RESUMEN
BACKGROUND: The Enhanced Liver Fibrosis (ELFTM) Test comprises 3 direct serum markers of fibrosis-hyaluronic acid (HA), amino-terminal pro-peptide of type III procollagen (PIIINP), and tissue inhibitor of matrix metalloproteinase 1 (TIMP-1)-whose results are combined in an algorithm to generate the ELF score. Outside the U.S., the ELF Test and score are CE marked for assessment of liver fibrosis severity in patients with signs, symptoms, or risk factors of chronic liver disease to support diagnosis of fibrosis staging or prognosis for likelihood of progression to cirrhosis and liver-related clinical events. In the U.S., the FDA granted de novo marketing authorization to aid prognostic evaluation of disease progression (to cirrhosis and liver-related clinical events) in nonalcoholic steatohepatitis patients with advanced liver fibrosis. We describe the analytical performance of the ELF analytes and score on the Atellica® IM Analyzer. METHODS: Clinical and Laboratory Standards Institute protocols were followed for detection capability (limits of blank [LoB], detection [LoD], and quantitation [LoQ]), precision, interference, linearity, hook effect, and ELF reference interval. RESULTS: All parameters met predetermined requirements: HA (LoB 1.00 ng/mL, LoD 2.00 ng/mL, LoQ 3.00 ng/mL); PIIINP (LoB 0.50 ng/mL, LoD 0.75 ng/mL, LoQ 1.00 ng/mL); TIMP-1 (LoB 3.0 ng/mL, LoD 4.0 ng/mL, LoQ 5.0 ng/mL). Across the 3 assays, repeatability was ≤5.4% CV; within-lab precision was ≤8.5% CV. ELF score repeatability was ≤0.6% CV, within-lab precision ≤1.3% CV, and reproducibility ≤1.1% CV. Good correlation was obtained between the Atellica IM ELF and ADVIA Centaur ELF Tests (y = 1.01x - 0.22, r = 0.997). Assays were linear across analytical measuring ranges. CONCLUSIONS: Analytical performance validation results for the ELF Test and ELF score were excellent making the test acceptable for routine clinical use.
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Cirrosis Hepática , Inhibidor Tisular de Metaloproteinasa-1 , Humanos , Reproducibilidad de los Resultados , Fibrosis , Hígado/patología , Biomarcadores , Ácido HialurónicoRESUMEN
BACKGROUND: Current serological methods for SARS-CoV-2 lack adequate standardization to a universal standard reference material. Standardization will allow comparison of results across various lab-developed and commercial assays and publications. SARS-CoV-2 EURM-017 is human sera reference material containing antibodies directed against SARS-CoV-2 proteins, S1/S2 (full-length spike [S]), S1 receptor-binding domain (S1 RBD), S1, S2, and nucleocapsid (N) protein. The goal of this study was to characterize five antigen-specific serum fractions in EURM-017 for standardization of serology assays. METHODS: Five antigen-specific serum fractions were affinity purified, quantified, and PRNT50 titers compared. Standardization methods were established for two anti-S1 RBD (IgG and Total Ig) and one N protein assay. For the anti-S1 RBD assays, standardization involved determining assay index values for serial dilutions of S1-RBD anti-sera. Index values for the anti-S1 RBD IgG assay and PRNT50 titers were determined for 44 symptomatic COVID-19 patient sera. The index values were converted to EURM-017 ug/mL. RESULTS: Anti-sera protein content was as follows: S1 (17.7 µg/mL), S1 RBD (17.4 µg/mL), S1/S2 (full-length S) (34.1 µg/mL), S2 (29.7 µg/mL), and N protein (72.5 µg/mL). S1 anti-serum had the highest neutralization activity. A standardization method for S1 RBD anti-serum and an anti-S1 RBD IgG assay yielded the linear equation (y = 0.75x-0.10; y = index, x=µg/mL anti-serum). Patient sample index values for the S1-RBD IgG assay correlated well with PRNT50 titers (Pearson r = 0.84). Using the equation above, patient index values were converted to standardized µg/mL. CONCLUSIONS: Standardization of different lab-developed and commercial assays to EURM-017 antigen-specific anti-sera will allow comparison of results across studies globally due to traceability to a single standard reference material.
