essential and any deviation can lead to several acute and chronic disease states. To maintain normal physiological levels, phosphate needs to be tightly regulated. This is achieved through a complex relationship of organ cross-talk via hormonal regulation of the type II sodium-dependent phosphate co-transporters. This editorial provides evidence of the importance of intestinal NPT2b in health and chronic kidney disease (CKD). RECENT FINDINGS: The advent of the different Npt2b knockout mice has increased our understanding of how the intestinal phosphate co-transporter contributes to the regulation of systemic phosphate. In addition, these studies have suggested that Npt2b may participate in the phosphate-sensing machinery important for organ cross-talk. Studies using Drosophila have expanded our knowledge of phosphate sensing mechanisms and may provide a foundation for delineating these pathways in humans. Several preclinical studies using different agents to modulate Npt2b, and clinical studies using nicotinamide, have provided evidence that Npt2b is a viable therapeutic target for the management of hyperphosphatemia. SUMMARY: Over the last couple of years, new experimental approaches have increased our understanding of the important role of Npt2b in maintaining phosphate homeostasis. In addition, several clinical studies have associated the detrimental effects of elevated phosphate with cardiovascular events, and decreased lifespan. Although several key questions about intestinal phosphate transport remain to be answered, it is clear that the intestine is an important player, with current evidence suggesting that it is a prime target for regulating phosphate uptake and improving health outcomes in CKD.
Assuntos
Hiperfosfatemia/fisiopatologia , Mucosa Intestinal/metabolismo , Fosfatos/metabolismo , Insuficiência Renal Crônica/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/metabolismo , Animais , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/metabolismo , Homeostase , Humanos , Hiperfosfatemia/tratamento farmacológico , Receptor Cross-TalkRESUMO
The Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) ectoenzyme regulates vascular intimal proliferation and mineralization of bone and soft tissues. ENPP1 variants cause Generalized Arterial Calcification of Infancy (GACI), a rare genetic disorder characterized by ectopic calcification, intimal proliferation, and stenosis of large- and medium-sized arteries. ENPP1 hydrolyzes extracellular ATP to pyrophosphate (PPi) and AMP. AMP is the precursor of adenosine, which has been implicated in the control of neointimal formation. Herein, we demonstrate that an ENPP1-Fc recombinant therapeutic inhibits proliferation of vascular smooth muscle cells (VSMCs) in vitro and in vivo. Addition of ENPP1 and ATP to cultured VSMCs generated AMP, which was metabolized to adenosine. It also significantly decreased cell proliferation. AMP or adenosine alone inhibited VSMC growth. Inhibition of ecto-5'-nucleotidase CD73 decreased adenosine accumulation and suppressed the anti-proliferative effects of ENPP1/ATP. Addition of AMP increased cAMP synthesis and phosphorylation of VASP at Ser157. This AMP-mediated cAMP increase was abrogated by CD73 inhibitors or by A2aR and A2bR antagonists. Ligation of the carotid artery promoted neointimal hyperplasia in wild-type mice, which was exacerbated in ENPP1-deficient ttw/ttw mice. Prophylactic or therapeutic treatments with ENPP1 significantly reduced intimal hyperplasia not only in ttw/ttw but also in wild-type mice. These findings provide the first insight into the mechanism of the anti-proliferative effect of ENPP1 and broaden its potential therapeutic applications beyond enzyme replacement therapy.
