RESUMEN
Many peptide hormones form an α-helix on binding their receptors1-4, and sensitive methods for their detection could contribute to better clinical management of disease5. De novo protein design can now generate binders with high affinity and specificity to structured proteins6,7. However, the design of interactions between proteins and short peptides with helical propensity is an unmet challenge. Here we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that by extending RFdiffusion8 to enable binder design to flexible targets, and to refining input structure models by successive noising and denoising (partial diffusion), picomolar-affinity binders can be generated to helical peptide targets by either refining designs generated with other methods, or completely de novo starting from random noise distributions without any subsequent experimental optimization. The RFdiffusion designs enable the enrichment and subsequent detection of parathyroid hormone and glucagon by mass spectrometry, and the construction of bioluminescence-based protein biosensors. The ability to design binders to conformationally variable targets, and to optimize by partial diffusion both natural and designed proteins, should be broadly useful.
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Diseño Asistido por Computadora , Aprendizaje Profundo , Péptidos , Proteínas , Técnicas Biosensibles , Difusión , Glucagón/química , Glucagón/metabolismo , Mediciones Luminiscentes , Espectrometría de Masas , Hormona Paratiroidea/química , Hormona Paratiroidea/metabolismo , Péptidos/química , Péptidos/metabolismo , Estructura Secundaria de Proteína , Proteínas/química , Proteínas/metabolismo , Especificidad por Sustrato , Modelos MolecularesRESUMEN
Studies on human parathyroids are generally limited to hyperfunctioning glands owing to the difficulty in obtaining normal human tissue. We therefore obtained non-human primate (NHP) parathyroids to provide a suitable alternative for sequencing that would bear a close semblance to human organs. Single-cell RNA expression analysis of parathyroids from four healthy adult M. mulatta reveals a continuous trajectory of epithelial cell states. Pseudotime analysis based on transcriptomic signatures suggests a progression from GCM2 hi progenitors to mature parathyroid hormone (PTH)-expressing epithelial cells with increasing core mitochondrial transcript abundance along pseudotime. We sequenced, as a comparator, four histologically characterized hyperfunctioning human parathyroids with varying oxyphil and chief cell abundance and leveraged advanced computational techniques to highlight similarities and differences from non-human primate parathyroid expression dynamics. Predicted cell-cell communication analysis reveals abundant endothelial cell interactions in the parathyroid cell microenvironment in both human and NHP parathyroid glands. We show abundant RARRES2 transcripts in both human adenoma and normal primate parathyroid cells and use coimmunostaining to reveal high levels of RARRES2 protein (also known as chemerin) in PTH-expressing cells, which could indicate that RARRES2 plays an unrecognized role in parathyroid endocrine function. The data obtained are the first single-cell RNA transcriptome to characterize nondiseased parathyroid cell signatures and to show a transcriptomic progression of cell states within normal parathyroid glands, which can be used to better understand parathyroid cell biology.
Asunto(s)
Macaca mulatta , Glándulas Paratiroides , Análisis de la Célula Individual , Análisis de la Célula Individual/métodos , Humanos , Glándulas Paratiroides/metabolismo , Animales , Transcriptoma , Quimiocinas/metabolismo , Quimiocinas/genética , Hormona Paratiroidea/metabolismo , Hormona Paratiroidea/genética , Comunicación Celular , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica/métodos , Transcripción GenéticaRESUMEN
O-glycosylation is a conserved posttranslational modification that impacts many aspects of organismal viability and function. Recent studies examining the glycosyltransferase Galnt11 demonstrated that it glycosylates the endocytic receptor megalin in the kidneys, enabling proper binding and reabsorption of ligands, including vitamin D-binding protein (DBP). Galnt11-deficient mice were unable to properly reabsorb DBP from the urine. Vitamin D plays an essential role in mineral homeostasis and its deficiency is associated with bone diseases such as rickets, osteomalacia, and osteoporosis. We therefore set out to examine the effects of the loss of Galnt11 on vitamin D homeostasis and bone composition. We found significantly decreased levels of serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, consistent with decreased reabsorption of DBP. This was accompanied by a significant reduction in blood calcium levels and a physiologic increase in parathyroid hormone (PTH) in Galnt11-deficient mice. Bones in Galnt11-deficient mice were smaller and displayed a decrease in cortical bone accompanied by an increase in trabecular bone and an increase in a marker of bone formation, consistent with PTH-mediated effects on bone. These results support a unified model for the role of Galnt11 in bone and mineral homeostasis, wherein loss of Galnt11 leads to decreased reabsorption of DBP by megalin, resulting in a cascade of disrupted mineral and bone homeostasis including decreased circulating vitamin D and calcium levels, a physiological increase in PTH, an overall loss of cortical bone, and an increase in trabecular bone. Our study elucidates how defects in O-glycosylation can influence vitamin D and mineral homeostasis and the integrity of the skeletal system.