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Anticuerpos Antivirales/sangre , Prueba Serológica para COVID-19/normas , COVID-19/diagnóstico , SARS-CoV-2/inmunología , COVID-19/sangre , Prueba Serológica para COVID-19/métodos , Humanos , Inmunoensayo/normas , Inmunoglobulina G/sangre , Estándares de ReferenciaRESUMEN
OBJECTIVES: The ability of current immunoassays to accurately measure equimolar amounts of 25(OH)D2 and 25(OH)D3 has been recently questioned. This study determined serum 25(OH)D2, 25(OH)D3 and total serum 25(OH)D concentrations in healthy vitamin D2-supplemented subjects by isotope dilution liquid chromatography mass spectrometry (ID-LC-MS/MS); and, evaluated the ability of the Siemens, DiaSorin, Roche, and Abbott Vitamin D Total assays to monitor total serum 25(OH)D concentrations compared to an ID-LC-MS/MS method traceable to the National Institute of Standards and Technology (NIST), and that has achieved certification from the Centers for Disease Control and Prevention (CDC) Vitamin D Standardization Certification Program (VDSCP). DESIGN AND METHODS: Twenty (20) healthy adults, with no history of prior vitamin D supplementation were administered oral vitamin D2 (2400IU/day for 6months). Serum samples (140) from baseline and monthly blood draws were tested. RESULTS: After one month, the mean serum 25(OH)D2 concentrations rose from 0.8 to 43.6nmol/L, whereas 25(OH)D3 concentrations declined from 84.0 to 63.4nmol/L; total serum 25(OH)D concentrations rose from 86.6 to 107.0nmol/L. The overall mean bias to ID-LC-MS/MS was -7.1% for the Siemens ADVIA Centaur assay, -15.3% for the DiaSorin LIAISON assay; -8.4% for the Roche ELECSYS assay and -16.3% for the Abbott ARCHITECT assay. Correlation coefficients (r) were 0.94, 0.79, 0.74, and 0.73; the mean bias for baseline [25(OH) D3-containing] versus six-month [25(OH)D2- and 25(OH)D3-containing] samples was -13.4% and -5.7%; -3.5% and 20.3%, 9.6% and -12.1%, and 0.2% and -17.8%, respectively. CONCLUSIONS: The bias results obtained for the Siemens ADVIA Centaur assay and Roche ELECSYS assay were slightly lower than those for the DiaSorin LIAISON assay and the Abbott ARCHITECT assay, but all 25(OH)D assays demonstrated acceptable performance.
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25-Hidroxivitamina D 2/sangre , 25-Hidroxivitamina D 2/química , Bioensayo/métodos , Vitamina D/análogos & derivados , Adulto , Anciano , Cromatografía Liquida/métodos , Suplementos Dietéticos , Femenino , Humanos , Inmunoensayo/métodos , Masculino , Persona de Mediana Edad , Estándares de Referencia , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodos , Vitamina D/sangre , Vitamina D/química , Adulto JovenRESUMEN
Chronic kidney disease (CKD) is a worldwide health problem. Serum levels of FGF23, a phosphaturic hormone, increase at the earliest stages of CKD, and have been found to be independently associated with the mortality and morbidity of CKD patients. The purpose of this study was to evaluate whether FGF23 neutralization was able to improve bone quality and osseointegration of titanium implants. Uremia was induced by 5/6 nephrectomy in adult female mice. Postsurgery, the mice were injected with vehicle or FGF23 neutralizing antibody (5â mg/kg body weight) 3 times a week. Experimental titanium implants were inserted in the distal end of the femurs. FGF23 neutralization significantly increased serum phosphate, 1,25(OH)2D and BUN, and decreased serum PTH and FGF23, relative to vehicle-treated CKD mice. Histomorphometric analysis of the tibiae indicated that FGF23 neutralization normalized the osteoidosis observed in vehicle-treated CKD mice. Although bone-implant contact ratio remained unchanged by anti-FGF23 antibody treatment, the strength of osseointegration, as evidenced by a biomechanical push-in test, was significantly improved by FGF23 neutralization. Our findings revealed that FGF23 neutralization effectively improves bone quality and osseointegration of titanium implants in CKD mice, suggesting FGF23 as a key factor of CKD related bone diseases.
