RESUMO
Pharmaceuticals are of increasing environmental concern as they emerge and accumulate in surface- and groundwater systems around the world, endangering the overall health of aquatic ecosystems. Municipal wastewater discharge is a significant vector for pharmaceuticals and their metabolites to enter surface waters as humans incompletely absorb prescription drugs and excrete up to 50% into wastewater, which are subsequently incompletely removed during wastewater treatment. Microalgae present a promising target for improving wastewater treatment due to their ability to remove some pollutants efficiently. However, their inherent metabolic pathways limit their capacity to degrade more recalcitrant organic compounds such as pharmaceuticals. The human liver employs enzymes to break down and absorb drugs, and these enzymes are extensively researched during drug development, meaning the cytochrome P450 enzymes responsible for metabolizing each approved drug are well studied. Thus, unlocking or increasing cytochrome P450 expression in endogenous wastewater microalgae could be a cost-effective strategy to reduce pharmaceutical loads in effluents. Here, we discuss the challenges and opportunities associated with introducing cytochrome P450 enzymes into microalgae. We anticipate that cytochrome P450-engineered microalgae can serve as a new drug removal method and a sustainable solution that can upgrade wastewater treatment facilities to function as "mega livers".
Assuntos
Sistema Enzimático do Citocromo P-450 , Microalgas , Águas Residuárias , Purificação da Água , Microalgas/metabolismo , Microalgas/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Águas Residuárias/química , Águas Residuárias/microbiologia , Preparações Farmacêuticas/metabolismo , Purificação da Água/métodos , Poluentes Químicos da Água/metabolismo , Humanos , Biodegradação AmbientalRESUMO
AIMS: Non-renal extravasation of phosphate from the circulation and transient accumulation into tissues and extracellular fluid is a regulated process of acute phosphate homeostasis that is not well understood. This process is especially relevant in the setting of chronic kidney disease (CKD), where exposure to increased phosphate is prolonged due to inefficient kidney excretion. Furthermore, CKD-associated mineral dysregulation induces pathological accumulation of phosphate causing vascular calcification (VC). Our objective was to determine whether the systemic response to acute phosphate challenges is altered by VC. METHODS AND RESULTS: After bolus phosphate administration, circulating and tissue deposition of this challenge was assessed in two rat models of VC using a radiolabelled phosphate tracer. In an adenine-induced model of CKD (N = 70), animals with VC had a blunted elevation of circulating 33PO4 following oral phosphate administration (P < 0.01), and the discordant deposition could be traced to the calcified arteries (11.4 [7.5-13.1] vs.43.0 [35.5-53.7] pmol/ng tissue, P < 0.001). In a non-CKD model of VC, calcification was induced with 0.5 ug/kg calcitriol and then withdrawn (N = 24). New phosphate uptake by the calcified vasculature correlated to the pre-existing burden of calcification (r = 38, P < 0.001) and was substantially attenuated in the absence of calcification stimulus (P < 0.01). Phosphate accrual was stimulated by the phosphate challenge and not present to the same degree during passive disposition of circulating phosphate. Further, the form of phosphate that deposited to the vasculature was predominately amorphous inorganic phosphate and not that which was bound in matured calciprotein particles. CONCLUSIONS: In the process of calcification, arteries acutely deposit substantial amorphous phosphate while blunting the elevation in the circulation, thereby altering the systemic disposition of phosphate and identifying VC as a participatory mineral homeostatic organ. This study demonstrates the negative vascular consequence of acute fluctuations in circulating phosphate, and supports the importance of phosphate bioavailability and diet management in CKD patients as a mediator of cardiovascular risk.