Assuntos
5'-Nucleotidase , Adenosina , Proliferação de Células , Músculo Liso Vascular , Miócitos de Músculo Liso , Diester Fosfórico Hidrolases , Pirofosfatases , Transdução de Sinais , Diester Fosfórico Hidrolases/metabolismo , Diester Fosfórico Hidrolases/genética , Pirofosfatases/metabolismo , Pirofosfatases/genética , 5'-Nucleotidase/metabolismo , 5'-Nucleotidase/genética , Animais , Proliferação de Células/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Adenosina/metabolismo , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Camundongos , Humanos , Monofosfato de Adenosina/metabolismo , Camundongos Endogâmicos C57BL , AMP Cíclico/metabolismo , Masculino , Calcificação Vascular/metabolismo , Calcificação Vascular/patologia , Calcificação Vascular/genéticaRESUMO
Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) codes for a type 2 transmembrane glycoprotein which hydrolyzes extracellular phosphoanhydrides into bio-active molecules that regulate, inter alia, ectopic mineralization, bone formation, vascular endothelial proliferation, and the innate immune response. The clinical phenotypes produced by ENPP1 deficiency are disparate, ranging from life-threatening arterial calcifications to cutaneous hypopigmentation. To investigate associations between disease phenotype and enzyme activity we quantified the enzyme velocities of 29 unique ENPP1 pathogenic variants in 41 patients enrolled in an NIH study along with 33 other variants reported in literature. We correlated the relative enzyme velocities with the presenting clinical diagnoses, performing the catalytic velocity measurements simultaneously in triplicate using a high-throughput assay to reduce experimental variation. We found that ENPP1 variants associated with autosomal dominant phenotypes reduced enzyme velocities by 50 % or more, whereas variants associated with insulin resistance had non-significant effects on enzyme velocity. In Cole disease the catalytic velocities of ENPP1 variants associated with AD forms trended to lower values than those associated with autosomal recessive forms - 8-32 % vs. 33 % of WT, respectively. Additionally, ENPP1 variants leading to life-threatening vascular calcifications in GACI patients had widely variable enzyme activities, ranging from no significant differences compared to WT to the complete abolishment of enzyme velocity. Finally, disease severity in GACI did not correlate with the mean enzyme velocity of the variants present in affected compound heterozygotes but did correlate with the more severely damaging variant. In summary, correlation of ENPP1 enzyme velocity with disease phenotypes demonstrate that enzyme velocities below 50 % of WT levels are likely to occur in the context of autosomal dominant disease (due to a monoallelic variant), and that disease severity in GACI infants correlates with the more severely damaging ENPP1 variant in compound heterozygotes, not the mean velocity of the pathogenic variants present.
Assuntos
Fenótipo , Diester Fosfórico Hidrolases , Pirofosfatases , Pirofosfatases/genética , Humanos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Feminino , Variação Genética , Masculino , Mutação/genéticaRESUMO
We recently discovered that chronic high fructose intake by lactating rats prevented adaptive increases in rates of active intestinal Ca(2+) transport and in levels of 1,25-(OH)2D3, the active form of vitamin D. Since sufficient Ca(2+) absorption is essential for skeletal growth, our discovery may explain findings that excessive consumption of sweeteners compromises bone integrity in children. We tested the hypothesis that 1,25-(OH)2D3 mediates the inhibitory effect of excessive fructose intake on active Ca(2+) transport. First, compared with those fed glucose or starch, growing rats fed fructose for 4 wk had a marked reduction in intestinal Ca(2+) transport rate as well as in expression of intestinal and renal Ca(2+) transporters that was tightly associated with decreases in circulating levels of 1,25-(OH)2D3, bone length, and total bone ash weight but not with serum parathyroid hormone (PTH). Dietary fructose increased the expression of 24-hydroxylase (CYP24A1) and decreased that of 1α-hydroxylase (CYP27B1), suggesting that fructose might enhance the renal catabolism and impair the synthesis, respectively, of 1,25-(OH)2D3. Serum FGF23, which is secreted by osteocytes and inhibits CYP27B1 expression, was upregulated, suggesting a potential role of bone in mediating the fructose effects on 1,25-(OH)2D3 synthesis. Second, 1,25-(OH)2D3 treatment rescued the fructose effect and normalized intestinal and renal Ca(2+) transporter expression. The mechanism underlying the deleterious effect of excessive fructose intake on intestinal and renal Ca(2+) transporters is a reduction in serum levels of 1,25-(OH)2D3. This finding is significant because of the large amounts of fructose now consumed by Americans increasingly vulnerable to Ca(2+) and vitamin D deficiency.