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Huesos , Homeostasis , Polipéptido N-Acetilgalactosaminiltransferasa , Vitamina D , Animales , Masculino , Ratones , Huesos/anatomía & histología , Huesos/química , Huesos/metabolismo , Calcio/metabolismo , Glicosilación , Homeostasis/genética , Hormona Paratiroidea/metabolismo , Vitamina D/metabolismo , Vitamina D/análogos & derivados , Proteína de Unión a Vitamina D/metabolismoRESUMEN
Calcitriol and calcimimetics are used to treat hyperparathyroidism secondary to chronic kidney disease (CKD). Calcitriol administration and the subsequent increase in serum calcium concentration decrease parathyroid hormone (PTH) levels, which should reduce bone remodeling. We have previously reported that, when maintaining a given concentration of PTH, the addition of calcimimetics is associated with an increased bone cell activity. Whether calcitriol administration affects bone cell activity while PTH is maintained constant should be evaluated in an animal model of renal osteodystrophy. The aim of the present study was to compare in CKD PTH-clamped rats the bone effects of calcitriol and calcimimetic administration. The results show that the administration of calcitriol and calcimimetic at doses that induced a similar reduction in PTH secretion produced dissimilar effects on osteoblast activity in 5/6 nephrectomized (Nx) rats with secondary hyperparathyroidism and in Nx rats with clamped PTH. Remarkably, in both rat models, the administration of calcitriol decreased osteoblastic activity, whereas calcimimetic increased bone cell activity. In vitro, calcitriol supplementation inhibited nuclear translocation of ß-catenin and reduced proliferation, osteogenesis, and mineralization in mesenchymal stem cells differentiated into osteoblasts. In conclusion, besides the action of calcitriol and calcimimetics at parathyroid level, these treatments have specific effects on bone cells that are independent of the PTH level.
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Calcimiméticos , Calcitriol , Osteoblastos , Hormona Paratiroidea , Animales , Calcitriol/farmacología , Ratas , Calcimiméticos/farmacología , Calcimiméticos/uso terapéutico , Hormona Paratiroidea/farmacología , Masculino , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Hiperparatiroidismo Secundario/tratamiento farmacológico , Hiperparatiroidismo Secundario/etiología , Hiperparatiroidismo Secundario/metabolismo , Huesos/metabolismo , Huesos/efectos de los fármacos , Ratas Wistar , Insuficiencia Renal/tratamiento farmacológico , Insuficiencia Renal/metabolismo , Osteogénesis/efectos de los fármacos , Insuficiencia Renal Crónica/tratamiento farmacológico , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/complicaciones , Diferenciación Celular/efectos de los fármacos , Calcio/metabolismoRESUMEN
BACKGROUND: Thyroidectomy causes impaired blood supply to the parathyroid glands, which leads to hypoparathyroidism. Tanshinone IIA (Tan IIA) is helpful in blood activation and cardiovascular protection. Therefore, the efficacy of Tan IIA in improving hypoparathyroidism was explored in this study. METHODS: New Zealand white rabbits were utilized to establish a unilateral parathyroid gland ischemia injury model. The model was created by selectively ligating the main blood supply vessel of one parathyroid gland, and the rabbits were then divided into three groups receiving 1, 5, and 10 mg/kg of Tan IIA. Serum calcium and parathyroid hormone (PTH) levels were measured using specialized assay kits. Immunohistochemistry was used to assess the microvessel density (MVD) in parathyroid glands. Western blotting (WB) was used to analyze protein expression related to the PI3K/AKT signaling pathway and the pathway-associated HIF-1α and VEGF. Moreover, MMP-2 and MMP-9 involved in angiogenesis were detected by WB. RESULTS: Tan IIA treatment effectively restored serum calcium and PTH levels in a dose-dependent manner. Notably, MVD in the parathyroid glands increased significantly, especially at higher doses. The Tan IIA treatment also elevated the p-PI3K/PI3K and p-AKT/AKT ratios, indicating that the PI3K/AKT pathway was reactivated. Moreover, Tan IIA significantly restored the decreased expression levels of VEGF and HIF-1α caused by parathyroid surgery. Additionally, Tan IIA increased MMP-2 and MMP-9 levels. CONCLUSION: Tan IIA activates the PI3K/AKT pathway, promotes angiogenesis by modulating VEGF, HIF-1α, MMP-2, and MMP-9, thereby further enhancing MVD within the parathyroid glands. This study demonstrates that Tan IIA improved post-thyroidectomy hypoparathyroidism.