Assuntos
Insuficiência Renal Crônica , Calcificação Vascular , Ratos , Animais , Calcificação Vascular/patologia , Insuficiência Renal Crônica/metabolismo , Minerais , Homeostase , Fosfatos/metabolismoRESUMO
Calcitriol, and other vitamin D receptor activators, remain a primary treatment for elevated parathyroid hormone levels in patients with end stage kidney disease. The objective of this study was to assess the 24-hydroxylation-mediated metabolism of 25(OH)D3 and 1,25(OH)2D3 in rats with experimental kidney disease treated with calcitriol and in a cross-sectional analysis of patients requiring hemodialysis. Methods: Animals were stratified by creatinine into a time control group or calcitriol (20 ng/kg/day) for 3 weeks following CKD induction using a dietary adenine model (0.25% adenine). Hemodialysis patients were recruited and demographic data including calcitriol prescription was obtained by chart review and participant interview. Vitamin D metabolites were assessed using LC-MS/MS. In the rat model, 1,25(OH)2D3 levels increased substantially in calcitriol-treated rats yet there was no increase in its primary metabolite: 1,24,25(OH)2D3. A lower ratio of 1,24,25(OH)2D3:1,25(OH)2D3 (1,25-VMR) was associated with increased calcium levels in calcitriol treated rats. In hemodialysis patients (N = 86), the level of 1,25(OH)2D3 was substantially higher in calcitriol-treated patients yet there was no difference between groups in 1,24,25(OH)3D3, resulting in a marked decrease in the 1,25-VMR in calcitriol treated patients. In hemodialysis patients treated with calcitriol, 1,25(OH)2D3 and a lower ratio between 1,24,25(OH)3D3 and 1,25(OH)2D3 were associated with higher serum calcium levels. Impaired metabolism of exogenous calcitriol may contribute to the adverse effects associated with this treatment. A better understanding of the uniquely dysfunctional catabolic vitamin D profile in CKD may guide more effective treatment strategies.
Assuntos
Calcitriol , Falência Renal Crônica , Humanos , Ratos , Animais , Cromatografia Líquida , Cálcio , Estudos Transversais , Espectrometria de Massas em Tandem , Vitamina D , Receptores de Calcitriol , Falência Renal Crônica/tratamento farmacológicoRESUMO
Megalin is an endocytic receptor abundantly expressed in proximal tubular epithelial cells and other calciotropic extrarenal cells expressing vitamin D metabolizing enzymes, such as bone and parathyroid cells. The receptor functions in the uptake of the vitamin D-binding protein (DBP) complexed to 25 hydroxyvitamin D3 (25(OH)D3), facilitating the intracellular conversion of precursor 25(OH)D3 to the active 1,25 dihydroxyvitamin D3 (1,25(OH)2D3). The significance of renal megalin-mediated reabsorption of 25(OH)D3 and 1,25(OH)2D3 has been well established experimentally, and other studies have demonstrated relevant roles of extrarenal megalin in regulating vitamin D homeostasis in mammary cells, fat, muscle, bone, and mesenchymal stem cells. Parathyroid gland megalin may regulate calcium signaling, suggesting intriguing possibilities for megalin-mediated cross-talk between calcium and vitamin D regulation in the parathyroid; however, parathyroid megalin functionality has not been assessed in the context of vitamin D. Within various models of chronic kidney disease (CKD), megalin expression appears to be downregulated; however, contradictory results have been observed between human and rodent models. This review aims to provide an overview of the current knowledge of megalin function in the context of vitamin D metabolism, with an emphasis on extrarenal megalin, an area that clearly requires further investigation.
Assuntos
Nefropatias , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Calcitriol/metabolismo , Cálcio/metabolismo , Humanos , Rim/metabolismo , Nefropatias/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Vitamina D/metabolismo , Proteína de Ligação a Vitamina D/metabolismoRESUMO
For almost a century, vitamin A has been known as a nutrient critical for normal development, differentiation, and homeostasis; accordingly, there has been much interest in understanding its mechanism of action. This review is about the discovery of specific receptors for the vitamin A derivative, retinoic acid (RA), which launched extensive molecular, genetic, and structural investigations into these new members of the nuclear receptor superfamily of transcriptional regulators. These included two families of receptors, the RAR isotypes (α, ß, and γ) along with three RXR isotypes (α, ß, and γ), which bind as RXR/RAR heterodimers to cis-acting response elements of RA target genes to generate a high degree of complexity. Such studies have provided deep molecular insight into how the widespread pleiotropic effects of RA can be generated.
Assuntos
Receptores do Ácido Retinoico , Vitamina A , Proteínas de Transporte , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/genética , Receptores X de Retinoides/metabolismo , Tretinoína/metabolismo , Vitamina A/metabolismoRESUMO
Chronic kidney disease (CKD) results in calcitriol deficiency and altered vitamin D metabolism. The objective of this study was to assess the 24-hydroxylation-mediated metabolism of 25(OH)D3 and 1,25(OH)2D3 in a cross-sectional analysis of participants with a range of kidney function assessed by precise measured GFR (mGFR) (N = 143) and in rats with the induction and progression of experimental kidney disease. Vitamin D metabolites were assessed with LC-MS/MS. Circulating measures of 24-hydroxylation of 25(OH)D3 (24,25(OH)2D3:25(OH)D3) precisely decreased according to mGFR in humans and progressively in rats with developing CKD. In contrast, the 1,24,25(OH)3D3: 1,25(OH)2D3 vitamin D metabolite ratio increased in humans as the mGFR decreased and in rats with the induction and progression of CKD. Human participants taking cholecalciferol had higher circulating 1,24,25(OH)3D3, despite no increase of 1,25(OH)2D3. This first report of circulating 1,24,25(OH)3D3 in the setting of CKD provides novel insight into the uniquely altered vitamin D metabolism in this setting. A better understanding of the uniquely dysfunctional catabolic vitamin D profile in CKD may guide more effective treatment strategies. The potential that 24-hydroxylated products have biological activity of is an important area of future research.