Assuntos
Calcitriol/metabolismo , Cálcio/metabolismo , Frutose/efeitos adversos , Absorção Intestinal/efeitos dos fármacos , Absorção Intestinal/fisiologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Fatores Etários , Animais , Desenvolvimento Ósseo/efeitos dos fármacos , Desenvolvimento Ósseo/fisiologia , Osso e Ossos/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Ácido Glucárico/farmacologia , Mucosa Intestinal/metabolismo , Intestinos/crescimento & desenvolvimento , Rim/crescimento & desenvolvimento , Rim/metabolismo , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Esteroide Hidroxilases/genética , Deficiência de Vitamina D/metabolismo , Vitamina D3 24-HidroxilaseRESUMO
We recently showed that excessive fructose consumption, already associated with numerous metabolic abnormalities, reduces rates of intestinal Ca(2+) transport. Using a rat lactation model with increased Ca(2+) requirements, we tested the hypothesis that mechanisms underlying these inhibitory effects of fructose involve reductions in renal synthesis of 1,25-(OH)(2)D(3). Pregnant and virgin (control) rats were fed isocaloric fructose or, as controls, glucose, and starch diets from d 2 of gestation to the end of lactation. Compared to virgins, lactating dams fed glucose or starch had higher rates of intestinal transcellular Ca(2+) transport, elevated intestinal and renal expression of Ca(2+) channels, Ca(2+)-binding proteins, and CaATPases, as well as increased levels of 25-(OH)D(3) and 1,25-(OH)(2)D(3). Fructose consumption prevented almost all of these lactation-induced increases, and reduced vitamin D receptor binding to promoter regions of Ca(2+) channels and binding proteins. Changes in 1,25-(OH)(2)D(3) level were tightly correlated with alterations in expression of 1α-hydroxylase but not with levels of parathyroid hormone and of 24-hydroxylase. Bone mineral density, content, and mechanical strength each decreased with lactation, but then fructose exacerbated these effects. When Ca(2+) requirements increase during lactation or similar physiologically challenging conditions, excessive fructose consumption may perturb Ca(2+) homeostasis because of fructose-induced reductions in synthesis of 1,25-(OH)(2)D(3).
Assuntos
Calcitriol/biossíntese , Sinalização do Cálcio/efeitos dos fármacos , Carboidratos da Dieta/efeitos adversos , Frutose/efeitos adversos , Lactação/efeitos dos fármacos , Lactação/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Adaptação Fisiológica/efeitos dos fármacos , Animais , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea/etiologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Carboidratos da Dieta/administração & dosagem , Feminino , Frutose/administração & dosagem , Histonas/metabolismo , Hipertrofia , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Lactação/genética , Fosfatos/metabolismo , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Calcitriol/metabolismo , Edulcorantes/administração & dosagem , Edulcorantes/efeitos adversos , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismoRESUMO
In prokaryotes and eukaryotes inorganic phosphate plays a vital role in many cellular and biological processes. Maintenance of proper phosphate homeostasis is therefore essential and any deviation from that state can lead to several acute and chronic disease states. In order to maintain physiological levels, a tightly regulated phosphate sensing and signaling mechanism needs to exist. The earliest and best characterized mechanisms of phosphate sensing and signaling have been described in yeast and bacteria involving the Pho regulon. The Pho regulon has been shown to function in a coordinated fashion in order to meet the cellular needs of the organism. Studies have also shown that the protein kinase A (PKA) signaling pathway is involved in phosphate metabolism. In eukaryotes, due to the complexity of the regulatory mechanisms and involvement of several key regulators of phosphate secreted from different organs, it has been difficult to identify the phosphate sensor. Nonetheless the crosstalk between organs in response to phosphate provides strong evidence to support the existence of such a mechanism. This review will focus on this evidence and highlight the parallels tha exist with the Pho regulon. We will focus on the kidney-arathyroid, kidney-intestinal, parathyroid-intestinal, kidney-bone, and finally the parathyroid-bone axes.