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Abietanos , Modelos Animales de Enfermedad , Hipoparatiroidismo , Glándulas Paratiroides , Tiroidectomía , Animales , Hipoparatiroidismo/tratamiento farmacológico , Hipoparatiroidismo/etiología , Hipoparatiroidismo/metabolismo , Abietanos/farmacología , Abietanos/uso terapéutico , Tiroidectomía/efectos adversos , Conejos , Glándulas Paratiroides/metabolismo , Glándulas Paratiroides/efectos de los fármacos , Glándulas Paratiroides/cirugía , Transducción de Señal/efectos de los fármacos , Humanos , Calcio/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Masculino , Hormona Paratiroidea/metabolismo , Hormona Paratiroidea/sangreRESUMEN
SIGNIFICANCE STATEMENT: Patients with AKI suffer a staggering mortality rate of approximately 30%. Fibroblast growth factor 23 (FGF23) and phosphate (P i ) rise rapidly after the onset of AKI and have both been independently associated with ensuing morbidity and mortality. This study demonstrates that dietary P i restriction markedly diminished the early rise in plasma FGF23 and prevented the rise in plasma P i , parathyroid hormone, and calcitriol in mice with folic acid-induced AKI (FA-AKI). Furthermore, the study provides evidence for P i -sensitive osseous Fgf23 mRNA expression and reveals that P i restriction mitigated calciprotein particles (CPPs) formation, inflammation, acidosis, cardiac electrical disturbances, and mortality in mice with FA-AKI. These findings suggest that P i restriction may have a prophylactic potential in patients at risk for AKI. BACKGROUND: In AKI, plasma FGF23 and P i rise rapidly and are independently associated with disease severity and outcome. METHODS: The effects of normal (NP) and low (LP) dietary P i were investigated in mice with FA-AKI after 3, 24, and 48 hours and 14 days. RESULTS: After 24 hours of AKI, the LP diet curbed the rise in plasma FGF23 and prevented that of parathyroid hormone and calcitriol as well as of osseous but not splenic or thymic Fgf23 mRNA expression. The absence of Pth prevented the rise in calcitriol and reduced the elevation of FGF23 in FA-AKI with the NP diet. Furthermore, the LP diet attenuated the rise in renal and plasma IL-6 and mitigated the decline in renal α -Klotho. After 48 hours, the LP diet further dampened renal IL-6 expression and resulted in lower urinary neutrophil gelatinase-associated lipocalin. In addition, the LP diet prevented the increased formation of CPPs. Fourteen days after AKI induction, the LP diet group maintained less elevated plasma FGF23 levels and had greater survival than the NP diet group. This was associated with prevention of metabolic acidosis, hypocalcemia, hyperkalemia, and cardiac electrical disturbances. CONCLUSIONS: This study reveals P i -sensitive FGF23 expression in the bone but not in the thymus or spleen in FA-AKI and demonstrates that P i restriction mitigates CPP formation, inflammation, acidosis, and mortality in this model. These results suggest that dietary P i restriction could have prophylactic potential in patients at risk for AKI.
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Acidosis , Lesión Renal Aguda , Animales , Humanos , Ratones , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/prevención & control , Calcitriol , Ácido Fólico , Inflamación , Interleucina-6 , Hormona Paratiroidea , Fosfatos , ARN MensajeroRESUMEN
Secondary hyperparathyroidism has a significant impact on the overall well-being of the body. Capsiates, known for their antioxidant and metabolic properties, have emerged as a promising alternative treatment for secondary hyperparathyroidism. This study aims to evaluate the effects and mechanisms of capsiates in the treatment of secondary hyperparathyroidism. To achieve our research objectives, we conducted a study on patients' serum and examined changes in metabolic markers using serum metabolomics. We induced secondary hyperparathyroidism in rat through dietary intervention and divided them into four groups. The first group, referred to as the Parathyroid Hormone (PTH) group, received a low-calcium and high-phosphate diet (0.2% calcium, 1.2% phosphorus). The second group served as the control group, receiving a standard phosphate and calcium diet (0.6% calcium, 0.6% phosphorus). The third group, called the capsiates group, consisted of rat from the control group treated with capsiates (intraperitoneal injection of 2 mg/kg capsiates for 2 weeks after 2 weeks of dietary intervention). The fourth group was the capsiates-treated PTH group. Subsequently, we conducted ribose nucleic acid (RNA) sequencing on parathyroid gland cells and evaluated serum thyroxine levels, oxidative stress, expression of proteins associated with vascular neogenesis, measurement of SOD, GSH and 3-nitrotyrosine, micro-CT and histological staining. The serum metabolomic data revealed a significant decrease in capsiate levels in the secondary hyperparathyroidism group. Administration of capsiates to PTH rat resulted in increased calcium levels compared to the PTH group. Additionally, the PTH + Capsiates group showed significantly lower levels of PTH and phosphate compared to the PTH group. The PTH group exhibited a notable increase in the quantity and size of mitochondria compared to the control group. Following capsiates administration to the PTH group, there was a significant reduction in the number of mitochondria and length of microvilli, but an increase in the size of mitochondria compared to the PTH group. Sequencing analysis revealed that vascular endothelial growth factor (VEGF) and Vascular Endothelial Growth Factor Receptor 1 (VEGFR1) play crucial roles in this process. Vascular-related variables and downstream signalling were significantly elevated in hyperthyroidism and were alleviated with capsaicin treatment. Finally, combining capsiates with the PTH group improved bone mineral density, Tb.N, BV.TV, Cs.Th, Tt.Ar, OPG, Ob.TV and Oc.TV, as well as the mineral apposition rate, but significantly decreased Tb.Sp and Receptor Activator for Nuclear Factor-κ B Ligand (RANKL) compared to the PTH group. The findings suggest that capsiates can improve secondary hyperparathyroidism and ameliorated osteoporosis outcomes by inhibiting angiogenesis and reducing oxidative stress.