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Anticuerpos Neutralizantes/farmacología , Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Implantes Experimentales , Oseointegración/efectos de los fármacos , Insuficiencia Renal Crónica/metabolismo , Titanio , Animales , Femenino , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/metabolismo , Ratones , Insuficiencia Renal Crónica/patología , Tibia/metabolismo , Tibia/patologíaRESUMEN
[This corrects the article DOI: 10.1155/2014/691679.].
RESUMEN
Vitamin D status in different populations relies on accurate measurement of total serum 25-hydroxyvitamin D [25(OH)D] concentrations [i.e., 25(OH)D3 and 25(OH)D2]. This study evaluated agreement between the ADVIA Centaur Vitamin D Total assay for 25(OH)D testing (traceable to the NIST-Ghent reference method procedure) and a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for various populations with different levels of vitamin D binding protein (DBP). Total serum 25(OH)D concentrations were measured for 36 pregnant women, 40 hemodialysis patients, and 30 samples (DBP-spiked or not) from healthy subjects. ELISA measured DBP levels. The mean serum DBP concentrations were higher for pregnancy (415 µg/mL) and lower for hemodialysis subjects (198 µg/mL) than for healthy subjects and were highest for spiked serum (545 µg/mL). The average bias between the ADVIA Centaur assay and the LC-MS/MS method was -1.4% (healthy), -6.1% (pregnancy), and 4.4% (hemodialysis). The slightly greater bias for samples from some pregnancy and hemodialysis subjects with serum DBP levels outside of the normal healthy range fell within a clinically acceptable range-reflected by analysis of their low-range (≤136 µg/mL), medium-range (137-559 µg/mL), and high-range (≥560 µg/mL) DBP groups. Thus, the ADVIA Centaur Vitamin D Total assay demonstrates acceptable performance compared with an LC-MS/MS method for populations containing different amounts of DBP.
RESUMEN
Fibroblast growth factor-23 (FGF23) is a bone-derived hormone regulating renal phosphate reabsorption and vitamin D synthesis in renal proximal tubules. Here, we show that FGF23 directly regulates the membrane abundance of the Na(+):Cl(-) co-transporter NCC in distal renal tubules by a signaling mechanism involving the FGF receptor/αKlotho complex, extracellular signal-regulated kinase 1/2 (ERK1/2), serum/glucocorticoid-regulated kinase 1 (SGK1), and with-no lysine kinase-4 (WNK4). Renal sodium (Na(+)) reabsorption and distal tubular membrane expression of NCC are reduced in mouse models of Fgf23 and αKlotho deficiency. Conversely, gain of FGF23 function by injection of wild-type mice with recombinant FGF23 or by elevated circulating levels of endogenous Fgf23 in Hyp mice increases distal tubular Na(+) uptake and membrane abundance of NCC, leading to volume expansion, hypertension, and heart hypertrophy in a αKlotho and dietary Na(+)-dependent fashion. The NCC inhibitor chlorothiazide abrogates FGF23-induced volume expansion and heart hypertrophy. Our findings suggest that FGF23 is a key regulator of renal Na(+) reabsorption and plasma volume, and may explain the association of FGF23 with cardiovascular risk in chronic kidney disease patients.
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Presión Sanguínea , Factores de Crecimiento de Fibroblastos/metabolismo , Hipertensión/metabolismo , Riñón/metabolismo , Sodio/metabolismo , Animales , Antihipertensivos/uso terapéutico , Cardiomegalia/metabolismo , Clorotiazida/uso terapéutico , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/administración & dosificación , Factores de Crecimiento de Fibroblastos/genética , Eliminación de Gen , Glucuronidasa/genética , Humanos , Hipertensión/tratamiento farmacológico , Hipertensión/genética , Proteínas Klotho , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Inhibidores de los Simportadores del Cloruro de Sodio/uso terapéutico , Miembro 3 de la Familia de Transportadores de Soluto 12/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Regulación hacia ArribaRESUMEN
αKlotho is thought to activate the epithelial calcium channel Transient Receptor Potential Vanilloid-5 (TRPV5) in distal renal tubules through its putative glucuronidase/sialidase activity, thereby preventing renal calcium loss. However, αKlotho also functions as the obligatory co-receptor for fibroblast growth factor-23 (FGF23), a bone-derived phosphaturic hormone. Here, we show that renal calcium reabsorption and renal membrane abundance of TRPV5 are reduced in Fgf23 knockout mice, similar to what is seen in αKlotho knockout mice. We further demonstrate that αKlotho neither co-localizes with TRPV5 nor is regulated by FGF23. Rather, apical membrane abundance of TRPV5 in renal distal tubules and thus renal calcium reabsorption are regulated by FGF23, which binds the FGF receptor-αKlotho complex and activates a signaling cascade involving ERK1/2, SGK1, and WNK4. Our data thereby identify FGF23, not αKlotho, as a calcium-conserving hormone in the kidney.