Assuntos
Bactérias/genética , Transporte Biológico Ativo/fisiologia , Regulação da Expressão Gênica , Fosfatos/metabolismo , Leveduras/genética , Animais , Bactérias/metabolismo , Homeostase , Humanos , Fenômenos Microbiológicos , Regulon , Transdução de Sinais , Leveduras/metabolismoRESUMO
BACKGROUND: In vivo models of uremia are important tools to study numerous aspects of acute and chronic kidney disease. Mouse models are pivotal because most genetically engineered animal models are mice, which allow dissecting the impact of selected target genes in renal failure. Adenine-based protocols to induce renal failure are available in rats, but have not been adapted in mice due to their reluctance to consume adenine. In the current paper we developed a novel method for induction of renal failure through dietary delivery of adenine mixed in a casein-based diet. RESULTS: After an induction phase, a stable model of renal impairment was obtained (target urea range 80-100 mg/dL), mimicking several aspects of chronic kidney disease - mineral and bone disorder including secondary hyperparathyroidism, bone abnormalities and pathological elevation of FGF23. No deaths occurred and the level of uremia was adaptable through adjustments of the adenine content, providing significant advantages compared to existing models. In an 8-week proof-of-concept study, renal histology showed mainly a tubulointerstitial damage with infiltrating leukocytes, interstitial edema and widening of the Bownman's space. Fibrosis was present in most animals as defined by histology and gene expression changes of fibrosis markers. Parathyroid cell proliferation was markedly increased but without signs of glandular hypertrophy. Skeletal histology showed increased trabecular bone and bone marrow adiposity whereas bone biomarkers (CTX and PINP) suggested higher bone formation, but surprisingly, lower bone resorption and perturbations in mineral metabolism. CONCLUSIONS: We present a novel, non-surgical method for induction of renal failure in mice. This is an important complement to existing uremic models for pathophysiological studies in acute and chronic kidney disease, especially in terms of tubulointerstitial lesions.
Assuntos
Adenina/administração & dosagem , Adenina/toxicidade , Modelos Animais de Doenças , Nefrite Intersticial/induzido quimicamente , Nefrite Intersticial/patologia , Animais , Fator de Crescimento de Fibroblastos 23 , Camundongos , Camundongos Endogâmicos C57BL , Nefrite Intersticial/metabolismoRESUMO
The incidence of cardiovascular events and mortality strongly correlates with serum phosphate in individuals with CKD. The Npt2b transporter contributes to maintaining phosphate homeostasis in the setting of normal renal function, but its role in CKD-associated hyperphosphatemia is not well understood. Here, we used adenine to induce uremia in both Npt2b-deficient and wild-type mice. Compared with wild-type uremic mice, Npt2b-deficient uremic mice had significantly lower levels of serum phosphate and attenuation of FGF23. Treating Npt2b-deficient mice with the phosphate binder sevelamer carbonate further reduced serum phosphate levels. Uremic mice exhibited high turnover renal osteodystrophy; treatment with sevelamer significantly decreased the number of osteoclasts and the rate of mineral apposition in Npt2b-deficient mice, but sevelamer did not affect bone formation and rate of mineral apposition in wild-type mice. Taken together, these data suggest that targeting Npt2b in addition to using dietary phosphorus binders may be a therapeutic approach to modulate serum phosphate in CKD.
Assuntos
Hiperfosfatemia/etiologia , Insuficiência Renal Crônica/complicações , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/deficiência , Animais , Distúrbio Mineral e Ósseo na Doença Renal Crônica/tratamento farmacológico , Distúrbio Mineral e Ósseo na Doença Renal Crônica/etiologia , Distúrbio Mineral e Ósseo na Doença Renal Crônica/metabolismo , Modelos Animais de Doenças , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Hiperfosfatemia/metabolismo , Camundongos , Camundongos Knockout , Poliaminas/farmacologia , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/metabolismo , Sevelamer , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/genética , Uremia/complicações , Uremia/metabolismoRESUMO
BACKGROUND: Inorganic pyrophosphate (PPi) is highly regulated as it plays a critical role in the regulation of physiological mineralization. Dysregulation of plasma PPi is associated with skeletal hypomineralization and pathogenic mineralization in soft connective tissue, arteries, and heart valves. There is no standard approach to measuring PPi, making it difficult to establish PPi as a biomarker of mineralization disorders. This study aims to determine the impact of time of day, meals, or exercise on plasma PPi homeostasis using a highly sensitive PPi assay. METHODS: In this single-center trial, a clinical laboratory improvement amendment (CLIA) validated modified sulfurylase-based adenosine 5-triphosphate (ATP) assay was used to measure PPi levels throughout the day in 10 healthy adults under 3 conditions; normal diet (non-fasting), fasting, and normal diet with exercise. Serum ectonucleotide pyrophosphatase/phosphodiesterase 1 activity (ENPP1; an enzyme that produces PPi) was also measured to determine whether these conditions influence PPi levels through ENPP1 activity. RESULTS: There is a circadian increase in mean PPi levels under fasting and non-fasting conditions between 8 am and 6 pm, followed by a rapid return to baseline overnight. A circadian increase in ENPP1 activity was also measured under fasting but was lost under non-fasting conditions. Meals increased the individual variability of PPi levels when compared to the same individual fasting. PPi levels and ENPP1 activity exhibited a short-term increase after intense exercise. We found PPi ranges from 1465 nM to 2969 nM (mean 2164 nM) after fasting overnight. Within this range, there was lower intra-subject variability in PPi, suggesting that each individual has a uniquely regulated normal PPi range. CONCLUSION: Plasma levels of PPi can be reliably measured after an overnight fast and show promise as a biomarker of mineralization disorders.