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Capsaicina/análogos & derivados , Hiperparatiroidismo Secundario , Resistencia a la Insulina , Humanos , Ratas , Animales , Calcio , Angiogénesis , Factor A de Crecimiento Endotelial Vascular , Hiperparatiroidismo Secundario/tratamiento farmacológico , Hiperparatiroidismo Secundario/etiología , Hormona Paratiroidea , Fósforo , FosfatosRESUMEN
Parathyroid hormone (PTH) serves dual roles in bone metabolism, exhibiting both anabolic and catabolic effects. The anabolic properties of PTH have been utilized in the treatment of osteoporosis with proven efficacy in preventing fractures. Despite these benefits, PTH can be administered therapeutically for up to 2 years, and its use in patients with underlying malignancies remains a subject of ongoing debate. These considerations underscore the need for a more comprehensive understanding of the underlying mechanisms. p21-activated kinase 4 (PAK4) is involved in bone resorption and cancer-associated osteolysis; however, its role in osteoblast function and PTH action remains unknown. Therefore, in this study, we aimed to clarify the role of PAK4 in osteoblast function and its effects on PTH-induced anabolic activity. PAK4 enhanced MC3T3-E1 osteoblast viability and proliferation and upregulated cyclin D1 expression. PAK4 also augmented osteoblast differentiation, as indicated by increased mineralization found by alkaline phosphatase and Alizarin Red staining. Treatment with PTH (1-34), an active PTH fragment, stimulated PAK4 expression and phosphorylation in a protein kinase A-dependent manner. In addition, bone morphogenetic protein-2 (which is known to promote bone formation) increased phosphorylated PAK4 (p-PAK4) and PAK4 levels. PAK4 regulated the expression of both phosphorylated and total ß-catenin, which are critical for osteoblast proliferation and differentiation. Moreover, p-PAK4 directly interacted with ß-catenin, and disruption of ß-catenin's binding to T-cell factor impaired PAK4- and PTH-induced osteoblast differentiation. Our findings elucidate the effect of PAK4 on enhancing bone formation in osteoblasts and its pivotal role in the anabolic activity of PTH mediated through its interaction with ß-catenin. These insights improve the understanding of the mechanisms underlying PTH activity and should inform the development of more effective and safer osteoporosis treatments.
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Diferenciación Celular , Proliferación Celular , Osteoblastos , Hormona Paratiroidea , beta Catenina , Quinasas p21 Activadas , Animales , Humanos , Ratones , beta Catenina/metabolismo , beta Catenina/genética , Calcificación Fisiológica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclina D1/metabolismo , Ciclina D1/genética , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Quinasas p21 Activadas/metabolismo , Quinasas p21 Activadas/genética , Hormona Paratiroidea/farmacología , Hormona Paratiroidea/metabolismo , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células CultivadasRESUMEN
Teriparatide is a peptide derived from a parathyroid hormone (PTH) and an osteoporosis therapeutic drug with potent bone formation-promoting activity. To identify novel druggable genes that act downstream of PTH signaling and are potentially involved in bone formation, we screened PTH target genes in mouse osteoblast-like MC3T3-E1 cells. Here we show that Gprc5a, encoding an orphan G protein-coupled receptor, is a novel PTH-inducible gene and negatively regulates osteoblast proliferation and differentiation. PTH treatment induced Gprc5a expression in MC3T3-E1 cells, rat osteosarcoma ROS17/2.8 cells, and mouse femurs. Induction of Gprc5a expression by PTH occurred in the absence of protein synthesis and was mediated primarily via the cAMP pathway, suggesting that Gprc5a is a direct target of PTH signaling. Interestingly, Gprc5a expression was induced additively by co-treatment with PTH and 1α, 25-dihydroxyvitamin D3 (calcitriol), or retinoic acid in MC3T3-E1 cells. Reporter analysis of a 1 kb fragment of human GPRC5A promoter revealed that the promoter fragment showed responsiveness to PTH via the cAMP response element, suggesting that GPRC5A is also a PTH-inducible gene in humans. Gprc5a knockdown promoted cell viability and proliferation, as demonstrated by MTT and BrdU assays. Gprc5a knockdown also promoted osteoblast differentiation, as indicated by gene expression analysis and mineralization assay. Mechanistic studies showed that Gprc5a interacted with BMPR1A and suppressed BMP signaling induced by BMP-2 and constitutively active BMP receptors, ALK2 (ACVR1) Q207D and ALK3 (BMPR1A) Q233D. Thus, our results suggest that Gprc5a is a novel gene induced by PTH that acts in an inhibitory manner on both cell proliferation and osteoblast differentiation and is a candidate for drug targets for osteoporosis.