Assuntos
Insuficiência Renal Crônica , Deficiência de Vitamina D , Animais , Cromatografia Líquida , Estudos Transversais , Humanos , Ratos , Espectrometria de Massas em Tandem , Vitamina D , Deficiência de Vitamina D/tratamento farmacológico , VitaminasRESUMO
Vitamin D receptor agonist (VDRA) therapy for PTH suppression is a mainstay for patients with severe CKD. Calcitriol (1,25-(OH)2 D3 ) is a former first-line VDRA in CKD treatment. However, a consequence of its use in CKD is accelerated vascular calcification (VC). An experimental CKD model was used to determine whether altering the calcitriol delivery profile to obtain different PTH suppression levels could improve vascular health outcomes. High adenine diet (0.25%) was used to generate experimental CKD in rats. CKD rats were treated using different calcitriol dosing strategies: (a) 20 ng/kg SD (n = 8), (b) 80 ng/kg SD (n = 8), (c) 5 ng/kg QID (n = 9), or (d) 20 ng/kg QID (n = 9). Multiple targets of calcitriol were assessed which include arterial calcium and phosphate as well as circulating calcium, phosphate, PTH, FGF-23, VWF, and vitamin D metabolome. PTH suppression occurred dose-dependently after 1-week calcitriol treatment (P < .01), but the suppressive effect was lost over time. Both VC and circulating FGF-23 increased > 10× in all calcitriol-treated rats (P < .05 and P < .001, respectively); similarly, circulating VWF increased at all time points (P < .05). Ad-hoc analysis of CKD morbidities in treated rats indicated no differences in negative outcomes based on PTH suppression level (minimal-, target-, and over-). Comparing different calcitriol dosing strategies revealed the following: (a) despite initial calcitriol-influenced PTH suppression across all treatments, the ability to continually suppress PTH was markedly reduced by study conclusion and (b) PTH suppression level is not an adequate proxy for improvements in overall CKD morbidity. These findings show (a) a more holistic approach to evaluate CKD treatment efficacy aside from PTH suppression is needed and (b) that other VDRA therapies should be examined in CKD treatment.
Assuntos
Calcitriol/farmacologia , Hormônio Paratireóideo/metabolismo , Receptores de Calcitriol/agonistas , Insuficiência Renal Crônica/tratamento farmacológico , Animais , Calcitriol/administração & dosagem , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Masculino , Ratos , Ratos Sprague-Dawley , Insuficiência Renal Crônica/fisiopatologia , Fatores de TempoAssuntos
Antineoplásicos/farmacologia , Família 26 do Citocromo P450/genética , Resistencia a Medicamentos Antineoplásicos/genética , Retinoides/farmacologia , Tretinoína/farmacologia , Animais , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Receptores do Ácido Retinoico/agonistas , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Vitamin D has emerged as a potentially important molecule in ophthalmology. To date, all ophthalmic data pertaining to vitamin D has been restricted primarily to tear and serum analysis in human patients. Considering the isolated nature of the eye, we sought to determine the presence of intraocular vitamin D in ocular disease. METHODS: 25-Hydroxyvitamin D3 (25(OH)D3) concentrations were measured in the eye and blood of 120 participants undergoing ophthalmic procedures. Ocular localization of the 1,25-dihydroxyvitamin D3-generating (CYP27B1) and deactivating (CYP24A1) hydroxylases was performed by immunohistochemistry. Gene expression of CYP27B1, CYP24A1 and VEGF-A was measured in eyes from patients with and without disease. RESULTS: 25(OH)D3 was quantified in 112 ocular samples. In 40 cataract patient samples, the average 25(OH)D3 concentration was 0.057â¯ng/mL, compared to 72 retinal disease patient samples, average of 0.502â¯ng/mL (pâ¯<â¯0.001). Intraocular 25(OH)D3 did not correlate with serum levels of 25(OH)D3. There was no difference between the level of 25(OH)D3 measured in the aqueous and vitreous humour. The vitamin D-specific CYPs 27B1 and 24A1, strongly localized to complementary regions of the ciliary body, retinal pigment epithelium and neural retina. Gene expression analysis confirmed retinal CYP27B1 correlated strongly with VEGF-A in eyes from diabetic patients (râ¯=â¯0.92, pâ¯<â¯0.001). CONCLUSIONS: Our data confirms that vitamin D is present in the humours of the human eye and that local synthesis/degradation is possible via the ocular CYP27B1 and CYP24A1. This argues for a functional role for local vitamin D production and signaling in the eye and suggests that vitamin D may be an important intraocular mediator in disease pathogenesis.