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Calcio/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Riñón/metabolismo , Receptores de Superficie Celular/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Membrana Celular/metabolismo , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/genética , Glucuronidasa , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Klotho , Masculino , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de SeñalRESUMEN
Chronic kidney disease-mineral and bone disorder (CKD-MBD) is associated with secondary hyperparathyroidism (HPT) and serum elevations in the phosphaturic hormone FGF23, which may be maladaptive and lead to increased morbidity and mortality. To determine the role of FGF23 in the pathogenesis of CKD-MBD and development of secondary HPT, we developed a monoclonal FGF23 antibody to evaluate the impact of chronic FGF23 neutralization on CKD-MBD, secondary HPT, and associated comorbidities in a rat model of CKD-MBD. CKD-MBD rats fed a high-phosphate diet were treated with low or high doses of FGF23-Ab or an isotype control antibody. Neutralization of FGF23 led to sustained reductions in secondary HPT, including decreased parathyroid hormone, increased vitamin D, increased serum calcium, and normalization of bone markers such as cancellous bone volume, trabecular number, osteoblast surface, osteoid surface, and bone-formation rate. In addition, we observed dose-dependent increases in serum phosphate and aortic calcification associated with increased risk of mortality in CKD-MBD rats treated with FGF23-Ab. Thus, mineral disturbances caused by neutralization of FGF23 limited the efficacy of FGF23-Ab and likely contributed to the increased mortality observed in this CKD-MBD rat model.
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Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Hiperparatiroidismo Secundario/metabolismo , Insuficiencia Renal Crónica/metabolismo , Animales , Anticuerpos Monoclonales de Origen Murino/farmacología , Aorta/patología , Biomarcadores/metabolismo , Células CHO , Calcitriol/sangre , Calcio/sangre , Trastorno Mineral y Óseo Asociado a la Enfermedad Renal Crónica/sangre , Trastorno Mineral y Óseo Asociado a la Enfermedad Renal Crónica/metabolismo , Trastorno Mineral y Óseo Asociado a la Enfermedad Renal Crónica/fisiopatología , Cricetinae , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/inmunología , Factores de Crecimiento de Fibroblastos/metabolismo , Genes Reporteros , Tasa de Filtración Glomerular , Hemodinámica , Hiperparatiroidismo Secundario/sangre , Hiperparatiroidismo Secundario/fisiopatología , Riñón/patología , Riñón/fisiopatología , Luciferasas/biosíntesis , Luciferasas/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Miocardio/patología , Hormona Paratiroidea/sangre , Fosfatos/sangre , Ratas , Ratas Sprague-Dawley , Insuficiencia Renal Crónica/sangre , Insuficiencia Renal Crónica/fisiopatología , Tibia/metabolismo , Tibia/patología , Calcificación Vascular/patologíaRESUMEN
Fibroblast growth factor 23 (FGF23) modulates mineral metabolism by promoting phosphaturia and decreasing the production of 1,25-dihydroxyvitamin D(3). FGF23 decreases parathyroid hormone (PTH) mRNA and secretion, but despite a marked elevation in FGF23 in uremia, PTH production increases. Here, we investigated the effect of FGF23 on parathyroid function in normal and uremic hyperplastic parathyroid glands in rats. In normal parathyroid glands, FGF23 decreased PTH production, increased expression of both the parathyroid calcium-sensing receptor and the vitamin D receptor, and reduced cell proliferation. Furthermore, FGF23 induced phosphorylation of extracellular signal-regulated kinase 1/2, which mediates the action of FGF23. In contrast, in hyperplastic parathyroid glands, FGF23 did not reduce PTH production, did not affect expression of the calcium-sensing receptor or vitamin D receptor, and did not affect cell proliferation. In addition, FGF23 failed to activate the extracellular signal-regulated kinase 1/2-mitogen-activated protein kinase pathway in hyperplastic parathyroid glands. We observed very low expression of the FGF23 receptor 1 and the co-receptor Klotho in uremic hyperplastic parathyroid glands, which may explain the lack of response to FGF23 in this tissue. In conclusion, in hyperparathyroidism secondary to renal failure, the parathyroid cells resist the inhibitory effects of FGF23, perhaps as a result of the low expression of FGF23 receptor 1 and Klotho in this condition.