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Diferenciación Celular , Proliferación Celular , Osteoblastos , Hormona Paratiroidea , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Hormona Paratiroidea/farmacología , Ratones , Ratas , Humanos , Transducción de Señal/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Regiones Promotoras Genéticas/genética , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , AMP Cíclico/metabolismo , Tretinoina/farmacología , Calcitriol/farmacologíaRESUMEN
The Calcium-sensing receptor (CaSR) senses extracellular calcium, regulates parathyroid hormone (PTH) secretion, and has additional functions in various organs related to systemic and local calcium and mineral homeostasis. Familial hypocalciuric hypercalcemia type I (FHH1) is caused by heterozygous loss-of-function mutations in the CaSR gene, and is characterized by the combination of hypercalcemia, hypocalciuria, normal to elevated PTH, and facultatively hypermagnesemia and mild bone mineralization defects. To date, only heterozygous Casr null mice have been available as model for FHH1. Here we present a novel mouse FHH1 model identified in a large ENU-screen that carries an c.2579 T > A (p.Ile859Asn) variant in the Casr gene (CasrBCH002 mice). In order to dissect direct effects of the genetic variant from PTH-dependent effects, we crossed CasrBCH002 mice with PTH deficient mice. Heterozygous CasrBCH002 mice were fertile, had normal growth and body weight, were hypercalcemic and hypermagnesemic with inappropriately normal PTH levels and urinary calcium excretion replicating some features of FHH1. Hypercalcemia and hypermagnesemia were independent from PTH and correlated with higher expression of claudin 16 and 19 in kidneys. Likewise, reduced expression of the renal TRPM6 channel in CasrBCH002 mice was not dependent on PTH. In bone, mutations in Casr rescued the bone phenotype observed in Pth null mice by increasing osteoclast numbers and improving the columnar pattern of chondrocytes in the growth zone. In summary, CasrBCH002 mice represent a new model to study FHH1 and our results indicate that only a part of the phenotype is driven by PTH.
Asunto(s)
Hipercalcemia , Hormona Paratiroidea , Receptores Sensibles al Calcio , Animales , Masculino , Ratones , Calcio/metabolismo , Modelos Animales de Enfermedad , Hipercalcemia/genética , Hipercalcemia/metabolismo , Hipercalcemia/congénito , Ratones Endogámicos C57BL , Hormona Paratiroidea/metabolismo , Hormona Paratiroidea/genética , Receptores Sensibles al Calcio/genética , Receptores Sensibles al Calcio/metabolismoRESUMEN
Sclerostin (SOST) is produced by osteocytes and is known as a negative regulator of bone homeostasis. Parathyroid hormone (PTH) regulates calcium, phosphate as well as vitamin D metabolism, and is a strong inhibitor of SOST synthesis in vitro and in vivo. PTH has two methionine amino acids (positions 8 and 18) which can be oxidized. PTH oxidized at Met18 (Met18(ox)-PTH) continues to be bioactive, whereas PTH oxidized at Met8 (Met8(ox)-PTH) or PTH oxidized at Met8 and Met18 (Met8, Met18(di-ox)-PTH) has minor bioactivity. How non-oxidized PTH (n-oxPTH) and oxidized forms of PTH act on sclerostin synthesis is unknown. The effects of n-oxPTH and oxidized forms of PTH on SOST gene expression were evaluated in UMR106 osteoblast-like cells. Moreover, we analyzed the relationship of SOST with n-oxPTH and all forms of oxPTH in 516 stable kidney transplant recipients using an assay system that can distinguish in clinical samples between n-oxPTH and the sum of all oxidized PTH forms (Met8(ox)-PTH, Met18(ox)-PTH, and Met8, Met18(di-ox)-PTH). We found that both n-oxPTH and Met18(ox)-PTH at doses of 1, 3, 20, and 30 nmol/L significantly inhibit SOST gene expression in vitro, whereas Met8(ox)-PTH and Met8, Met18(di-ox)-PTH only have a weak inhibitory effect on SOST gene expression. In the clinical cohort, multivariate linear regression showed that only n-oxPTH, but not intact PTH (iPTH) nor oxPTH, is independently associated with circulating SOST after adjusting for known confounding factors. In conclusion, only bioactive PTH forms such as n-oxPTH and Met18(ox)-PTH, inhibit SOST synthesis.
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Proteínas Adaptadoras Transductoras de Señales , Proteínas Morfogenéticas Óseas , Hormona Paratiroidea , Hormona Paratiroidea/metabolismo , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Marcadores Genéticos , Animales , Osteoblastos/metabolismo , Osteoblastos/efectos de los fármacos , Masculino , Oxidación-Reducción , Femenino , Ratas , Metionina/metabolismo , Metionina/farmacología , Línea Celular , Persona de Mediana EdadRESUMEN
Parathyroid hormone 1 receptor (PTH1R) plays a key role in mediating calcium homeostasis and bone development, and aberrant PTH1R activity underlies several human diseases. Peptidic PTH1R antagonists and inverse agonists have therapeutic potential in treating these diseases, but their poor pharmacokinetics and pharmacodynamics undermine their in vivo efficacy. Herein, we report the use of a backbone-modification strategy to design a peptidic PTH1R inhibitor that displays prolonged activity as an antagonist of wild-type PTH1R and an inverse agonist of the constitutively active PTH1R-H223R mutant both in vitro and in vivo. This peptide may be of interest for the future development of therapeutic agents that ameliorate PTH1R malfunction.