Assuntos
Calcinose , Sistema Cardiovascular , Adulto , Humanos , Trifosfato de Adenosina , Calcinose/patologia , Difosfatos , Diester Fosfórico Hidrolases , Pirofosfatases , Jejum , AlimentosAssuntos
Medula Óssea/metabolismo , Osso e Ossos/metabolismo , Eritropoetina/farmacologia , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , CamundongosRESUMO
PURPOSE: Generalized arterial calcification of infancy, pseudoxanthoma elasticum, autosomal recessive hypophosphatemic rickets type 2, and hypophosphatasia are rare inherited disorders associated with altered plasma levels of inorganic pyrophosphate (PPi). In this study, we aimed to establish a reference range for plasma PPi in the pediatric population, which would be essential to support its use as a biomarker in children with mineralization disorders. METHODS: Plasma samples were collected from 200 children aged 1 day to 18 years who underwent blood testing for medical conditions not affecting plasma PPi levels. PPi was measured in proband plasma utilizing a validated adenosine triphosphate (ATP) sulfurylase method. RESULTS: The analytical sensitivity of the ATP sulfurylase assay consisted of 0.15 to 10 µM PPi. Inter- and intra-assay coefficients of variability on identical samples were below 10%. The standard range of PPi in the blood plasma of children and adolescents aged 0 to 18 years was calculated as 2.36 to 4.44 µM, with a median of 3.17 µM, with no difference between male and female probands. PPi plasma levels did not differ significantly in different pediatric age groups. MAIN CONCLUSIONS: Our results yielded no noteworthy discrepancy to the reported standard range of plasma PPi in adults (2-5 µM). We propose the described ATP sulfurylase method as a diagnostic tool to measure PPi levels in plasma as a biomarker in the pediatric population.
Assuntos
Raquitismo Hipofosfatêmico Familiar/diagnóstico , Hipofosfatasia/diagnóstico , Fosfatos/sangue , Pseudoxantoma Elástico/diagnóstico , Doenças Raras/diagnóstico , Adolescente , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Biomarcadores/sangue , Criança , Pré-Escolar , Ensaios Enzimáticos/métodos , Ensaios Enzimáticos/normas , Raquitismo Hipofosfatêmico Familiar/sangue , Raquitismo Hipofosfatêmico Familiar/genética , Feminino , Humanos , Hipofosfatasia/sangue , Hipofosfatasia/genética , Lactente , Recém-Nascido , Masculino , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Mutação , Fosfatos/metabolismo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Pseudoxantoma Elástico/sangue , Pseudoxantoma Elástico/genética , Pirofosfatases/genética , Pirofosfatases/metabolismo , Doenças Raras/sangue , Doenças Raras/genética , Valores de Referência , Sulfato Adenililtransferase/metabolismoRESUMO
We have previously shown that the retinoblastoma protein (pRb) can activate expression of Runx2-dependent, bone-specific genes in cultured cells. We now show that pRb also plays a role early in osteogenesis, and that in primary RB1(-/-) calvarial cells there is an increased osteoprogenitor pool. To understand pRb's function in vivo, we generated a conditional RB1-KO mouse in which pRb expression is efficiently extinguished in osteoblasts. These animals display an apparent developmental defect in bones, most strikingly in the calvaria. Cultured RB1(-/-) calvarial osteoblasts fail to cease proliferation upon reaching confluence or following differentiation. Re-plating assays of primary RB1(-/-) calvarial cells after differentiation showed a clear adipogenic ability with increased multipotency. RB1(-/-) osteoblasts display a severe reduction in levels of mRNAs expressed late in differentiation. In this study, we present strong evidence that pRb has multiple regulatory roles in osteogenesis. Furthermore, in the absence of RB1(-/-) there is a larger pool of multipotent cells compared with the WT counterpart. This increased pool of osteoprogenitor cells may be susceptible to additional transforming events leading to osteosarcoma, and is therefore key to understanding RB1 as a target in malignancy.