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Factores de Crecimiento de Fibroblastos/farmacología , Glándulas Paratiroides/metabolismo , Hormona Paratiroidea/metabolismo , Uremia/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Glucuronidasa/metabolismo , Hiperplasia/metabolismo , Hiperplasia/patología , Proteínas Klotho , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Glándulas Paratiroides/efectos de los fármacos , Glándulas Paratiroides/patología , Ratas , Ratas Wistar , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptores de Calcitriol/metabolismo , Receptores Sensibles al Calcio/metabolismo , Técnicas de Cultivo de Tejidos , Uremia/patologíaRESUMEN
Calcimimetics and vitamin D sterols reduce serum parathyroid hormone (PTH) in patients with secondary hyperparathyroidism receiving dialysis, a disease state associated with parathyroid hyperplasia, vascular calcification, bone disease, and increased mortality. The aim of this study was to determine the effects of the research calcimimetic AMG 641 (Amgen, Inc., Thousand Oaks, CA) or calcitriol (Sigma Aldrich Corporation, St. Louis, MO) on vascular calcification in a rodent model of progressive uremia with accompanying secondary hyperparathyroidism induced by dietary adenine. Treatment effects on parathyroid gland hyperplasia and bone loss were also investigated. Rats were treated daily with vehicle, calcitriol (10 ng), AMG 641 (3 mg/kg), or no treatment during the 4 week period the animals were fed adenine. The uremia-induced increases in serum PTH levels were significantly attenuated by both AMG 641 (>90%) and calcitriol (approximately 50%). AMG 641 significantly reduced calcium-phosphorus product (CaxP) and significantly attenuated the development of both parathyroid hyperplasia and vascular calcification. In addition, AMG 641 prevented the defects in trabecular bone volume, trabecular number, and bone mineralization, as well as increases in trabecular spacing in this rodent model of secondary hyperparathyroidism. Calcitriol (10 ng/rat) decreased osteoid surface/bone surface, but had no effects on other bone parameters, or parathyroid hyperplasia (likely due to the lower PTH suppressive effect of calcitriol at the dose used in this study). However, this dose of calcitriol significantly exacerbated vascular calcification. These results suggest that calcimimetics can reduce the development of vascular calcification, parathyroid hyperplasia and bone abnormalities associated with secondary hyperparathyroidism.
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Materiales Biomiméticos/farmacología , Compuestos de Bifenilo/farmacología , Huesos/efectos de los fármacos , Calcinosis/complicaciones , Calcio/metabolismo , Glándulas Paratiroides/irrigación sanguínea , Glándulas Paratiroides/efectos de los fármacos , Fenetilaminas/farmacología , Uremia/complicaciones , Adenina/farmacología , Animales , Materiales Biomiméticos/química , Compuestos de Bifenilo/química , Huesos/metabolismo , Calcinosis/sangre , Calcinosis/metabolismo , Calcinosis/patología , Cinacalcet , Relación Dosis-Respuesta a Droga , Hiperplasia/sangre , Hiperplasia/complicaciones , Hiperplasia/metabolismo , Hiperplasia/patología , Masculino , Peso Molecular , Naftalenos/farmacología , Tamaño de los Órganos/efectos de los fármacos , Glándulas Paratiroides/metabolismo , Glándulas Paratiroides/patología , Hormona Paratiroidea/sangre , Fenetilaminas/química , Ratas , Ratas Sprague-Dawley , Uremia/inducido químicamenteRESUMEN
The development of bone-rebuilding anabolic agents for potential use in the treatment of bone loss conditions, such as osteoporosis, has been a long-standing goal. Genetic studies in humans and mice have shown that the secreted protein sclerostin is a key negative regulator of bone formation, although the magnitude and extent of sclerostin's role in the control of bone formation in the aging skeleton is still unclear. To study this unexplored area of sclerostin biology and to assess the pharmacologic effects of sclerostin inhibition, we used a cell culture model of bone formation to identify a sclerostin neutralizing monoclonal antibody (Scl-AbII) for testing in an aged ovariectomized rat model of postmenopausal osteoporosis. Six-month-old female rats were ovariectomized and left untreated for 1 yr to allow for significant estrogen deficiency-induced bone loss, at which point Scl-AbII was administered for 5 wk. Scl-AbII treatment in these animals had robust anabolic effects, with marked increases in bone formation on trabecular, periosteal, endocortical, and intracortical surfaces. This not only resulted in complete reversal, at several skeletal sites, of the 1 yr of estrogen deficiency-induced bone loss, but also further increased bone mass and bone strength to levels greater than those found in non-ovariectomized control rats. Taken together, these preclinical results establish sclerostin's role as a pivotal negative regulator of bone formation in the aging skeleton and, furthermore, suggest that antibody-mediated inhibition of sclerostin represents a promising new therapeutic approach for the anabolic treatment of bone-related disorders, such as postmenopausal osteoporosis.