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Agonismo Inverso de Drogas , Receptor de Hormona Paratiroídea Tipo 1 , Humanos , Péptidos , Hormona Paratiroidea/farmacologíaRESUMEN
The kidney controls systemic inorganic phosphate (Pi) levels by adapting reabsorption to Pi intake. Renal Pi reabsorption is mostly mediated by sodium-phosphate cotransporters NaPi-IIa (SLC34A1) and NaPi-IIc (SLC34A3) that are tightly controlled by various hormones including parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23). PTH and FGF23 rise in response to Pi intake and decrease NaPi-IIa and NaPi-IIc brush border membrane abundance enhancing phosphaturia. Phosphaturia and transporter regulation occurs even in the absence of PTH and FGF23 signaling. The calcium-sensing receptor (CaSR) regulates PTH and FGF23 secretion, and may also directly affect renal Pi handling. Here, we combined pharmacological and genetic approaches to examine the role of the CaSR in the acute phosphaturic response to Pi loading. Animals pretreated with the calcimimetic cinacalcet were hyperphosphatemic, had blunted PTH levels upon Pi administration, a reduced Pi-induced phosphaturia, and no Pi-induced NaPi-IIa downregulation. The calcilytic NPS-2143 exaggerated the PTH response to Pi loading but did not abolish Pi-induced downregulation of NaPi-IIa. In mice with a dominant inactivating mutation in the Casr (CasrBCH002), baseline NaPi-IIa expression was higher, whereas downregulation of transporter expression was blunted in double CasrBCH002/PTH knockout (KO) transgenic animals. Thus, in response to an acute Pi load, acute modulation of the CaSR affects the endocrine and renal response, whereas chronic genetic inactivation, displays only subtle differences in the downregulation of NaPi-IIa and NaPi-IIc renal expression. We did not find evidence that the CaSR impacts on the acute renal response to oral Pi loading beyond its role in regulating PTH secretion.NEW & NOTEWORTHY Consumption of phosphate-rich diets causes an adaptive response of the body leading to the urinary excretion of phosphate. The underlying mechanisms are still poorly understood. Here, we examined the role of the calcium-sensing receptor (CaSR) that senses both calcium and phosphate. We confirmed that the receptor increases the secretion of parathyroid hormone involved in stimulating urinary phosphate excretion. However, we did not find any evidence for a role of the receptor beyond this function.
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Factor-23 de Crecimiento de Fibroblastos , Riñón , Ratones Noqueados , Hormona Paratiroidea , Fosfatos , Receptores Sensibles al Calcio , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc , Receptores Sensibles al Calcio/metabolismo , Receptores Sensibles al Calcio/genética , Animales , Hormona Paratiroidea/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genética , Fosfatos/metabolismo , Riñón/metabolismo , Riñón/efectos de los fármacos , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/genética , Ratones , Reabsorción Renal/efectos de los fármacos , Masculino , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Ratones Endogámicos C57BLRESUMEN
Chronic kidney disease mineral bone disorder (CKD-MBD) is a complex clinical syndrome responsible for the accelerated cardiovascular mortality seen in individuals afflicted with CKD. Current approaches to therapy have failed to improve clinical outcomes adequately, likely due to targeting surrogate biochemical parameters as articulated by the guideline developer, Kidney Disease: Improving Global Outcomes (KDIGO). We hypothesized that using a Systems Biology Approach combining machine learning with mathematical modeling, we could test a novel approach to therapy targeting the abnormal movement of mineral out of bone and into soft tissue that is characteristic of CKD-MBD. The mathematical model describes the movement of calcium and phosphate between body compartments in response to standard therapeutic agents. The machine-learning technique we applied is reinforcement learning (RL). We compared calcium, phosphate, parathyroid hormone (PTH), and mineral movement out of bone and into soft tissue under four scenarios: standard approach (KDIGO), achievement of KDIGO guidelines using RL (RLKDIGO), targeting abnormal mineral flux (RLFLUX), and combining achievement of KDIGO guidelines with minimization of abnormal mineral flux (RLKDIGOFLUX). We demonstrate through simulations that explicitly targeting abnormal mineral flux significantly decreases abnormal mineral movement compared with standard approach while achieving acceptable biochemical outcomes. These investigations highlight the limitations of current therapeutic targets, primarily secondary hyperparathyroidism, and emphasize the central role of deranged phosphate homeostasis in the genesis of the CKD-MBD syndrome.NEW & NOTEWORTHY Artificial intelligence is a powerful tool for exploration of complex processes but application to clinical syndromes is challenging. Using a mathematical model describing the movement of calcium and phosphate between body compartments combined with machine learning, we show the feasibility of testing alternative goals of therapy for Chronic Kidney Disease Mineral Bone Disorder while maintaining acceptable biochemical outcomes. These simulations demonstrate the potential for using this platform to generate and test hypotheses in silico rapidly, inexpensively, and safely.