Assuntos
Calcifediol/metabolismo , Oftalmopatias/metabolismo , Olho/metabolismo , Vitamina D/metabolismo , Vitaminas/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Estudos Transversais , Oftalmopatias/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Vitamina D3 24-Hidroxilase/metabolismo , Adulto JovemRESUMO
BACKGROUND: Vitamin D repletion is recommended for secondary hyperparathyroidism (SHPT) and associated vitamin D insufficiency (VDI) in chronic kidney disease (CKD), but optimal levels of serum total 25-hydroxyvitamin D remain undefined. Clinical practice guidelines target sufficiency, whereas recent data indicate that higher levels are required to control the elevation of intact parathyroid hormone (iPTH) as CKD advances. This secondary analysis of 2 randomized controlled trials seeks to identify the minimum level of mean serum 25-hydroxyvitamin D required to control SHPT arising from VDI in stage 3 or 4 CKD. METHODS: Adult subjects (n = 429) with SHPT, VDI, and stage 3 or 4 CKD were stratified by stage and treated daily with either extended-release calcifediol (ERC) or placebo in 2 identical, parallel, randomized, double-blind studies. After treatment for 26 weeks, all subjects were ranked by the level of serum total 25-hydroxyvitamin D and divided into quintiles in order to examine the relationships between the degree of vitamin D repletion and the associated changes in plasma iPTH, serum bone turnover markers, calcium, phosphorus, intact fibroblast growth factor 23 (FGF23) and vitamin D metabolites, estimated glomerular filtration rate (eGFR), and urine calcium:creatinine (Ca:Cr) ratio. RESULTS: Progressive increases in serum 1,25-dihydroxyvitamin D and reductions in plasma iPTH and serum bone turnover markers were observed as mean posttreatment serum 25-hydroxyvitamin D rose from 13.9 ng/mL (in Quintile 1) to 92.5 ng/mL (in Quintile 5), irrespective of CKD stage. Mean serum calcium, phosphorus and FGF23, eGFR, and urine Ca:Cr ratio (collectively "safety parameters") did not significantly change from Quintile 1. Suppression of iPTH and bone turnover markers was not observed until serum 25-hydroxyvitamin D rose to at least 50.8 ng/mL (Quintile 3). CONCLUSION: ERC therapy produced exposure-dependent reductions in plasma iPTH and bone turnover markers only when mean serum total 25-hydroxyvitamin D reached at least 50.8 ng/mL, indicating that current targets for vitamin D repletion therapy in CKD are too low. Gradual elevation of mean serum 25-hydroxyvitamin D to 92.5 ng/mL was not associated with significant adverse changes in safety parameters.