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Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Proteínas Morfogenéticas Óseas/inmunología , Huesos/efectos de los fármacos , Marcadores Genéticos/inmunología , Osteogénesis/efectos de los fármacos , Osteoporosis Posmenopáusica/tratamiento farmacológico , Animales , Bioensayo , Fenómenos Biomecánicos , Densidad Ósea/efectos de los fármacos , Huesos/patología , Linaje de la Célula/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Fémur/efectos de los fármacos , Fémur/patología , Humanos , Vértebras Lumbares/efectos de los fármacos , Vértebras Lumbares/patología , Ratones , Pruebas de Neutralización , Tamaño de los Órganos/efectos de los fármacos , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteocalcina/sangre , Osteoporosis Posmenopáusica/sangre , Osteoporosis Posmenopáusica/patología , Osteoporosis Posmenopáusica/fisiopatología , Ovariectomía , Ratas , Ratas Sprague-Dawley , Tibia/efectos de los fármacos , Tibia/patología , Tomografía Computarizada por Rayos XRESUMEN
UNLABELLED: Skeletal anabolism with PTH is achieved through daily injections that result in brief exposure to the peptide. We hypothesized that similar anabolic effects could be achieved with less frequent but more sustained exposures to PTH. A PTH-Fc fusion protein with a longer half-life than PTH(1-34) increased cortical and cancellous BMD and bone strength with once- or twice-weekly injections. INTRODUCTION: The anabolic effects of PTH are currently achieved with, and thought to require, daily injections that result in brief exposure to the peptide. We hypothesized that less frequent but more sustained exposures to PTH could also be anabolic for bone, provided that serum levels of PTH were not constant. MATERIALS AND METHODS: PTH(1-34) was fused to the Fc fragment of human IgG1 to increase the half-life of PTH. Skeletal anabolism was examined in mice and rats treated once or twice per week with this PTH-Fc fusion protein. RESULTS: PTH-Fc and PTH(1-34) had similar effects on PTH/PTHrP receptor activation, internalization, and signaling in vitro. However, PTH-Fc had a 33-fold longer mean residence time in the circulation of rats compared with that of PTH(1-34). Subcutaneous injection of PTH-Fc once or twice per week resulted in significant increases in bone volume, density, and strength in osteopenic ovariectomized mice and rats. These anabolic effects occurred in association with hypercalcemia and were significantly greater than those achievable with high concentrations of daily PTH(1-34). PTH-Fc also significantly improved cortical bone volume and density under conditions where daily PTH(1-34) did not. Antiresorptive co-therapy with estrogen further enhanced the ability of PTH-Fc to increase bone mass and strength in ovariectomized rats. CONCLUSIONS: These results challenge the notion that brief daily exposure to PTH is essential for its anabolic effects on cortical and cancellous bone. PTH-derived molecules with a sustained circulating half-life may represent a powerful and previously undefined anabolic regimen for cortical and cancellous bone.