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Trastorno Mineral y Óseo Asociado a la Enfermedad Renal Crónica , Humanos , Trastorno Mineral y Óseo Asociado a la Enfermedad Renal Crónica/metabolismo , Trastorno Mineral y Óseo Asociado a la Enfermedad Renal Crónica/tratamiento farmacológico , Trastorno Mineral y Óseo Asociado a la Enfermedad Renal Crónica/fisiopatología , Calcio/metabolismo , Hormona Paratiroidea/metabolismo , Inteligencia Artificial , Fosfatos/metabolismo , Biología de Sistemas , Aprendizaje Automático , Modelos Biológicos , Huesos/metabolismo , Huesos/efectos de los fármacos , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/fisiopatología , Insuficiencia Renal Crónica/terapiaRESUMEN
Conduit arterial disease in chronic kidney disease (CKD) is an important cause of cardiac complications. Cardiac function in CKD has not been studied in the absence of arterial disease. In an Alport syndrome model bred not to have conduit arterial disease, mice at 225 days of life (dol) had CKD equivalent to humans with CKD stage 4-5. Parathyroid hormone (PTH) and FGF23 levels were one log order elevated, circulating sclerostin was elevated, and renal activin A was strongly induced. Aortic Ca levels were not increased, and vascular smooth muscle cell (VSMC) transdifferentiation was absent. The CKD mice were not hypertensive, and cardiac hypertrophy was absent. Freshly excised cardiac tissue respirometry (Oroboros) showed that ADP-stimulated O2 flux was diminished from 52 to 22 pmol/mg (P = 0.022). RNA-Seq of cardiac tissue from CKD mice revealed significantly decreased levels of cardiac mitochondrial oxidative phosphorylation genes. To examine the effect of activin A signaling, some Alport mice were treated with a monoclonal Ab to activin A or an isotype-matched IgG beginning at 75 days of life until euthanasia. Treatment with the activin A antibody (Ab) did not affect cardiac oxidative phosphorylation. However, the activin A antibody was active in the skeleton, disrupting the effect of CKD to stimulate osteoclast number, eroded surfaces, and the stimulation of osteoclast-driven remodeling. The data reported here show that cardiac mitochondrial respiration is impaired in CKD in the absence of conduit arterial disease. This is the first report of the direct effect of CKD on cardiac respiration.NEW & NOTEWORTHY Heart disease is an important morbidity of chronic kidney disease (CKD). Hypertension, vascular stiffness, and vascular calcification all contribute to cardiac pathophysiology. However, cardiac function in CKD devoid of vascular disease has not been studied. Here, in an animal model of human CKD without conduit arterial disease, we analyze cardiac respiration and discover that CKD directly impairs cardiac mitochondrial function by decreasing oxidative phosphorylation. Protection of cardiac oxidative phosphorylation may be a therapeutic target in CKD.
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Cardiomegalia , Factor-23 de Crecimiento de Fibroblastos , Miocardio , Insuficiencia Renal Crónica , Animales , Factor-23 de Crecimiento de Fibroblastos/metabolismo , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Cardiomegalia/metabolismo , Cardiomegalia/patología , Miocardio/metabolismo , Miocardio/patología , Modelos Animales de Enfermedad , Activinas/metabolismo , Activinas/genética , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Ratones , Masculino , Fosforilación Oxidativa , Nefritis Hereditaria/metabolismo , Nefritis Hereditaria/patología , Nefritis Hereditaria/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Hormona Paratiroidea/metabolismoRESUMEN
Parathyroid hormone measurement is critical in managing chronic kidney disease-mineral bone disorders. However, there are several commercially available immunoassays with interassay variability. To address this, Cavalier et al. developed standardization of parathyroid hormone measurement by establishing regression equations of each assay against the liquid chromatography coupled to tandem mass spectrometry method. The recalibration successfully reduced interassay variability, allowing for more consistent interpretation. The proposed approach may pave the way for accurate interpretation of parathyroid hormone in clinical practice.
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Cromatografía Líquida con Espectrometría de Masas , Espectrometría de Masas en Tándem , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Hormona Paratiroidea , Inmunoensayo/métodos , Estándares de ReferenciaRESUMEN
Demand for kidney grafts outpaces supply, limiting kidney transplantation as a treatment for kidney failure. Xenotransplantation has the potential to make kidney transplantation available to many more patients with kidney failure, but the ability of xenografts to support human physiologic homeostasis has not been established. A brain-dead adult decedent underwent bilateral native nephrectomies followed by 10 gene-edited (four gene knockouts, six human transgenes) pig-to-human xenotransplantation. Physiologic parameters and laboratory values were measured for seven days in a critical care setting. Data collection aimed to assess homeostasis by measuring components of the renin-angiotensin-aldosterone system, parathyroid hormone signaling, glomerular filtration rate, and markers of salt and water balance. Mean arterial blood pressure was maintained above 60 mmHg throughout. Pig kidneys secreted renin (post-operative day three to seven mean and standard deviation: 47.3 ± 9 pg/mL). Aldosterone and angiotensin II levels were present (post-operative day three to seven, 57.0 ± 8 pg/mL and 5.4 ± 4.3 pg/mL, respectively) despite plasma renin activity under 0.6 ng/mL/hr. Parathyroid hormone levels followed ionized calcium. Urine output down trended from 37 L to 6 L per day with 4.5 L of electrolyte free water loss on post-operative day six. Aquaporin 2 channels were detected in the apical surface of principal cells, supporting pig kidney response to human vasopressin. Serum creatinine down trended to 0.9 mg/dL by day seven. Glomerular filtration rate ranged 90-240 mL/min by creatinine clearance and single-dose inulin clearance. Thus, in a human decedent model, xenotransplantation of 10 gene-edited pig kidneys provided physiologic balance for seven days. Hence, our in-human study paves the way for future clinical study of pig-to-human kidney xenotransplantation in living persons.