Assuntos
Calcifediol/administração & dosagem , Hiperparatireoidismo Secundário/tratamento farmacológico , Insuficiência Renal Crônica/complicações , Deficiência de Vitamina D/tratamento farmacológico , Vitamina D/análogos & derivados , Idoso , Método Duplo-Cego , Monitoramento de Medicamentos/normas , Feminino , Fator de Crescimento de Fibroblastos 23 , Humanos , Hiperparatireoidismo Secundário/sangue , Hiperparatireoidismo Secundário/diagnóstico , Hiperparatireoidismo Secundário/etiologia , Masculino , Pessoa de Meia-Idade , Hormônio Paratireóideo/sangue , Guias de Prática Clínica como Assunto , Valores de Referência , Insuficiência Renal Crônica/sangue , Vitamina D/sangue , Deficiência de Vitamina D/sangue , Deficiência de Vitamina D/diagnóstico , Deficiência de Vitamina D/etiologiaRESUMO
Mutation in the tyrosine kinase (TK) domain of the epidermal growth factor receptor ( EGFR) gene drives the development of lung cancer. EGFR tyrosine kinase inhibitors (EGFR TKIs), including erlotinib and afatinib, are initially effective in treating EGFR mutant nonsmall cell lung cancer (NSCLC). However, drug resistance quickly develops due to several mechanisms, including induction of the epithelial-mesenchymal transition (EMT). No effective therapies are currently available for patients who develop EMT-associated EGFR TKI resistance. 1,25-Dihydroxyvitamin D3 (1,25D3) promotes epithelial differentiation and inhibits growth of NSCLC cells. 1,25D3 thus represents a promising agent for the treatment of EMT-associated EGFR TKI resistance. However, 1,25D3 induces the expression of 24-hydroxylase (24OHase), which decreases 1,25D3 activity. CTA091, a potent and selective 24OHase inhibitor, has been developed to attenuate this adverse effect. CTA091 also suppresses renal 24OHase activity and so may promote hypercalcemia. To exploit favorable effects of 1,25D3 plus CTA091 in tumor cells while avoiding problematic systemic effects of 24OHase inhibition, we developed EGFR-targeted, liposomal nanoparticles (EGFR-LP) to offer tumor-targeted co-delivery of 1,25D3 and CTA091. We then established an EMT-associated model of EGFR TKI resistance, and showed that such nanoparticles improved cellular uptake of 1,25D3 and CTA091, drove pro-epithelial signaling by upregulating E-cadherin ( CDH1), and significantly inhibited the growth of EGFR TKI resistant cells. Our results demonstrated that the delivery of vitamin D-based drug payloads via tumor-targeted EGFR-LP has promise as a new therapy for EFGR TKI resistant lung cancer. Future studies will focus on in vivo evaluation of biological activity, therapeutic benefits, and systemic toxicity prior to clinical translation.
Assuntos
Calcitriol/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Combinação de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Receptores ErbB/genética , Cloridrato de Erlotinib/farmacologia , Cloridrato de Erlotinib/uso terapêutico , Humanos , Lipossomos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Nanopartículas/química , Inibidores de Proteínas Quinases/uso terapêutico , Vitamina D3 24-Hidroxilase/antagonistas & inibidores , Vitamina D3 24-Hidroxilase/metabolismoRESUMO
CYP24A1 (hereafter referred to as CYP24) enzymatic activity is pivotal in the inactivation of vitamin D metabolites. Basal renal and extrarenal CYP24 is usually low but is highly induced by its substrate 1,25-dihydroxyvitamin D. Unbalanced high and/or long-lasting CYP24 expression has been proposed to underlie diseases like chronic kidney disease, cancers, and psoriasis that otherwise should favorably respond to supplemental vitamin D. Using genetically modified mice, we have shown that renal phosphate wasting hypophosphatemic states arising from high levels of fibroblast growth factor 23 (FGF23) are also associated with increased renal Cyp24 expression, suggesting that elevated CYP24 activity is pivotal to the pathophysiology of these disorders. We therefore crossed 2 mouse strains, each with distinct etiology for high levels of circulating FGF23, onto a Cyp24-null background. Specifically, we evaluated Cyp24 deficiency in Hyp mice, the murine homolog of X-linked dominant hypophosphatemic rickets, and transgenic mice that overexpress a mutant FGF23 (FGF23R176Q) that is associated with the autosomal dominant form of hypophosphatemic rickets. Loss of Cyp24 in these murine models of human disease resulted in near-complete recovery of rachitic/osteomalacic bony abnormalities in the absence of any improvement in the serum biochemical profile. Moreover, treatment of Hyp and FGF23R1760-transgenic mice with the CYP24 inhibitor CTA102 also ameliorated their rachitic bones. Our results link CYP24 activity to the pathophysiology of FGF23-dependent renal phosphate wasting states and implicate pharmacologic CYP24 inhibition as a therapeutic adjunct for their treatment.