Assuntos
Desenvolvimento Ósseo , Células-Tronco Mesenquimais/citologia , Proteína do Retinoblastoma/fisiologia , Crânio/citologia , Animais , Proliferação de Células , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Proteína do Retinoblastoma/genéticaRESUMO
Renal disease leads to perturbations in calcium and phosphate homeostasis and vitamin D metabolism. Dietary fructose aggravates chronic kidney disease (CKD), but whether it also worsens CKD-induced derangements in calcium and phosphate homeostasis is unknown. Here, we fed rats diets containing 60% glucose or fructose for 1 mo beginning 6 wk after 5/6 nephrectomy or sham operation. Nephrectomized rats had markedly greater kidney weight, blood urea nitrogen, and serum levels of creatinine, phosphate, and calcium-phosphate product; dietary fructose significantly exacerbated all of these outcomes. Expression and activity of intestinal phosphate transporter, which did not change after nephrectomy or dietary fructose, did not correlate with hyperphosphatemia in 5/6-nephrectomized rats. Intestinal transport of calcium, however, decreased with dietary fructose, probably because of fructose-mediated downregulation of calbindin 9k. Serum calcium levels, however, were unaffected by nephrectomy and diet. Finally, only 5/6-nephrectomized rats that received dietary fructose demonstrated marked reductions in 25-hydroxyvitamin D(3) and 1,25-dihydroxyvitamin D(3) levels, despite upregulation of 1alpha-hydroxylase. In summary, excess dietary fructose inhibits intestinal calcium absorption, induces marked vitamin D insufficiency in CKD, and exacerbates other classical symptoms of the disease. Future studies should evaluate the relevance of monitoring fructose consumption in patients with CKD.
Assuntos
Cálcio/metabolismo , Carboidratos da Dieta/farmacologia , Frutose/farmacologia , Absorção Intestinal/efeitos dos fármacos , Nefropatias/metabolismo , Deficiência de Vitamina D/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Animais , Densidade Óssea , Calcifediol/metabolismo , Calcitriol/metabolismo , Doença Crônica , Carboidratos da Dieta/efeitos adversos , Modelos Animais de Doenças , Frutose/efeitos adversos , Glucose/farmacologia , Rim/metabolismo , Rim/cirurgia , Nefropatias/complicações , Masculino , Nefrectomia , Fosfatos/metabolismo , Ratos , Ratos Sprague-Dawley , Deficiência de Vitamina D/etiologiaRESUMO
Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is the major enzyme that cleaves extracellular adenosine triphosphate (ATP) to generate pyrophosphate (PPi), an inorganic metabolite with potent anticalcification activity. Loss-of-function mutations cause hypopyrophosphatemia and lead to a state of ENPP1 deficiency, which has an acute infantile phase known as generalized arterial calcification of infancy (GACI) and a pediatric to adult phase known as autosomal-recessive hypophosphatemic rickets type 2 (ARHR2). ENPP1 deficiency manifests as ectopic calcification of multiple tissues, neointimal proliferation, premature mortality, impaired growth, and bone deformities. INZ-701, a human ENPP1-Fc protein, is in clinical development as an enzyme replacement therapy for the treatment of ENPP1 deficiency. The pharmacokinetic and pharmacodynamic profile and therapeutic effect of INZ-701 were investigated in Enpp1asj/asj mice, a murine model of ENPP1 deficiency. Enpp1asj/asj mice have undetectable plasma PPi, lower plasma phosphate, and higher FGF23 levels compared with wild-type (WT) mice. Enpp1asj/asj mice on the acceleration diet, containing high phosphate and low magnesium, quickly develop clinical signs, including dehydration, rough hair coat, pinned ears, stiffed legs, and hunched back. Enpp1asj/asj mice treated with vehicle had aforementioned clinical signs plus severe ectopic calcification in multiple tissues and bone defects, characteristics of the clinical phenotype observed in GACI and ARHR2 patients. Our results showed a durable PPi response for more than 3 days after a single dose of INZ-701. Treatment of ENPP1-deficient mice every other day with INZ-701 for 8 weeks restored circulating levels of PPi, prevented pathological calcification in all the tested organs, restored growth parameters, corrected bone defects, improved clinical signs, and decreased mortality in Enpp1asj/asj mice, demonstrating the potential of INZ-701 to treat ENPP1 deficiency. © 2021 American Society for Bone and Mineral Research (ASBMR).