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Insuficiencia Renal , Renina , Adulto , Humanos , Animales , Porcinos , Trasplante Heterólogo , Riñón/fisiología , Sistema Renina-Angiotensina , Aldosterona , Homeostasis , Hormona Paratiroidea , AguaRESUMEN
OBJECTIVE: To investigate the effect of intermittent parathyroid hormone (iPTH) administration on pathological new bone formation during treatment of ankylosing spondylitis-related osteoporosis. METHODS: Animal models with pathological bone formation caused by hypothetical AS pathogenesis received treatment with iPTH. We determined the effects of iPTH on bone loss and the formation of pathological new bone with micro-computed tomography (micro-CT) and histological examination. In addition, the tamoxifen-inducible conditional knockout mice (CAGGCre-ERTM; PTHflox/flox, PTH-/-) was established to delete PTH and investigate the effect of endogenous PTH on pathological new bone formation. RESULTS: iPTH treatment significantly improved trabecular bone mass in the modified collagen-induced arthritis (m-CIA) model and unbalanced mechanical loading models. Meanwhile, iPTH treatment did not enhance pathological new bone formation in all types of animal models. Endogenous PTH deficiency had no effects on pathological new bone formation in unbalanced mechanical loading models. CONCLUSION: Experimental animal models of AS treated with iPTH show improvement in trabecular bone density, but not entheseal pathological bone formationï¼indicating it may be a potential treatment for inflammatory bone loss does in AS.
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Osteogénesis , Hormona Paratiroidea , Animales , Hormona Paratiroidea/administración & dosificación , Hormona Paratiroidea/farmacología , Hormona Paratiroidea/uso terapéutico , Osteogénesis/efectos de los fármacos , Ratones , Osteoporosis/tratamiento farmacológico , Osteoporosis/patología , Ratones Noqueados , Masculino , Microtomografía por Rayos X , Espondilitis Anquilosante/tratamiento farmacológico , Espondilitis Anquilosante/patología , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/patología , Densidad Ósea/efectos de los fármacosRESUMEN
PURPOSE OF REVIEW: Parathyroid hormone (PTH) is the major peptide hormone regulator of blood calcium homeostasis. Abnormal PTH levels can be observed in patients with various congenital and acquired disorders, including chronic kidney disease (CKD). This review will focus on rare human diseases caused by PTH mutations that have provided insights into the regulation of PTH synthesis and secretion as well as the diagnostic utility of different PTH assays. RECENT FINDINGS: Over the past years, numerous diseases affecting calcium and phosphate homeostasis have been defined at the molecular level that are responsible for reduced or increased serum PTH levels. The underlying genetic mutations impair parathyroid gland development, involve the PTH gene itself, or alter function of the calcium-sensing receptor (CaSR) or its downstream signaling partners that contribute to regulation of PTH synthesis or secretion. Mutations in the pre sequence of the mature PTH peptide can, for instance, impair hormone synthesis or intracellular processing, while amino acid substitutions affecting the secreted PTH(1-84) impair PTH receptor (PTH1R) activation, or cause defective cleavage of the pro-sequence and thus secretion of a pro- PTH with much reduced biological activity. Mutations affecting the secreted hormone can alter detection by different PTH assays, thus requiring detailed knowledge of the utilized diagnostic test. SUMMARY: Rare diseases affecting PTH synthesis and secretion have offered helpful insights into parathyroid biology and the diagnostic utility of commonly used PTH assays, which may have implications for the interpretation of PTH measurements in more common disorders such as CKD.
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Mutación , Hormona Paratiroidea , Humanos , Hormona Paratiroidea/metabolismo , Hormona Paratiroidea/sangre , Hormona Paratiroidea/genética , Receptores Sensibles al Calcio/genética , Receptores Sensibles al Calcio/metabolismo , Glándulas Paratiroides/metabolismo , Enfermedades Raras/diagnóstico , Enfermedades Raras/genética , Animales , Insuficiencia Renal Crónica/diagnóstico , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/metabolismo , Calcio/metabolismo , Predisposición Genética a la Enfermedad , Valor Predictivo de las Pruebas , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/genéticaRESUMEN
PURPOSE OF REVIEW: Defining the optimal parathyroid hormone (PTH) target in chronic kidney disease (CKD) is challenging, especially for bone outcomes, due to the substantial variability in the skeleton's response to PTH. Although PTH hyporesponsiveness is as integral a component of CKD-mineral bone disorder as elevated PTH levels, clinical awareness of this condition is limited. In this review, we will discuss factors and mechanisms contributing to PTH hyporesponsiveness in CKD. This knowledge may provide clues towards a personalized approach to treating secondary hyperparathyroidism in CKD. RECENT FINDINGS: Indicates a link between disturbed phosphate metabolism and impaired skeletal calcium sensing receptor signaling as an important mediator of PTH hyporesponsiveness in CKD. Further, cohort studies with diverse populations point towards differences in mineral metabolism control, rather than genetic or environmental factors, as drivers of the variability of PTH responsiveness. IN SUMMARY: Skeletal PTH hyporesponsiveness in CKD has a multifactorial origin, shows important interindividual variability, and is challenging to estimate in clinical practice. The variability in skeletal responsiveness compromises PTH as a biomarker of bone turnover, especially when considering populations that are heterogeneous in ethnicity, demography, kidney function, primary kidney disease and mineral metabolism control, and in patients treated with bone targeting drugs.