Assuntos
Inibidores das Enzimas do Citocromo P-450/farmacologia , Fatores de Crescimento de Fibroblastos/metabolismo , Fosfatos/urina , Insuficiência Renal Crônica , Vitamina D3 24-Hidroxilase/antagonistas & inibidores , Síndrome de Emaciação , Animais , Modelos Animais de Doenças , Feminino , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/genética , Humanos , Camundongos , Camundongos Knockout , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/urina , Vitamina D3 24-Hidroxilase/genética , Vitamina D3 24-Hidroxilase/metabolismo , Síndrome de Emaciação/tratamento farmacológico , Síndrome de Emaciação/genética , Síndrome de Emaciação/patologia , Síndrome de Emaciação/urinaRESUMO
The active metabolite of vitamin A, retinoic acid (RA), is known to be essential for spermatogenesis. Changes to RA levels within the seminiferous epithelium can alter the development of male germ cells, including blocking their differentiation completely. Excess RA has been shown to cause germ cell death in both neonatal and adult animals, yet the cells capable of degrading RA within the testis have yet to be investigated. One previous study alluded to a requirement for one of the RA degrading enzymes, CYP26B1, in Sertoli cells but no data exist to determine whether germ cells possess the ability to degrade RA. To bridge this gap, the roles of CYP26A1 and CYP26B1 within the seminiferous epithelium were investigated by creating single and dual conditional knockouts of these enzymes in either Sertoli or germ cells. Analysis of these knockout models revealed that deletion of both Cyp26a1 and Cyp26b1 in either cell type resulted in increased vacuolization within the seminiferous tubules, delayed spermatid release, and an increase in the number of STRA8-positive spermatogonia, but spermatozoa were still produced and the animals were found to be fertile. However, elimination of CYP26B1 activity within both germ and Sertoli cells resulted in severe male subfertility, with a loss of advanced germ cells from the seminiferous epithelium. These data indicate that CYP26 activity within either Sertoli or germ cells is essential for the normal progression of spermatogenesis and that its loss can result in reduced male fertility.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Epitélio Seminífero/enzimologia , Espermatogênese/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/genética , Células Germinativas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Ácido Retinoico 4 Hidroxilase , Células de Sertoli/metabolismo , Espermatogônias/metabolismo , Espermatozoides/metabolismoRESUMO
Hyperimmunized hens are an effective means of generating large quantities of antigen specific egg antibodies that have use as oral supplements. In this study, we attempted to create a peptide specific antibody that produced outcomes similar to those of the human pharmaceutical, sevelamer HCl, used in the treatment of hyperphosphatemia (a sequela of chronic renal disease). Egg antibodies were generated against 8 different human intestinal sodium-dependent phosphate cotransporter 2b (NaPi2b) peptides, and hNaPi2b peptide egg antibodies were screened for their ability to inhibit phosphate transport in human intestinal Caco-2 cell line. Antibody produced against human peptide sequence TSPSLCWT (anti-h16) was specific for its peptide sequence, and significantly reduced phosphate transport in human Caco-2 cells to 25.3±11.5% of control nonspecific antibody, when compared to nicotinamide, a known inhibitor of phosphate transport (P≤0.05). Antibody was then produced against the mouse-specific peptide h16 counterpart (mouse sequence TSPSYCWT, anti-m16) for further analysis in a murine model. When anti-m16 was fed to mice (1% of diet as dried egg yolk powder), egg yolk immunoglobulin (IgY) was detected using immunohistochemical staining in mouse ileum, and egg anti-m16 IgY colocalized with a commercial goat anti-NaPi2b antibody. The effectiveness of anti-m16 egg antibody in reducing serum phosphate, when compared to sevelamer HCl, was determined in a mouse feeding study. Serum phosphate was reduced 18% (P<0.02) in mice fed anti-m16 (1% as dried egg yolk powder) and 30% (P<0.0001) in mice fed sevelamer HCl (1% of diet) when compared to mice fed nonspecific egg immunoglobulin. The methods described and the findings reported show that oral egg antibodies are useful and easy to prepare reagents for the study and possible treatment of select diseases.