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Anabolizantes/administración & dosificación , Anabolizantes/farmacología , Huesos/efectos de los fármacos , Fragmentos Fc de Inmunoglobulinas/administración & dosificación , Hormona Paratiroidea/administración & dosificación , Hormona Paratiroidea/farmacología , Proteínas Recombinantes/farmacología , Envejecimiento/fisiología , Anabolizantes/farmacocinética , Animales , Arrestinas/metabolismo , Huesos/metabolismo , Línea Celular , Cricetinae , Relación Dosis-Respuesta a Droga , Estrógenos/farmacología , Semivida , Humanos , Masculino , Ratones , Ovariectomía , Hormona Paratiroidea/farmacocinética , Transporte de Proteínas , Ratas , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacocinética , Factores de Tiempo , beta-ArrestinasRESUMEN
Mutations affecting the activity of the Wnt co-receptors LRP5 and LRP6 that cause alterations in skeletal biology confirmed the involvement of Wnt signaling in bone formation. We evaluated the potential role of Dkk1, an inhibitor of LRP5/6 activity, in bone formation by examining the normal expression pattern of Dkk1 in normal young mice and by assessing the consequences of osteoblast overexpression of Dkk1 in transgenic mice. Endogenous Dkk1 expression was detected primarily in osteoblasts and osteocytes. Transgenic over-expression of Dkk1 using two different rat collagen 1A1 promoters resulted in distinct bone phenotypes. More widespread Dkk1 expression (driven by the Col1A1 3.6 kb promoter) yielded osteopenia with forelimb deformities and hairlessness, while expression restricted to osteoblasts (driven by the Col1A1 2.3 kb promoter) induced severe osteopenia without limb defects or alopecia. The decrease in bone mass in vivo resulted from a significant 49% reduction in osteoblast numbers and was reflected in a 45% reduction in serum osteocalcin concentration; an in vitro study revealed that Dkk1 caused a dose-dependent suppression of osteoblast matrix mineralization. These data indicate that Dkk1 may directly influence bone formation and suggest that osteopenia develops in mice over-expressing Dkk1 at least in part due to diminished bone formation resulting from reduced osteoblast numbers.
Asunto(s)
Enfermedades Óseas Metabólicas/fisiopatología , Huesos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas Wnt/fisiología , Células 3T3 , Animales , Densidad Ósea , Enfermedades Óseas Metabólicas/genética , Enfermedades Óseas Metabólicas/metabolismo , Huesos/patología , Huesos/fisiopatología , Células Cultivadas , Embrión de Mamíferos/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/fisiología , Proteínas Relacionadas con Receptor de LDL/metabolismo , Masculino , Ratones , Ratones Transgénicos , Osteoblastos/citología , Osteoblastos/metabolismo , Osteocalcina/sangre , Osteogénesis/genética , Osteogénesis/fisiología , Embarazo , Ratas , Proteínas Recombinantes/metabolismo , Transducción de Señal/fisiología , Proteínas Wnt/metabolismoRESUMEN
This study examined whether the calcium-sensing receptor (CaR) is expressed in normal adult human osteoblastic and osteoclastic cells in culture, and whether the calcimimetic, cinacalcet HCl (AMG 073), potentiates the effects of calcium (via CaR, or some other receptor/mechanism). When mouse or human osteoblastic cells were treated with higher concentrations of calcium (6.6 or 8.6 mM in alpha-MEM/10% FBS) than present in control cultures (1.6 mM), the previously well-documented increase in cell number was demonstrated. Cinacalcet HCl affected cell proliferation of CHO cells transfected with CaR, dose dependently, but had no effect on human or mouse osteoblastic cell proliferation in calcium-containing medium (1.6 or 8.6 mM). To test cinacalcet HCl and calcium on osteoclastic cells, peripheral blood mononuclear cells were cultured in medium containing RANK ligand and M-CSF, supplemented with calcium, and/or cinacalcet HCl. Tartrate-resistant acid phosphatase-positive multinucleated osteoclastic cells on plastic or bone were then counted at 11 and 21 days, respectively. Calcium (greater than 6.0 mM) inhibited osteoclast formation, but cinacalcet HCl (30-1000 nM) had no effect on osteoclastic formation or resorption in the presence of calcium (1.6 or 6.1 mM). RT-PCR did not detect CaR in human, rat, or mouse primary osteoblastic cells and cell lines or osteoclastic cells. In conclusion, these studies indicate that the calcium-induced increase in osteoblastic cell number, and the decrease in formation/function of osteoclastic cells, involves a mechanism or receptor other than CaR. In addition, the calcimimetic agent did not potentiate the effects of calcium on normal adult human bone cells in vitro.