Assuntos
Coristoma , Raquitismo Hipofosfatêmico Familiar , Calcificação Vascular , Adulto , Animais , Criança , Fator de Crescimento de Fibroblastos 23 , Humanos , Camundongos , Diester Fosfórico Hidrolases/genética , Pirofosfatases/genética , Calcificação Vascular/tratamento farmacológico , Calcificação Vascular/genéticaRESUMO
Osteogenesis imperfecta (OI), is a genetic disorder of bone fragility caused by mutations in collagen I or proteins involved in collagen processing. Previous studies in mice and human OI bones have shown that excessive activation of TGF-ß signaling plays an important role in dominant and recessive OI disease progression. Inhibition of TGF-ß signaling with a murine pan-specific TGF-ß neutralizing antibody (1D11) was shown to significantly increase trabecular bone volume and long bone strength in mouse models of OI. To investigate the frequency of dosing and dose options of TGF-ß neutralizing antibody therapy, we assessed the effect of 1D11 on disease progression in a dominant OI mouse model (col1a2 gene mutation at G610C). In comparison with OI mice treated with a control antibody, we attempted to define mechanistic effects of 1D11 measured via µCT, biomechanical, dynamic histomorphometry, and serum biomarkers of bone turnover. In addition, osteoblast and osteoclast numbers in histological bone sections were assessed to better understand the mechanism of action of the 1D11 antibody in OI. Here we show that 1D11 treatment resulted in both dose and frequency dependency, increases in trabecular bone volume fraction and ultimate force in lumbar bone, and ultimate force, bending strength, yield force, and yield strength in the femur (p ≤ 0.05). Suppression of serum biomarkers of osteoblast differentiation, osteocalcin, resorption, CTx-1, and bone formation were observed after 1D11 treatment of OI mice. Immunohistochemical analysis showed dose and frequency dependent decreases in runt-related transcription factor, and increase in alkaline phosphatase in lumbar bone sections. In addition, a significant decrease in TRACP and the number of osteoclasts to bone surface area was observed with 1D11 treatment. Our results show that inhibition of the TGF-ß pathway corrects the high-turnover aspects of bone disease and improves biomechanical properties of OI mice. These results highlight the potential for a novel treatment for osteogenesis imperfecta. © 2021 Sanofi-Genzyme. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
RESUMO
Intestinal phosphate absorption occurs through both a paracellular mechanism involving tight junctions and an active transcellular mechanism involving the type II sodium-dependent phosphate cotransporter NPT2b (SLC34a2). To define the contribution of NPT2b to total intestinal phosphate absorption, we generated an inducible conditional knockout mouse, Npt2b(-/-) (Npt2b(fl/fl):Cre(+/-)). Npt2b(-/-) animals had increased fecal phosphate excretion and hypophosphaturia, but serum phosphate remained unchanged. Decreased urinary phosphate excretion correlated with reduced serum levels of the phosphaturic hormone FGF23 and increased protein expression of the renal phosphate transporter Npt2a. These results demonstrate that the absence of Npt2b triggers compensatory renal mechanisms to maintain phosphate homeostasis. In animals fed a low phosphate diet followed by acute administration of a phosphate bolus, Npt2b(-/-) animals absorbed approximately 50% less phosphate than wild-type animals, confirming a major role of this transporter in phosphate regulation. In vitro analysis of active phosphate transport in ileum segments isolated from wild-type or Npt2b(-/-) mice demonstrated that Npt2b contributes to >90% of total active phosphate absorption. In summary, Npt2b is largely responsible for intestinal phosphate absorption and contributes to the maintenance of systemic phosphate homeostasis.