Assuntos
Imunoglobulinas/imunologia , Fosfatos/sangue , Proteínas Cotransportadoras de Sódio-Fosfato/genética , Animais , Anticorpos/metabolismo , Células CACO-2 , Embrião de Galinha , Galinhas , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulinas/sangue , Imunoglobulinas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óvulo/metabolismo , Análise de Sequência de Proteína , Proteínas Cotransportadoras de Sódio-Fosfato/imunologiaRESUMO
Vitamin D insufficiency is prevalent in chronic kidney disease (CKD) and associated with secondary hyperparathyroidism (SHPT) and increased risk of bone and vascular disease. Unfortunately, supplementation of stage 3 or 4 CKD patients with currently recommended vitamin D2 or D3 regimens does not reliably restore serum total 25-hydroxyvitamin D to adequacy (≥30ng/mL) or effectively control SHPT. Preclinical and clinical studies were conducted to evaluate whether the effectiveness of vitamin D repletion depends, at least in part, on the rate of repletion. A modified-release (MR) oral formulation of calcifediol (25-hydroxyvitamin D3) was developed which raised serum 25-hydroxyvitamin D3 and calcitriol levels gradually. Single doses of either bolus intravenous (IV) or oral MR calcifediol were administered to vitamin D deficient rats. Bolus IV calcifediol produced rapid increases in serum 25-hydroxyvitamin D3, calcitriol and FGF23, along with significant induction of CYP24A1 in both kidney and parathyroid gland. In contrast, oral MR calcifediol produced gradual increases in serum 25-hydroxyvitamin D3 and calcitriol and achieved similar hormonal exposure, yet neither CYP24A1 nor FGF23 were induced. A 10-fold greater exposure to bolus IV than oral MR calcifediol was required to similarly lower intact parathyroid hormone (iPTH). Single doses of oral MR (450 or 900µg) or bolus IV (450µg) calcifediol were administered to patients with stage 3 or 4 CKD, SHPT and vitamin D insufficiency. Changes in serum 25-hydroxyvitamin D3 and calcitriol and in plasma iPTH were determined at multiple time-points over the following 42 days. IV calcifediol produced abrupt and pronounced increases in serum 25-hydroxyvitamin D3 and calcitriol, but little change in plasma iPTH. As in animals, these surges triggered increased vitamin D catabolism, as evidenced by elevated production of 24,25-dihydroxyvitamin D3. In contrast, MR calcifediol raised serum 25-hydroxyvitamin D3 and calcitriol gradually, and meaningfully lowered plasma iPTH levels. Taken together, these studies indicate that rapid increases in 25-hydroxyvitamin D3 trigger CYP24A1 and FGF23 induction, limiting effective exposure to calcitriol and iPTH reduction in SHPT. They also support further investigation of gradual vitamin D repletion for improved clinical effectiveness. This article is part of a Special Issue entitled "17th Vitamin D Workshop".
Assuntos
Calcifediol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Insuficiência Renal Crônica/tratamento farmacológico , Deficiência de Vitamina D/prevenção & controle , Vitaminas/farmacologia , 24,25-Di-Hidroxivitamina D 3/sangue , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Administração Oral , Animais , Cálcio/sangue , Suplementos Nutricionais , Liberação Controlada de Fármacos , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Humanos , Masculino , Hormônio Paratireóideo/sangue , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Insuficiência Renal Crônica/metabolismo , Deficiência de Vitamina D/metabolismo , Vitamina D3 24-Hidroxilase/genéticaRESUMO
BACKGROUND/AIMS: Vitamin D insufficiency drives secondary hyperparathyroidism (SHPT) and is associated with increased cardiovascular mortality in patients with chronic kidney disease (CKD). SHPT is poorly addressed by current vitamin D repletion options. The present study evaluated a novel investigational vitamin D repletion therapy: a modified-release (MR) formulation of calcifediol designed to raise serum 25-hydroxyvitamin D in a gradual manner to minimize the induction of CYP24 and, thereby, improve the SHPT control. METHODS: This randomized, double-blind, placebo-controlled trial evaluated MR calcifediol in CKD subjects (n = 78) with plasma intact parathyroid hormone (iPTH) >70 pg/ml and serum total 25-hydroxyvitamin D <30 ng/ml. Subjects received daily treatment for six weeks with oral MR calcifediol (30, 60 or 90 µg) or a placebo. RESULTS: More than 90% of subjects treated with MR calcifediol achieved serum 25-hydroxyvitamin D levels ≥30 ng/ml versus 3% of subjects treated with placebo (p < 0.0001). Mean plasma iPTH decreased from baseline (140.3 pg/ml) by 20.9 ± 6.2% (SE), 32.8 ± 5.7 and 39.3 ± 4.3% in the 30, 60 and 90 µg dose groups, respectively, and increased 17.2 ± 7.8% in the pooled placebo group (p < 0.005). No clinically significant safety concerns arose during MR calcifediol treatment. CONCLUSION: Oral MR calcifediol appears safe and highly effective in treating SHPT associated with vitamin D insufficiency in CKD.
Assuntos
Calcifediol/administração & dosagem , Hiperparatireoidismo Secundário/tratamento farmacológico , Insuficiência Renal Crônica/complicações , Deficiência de Vitamina D/tratamento farmacológico , Vitamina D/análogos & derivados , Vitaminas/administração & dosagem , Adulto , Idoso , Calcifediol/sangue , Preparações de Ação Retardada , Método Duplo-Cego , Feminino , Humanos , Hiperparatireoidismo Secundário/sangue , Hiperparatireoidismo Secundário/etiologia , Masculino , Pessoa de Meia-Idade , Hormônio Paratireóideo/sangue , Insuficiência Renal Crônica/sangue , Vitamina D/sangue , Deficiência de Vitamina D/sangue , Deficiência de Vitamina D/etiologia , Vitaminas/sangueRESUMO
The vitamin A metabolite retinoic acid (RA) has potent immunomodulatory properties that affect T cell differentiation, migration and function. However, the precise role of RA metabolism in T cells remains unclear. Catabolism of RA is mediated by the Cyp26 family of cytochrome P450 oxidases. We examined the role of Cyp26b1, the T cell-specific family member, in CD4(+) T cells. Mice with a conditional knockout of Cyp26b1 in T cells (Cyp26b1 (-/-) mice) displayed normal lymphoid development but showed an increased sensitivity to serum retinoids, which led to increased differentiation under both inducible regulatory T (iTreg) cell- and TH17 cell-polarizing conditions in vitro. Further, Cyp26b1 expression was differentially regulated in iTreg and TH17 cells. Transfer of naïve Cyp26b1 (-/-) CD4(+) T cells into Rag1 (-/-) mice resulted in significantly reduced disease in a model of T cell-dependent colitis. Our results show that T cell-specific expression of Cyp26b1 is required for the development of T cell-mediated colitis and may be applicable to the development of therapeutics that target Cyp26b1 for the treatment of inflammatory bowel disease.
Assuntos
Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , Sistema Enzimático do Citocromo P-450/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Colite/imunologia , Camundongos , Camundongos Knockout , Ácido Retinoico 4 HidroxilaseRESUMO
OBJECTIVE: Hyperphosphatemia in animal models of human renal disease has been linked to increased risk of death. Phosphate binders (e.g., sevelamer hydrochloride) and plant-based, low phosphate diets are used to reduce dietary phosphate load; however, animal models show that treatment with active forms of vitamin D(3) (e.g., calcitriol, a renal disease therapy) renders plant phytate phosphate available for absorption. Using an established chick model, the effectiveness of sevelamer in preventing the apparent absorption of liberated phytate phosphate during active vitamin D use was investigated in two separate experiments. DESIGN: One-day-old chicks were fed ad libitum a basal diet containing deficient levels of inorganic phosphate (0.13%), but adequate in total phosphate (0.40%, 0.23% as phytate phosphate), with or without the inclusion of sevelamer hydrochloride (a phosphate binder), available inorganic phosphate, or active vitamin D as 1α-(OH) D(3). MAIN OUTCOME MEASURES: Plasma phosphate (mg/dL), total bone ash (%), and weight gain (g). RESULTS: Adding inorganic phosphate (0.36%) or 1α-(OH) D(3) increased plasma phosphate 49% and 48%, respectively (P < .0001), and bone ash 23% and 19%, respectively (P < .001). The addition of 1% sevelamer to the basal diet with added inorganic phosphate or 1α-(OH) D(3) significantly decreased plasma phosphate by 28% and 20%, respectively (P < .01). CONCLUSION: Active vitamin D increased the availability of phytate phosphate for intestinal absorption in an animal model; however, sevelamer effectively reduced the availability of phosphate liberated from phytate. These data imply that sevelamer has phytate phosphate binding efficacy.
Assuntos
Hidroxicolecalciferóis/administração & dosagem , Fosfatos/sangue , Ácido Fítico/metabolismo , Poliaminas/metabolismo , Ração Animal , Animais , Galinhas , Dieta , Hiperfosfatemia/tratamento farmacológico , Hiperfosfatemia/fisiopatologia , Masculino , Minerais/análise , Fósforo/análise , Fósforo/metabolismo , Fósforo na Dieta/administração & dosagem , Sevelamer , Aumento de Peso/efeitos dos fármacosRESUMO
Focal facial dermal dysplasia (FFDD) Type IV is a rare syndrome characterized by facial lesions resembling aplasia cutis in a preauricular distribution along the line of fusion of the maxillary and mandibular prominences. To identify the causative gene(s), exome sequencing was performed in a family with two affected siblings. Assuming autosomal recessive inheritance, two novel sequence variants were identified in both siblings in CYP26C1-a duplication of seven base pairs, which was maternally inherited, c.844_851dupCCATGCA, predicting p.Glu284fsX128 and a missense mutation, c.1433G>A, predicting p.Arg478His, that was paternally inherited. The duplication predicted a frameshift mutation that led to a premature stop codon and premature chain termination, whereas the missense mutation was not functional based on its in vitro expression in mammalian cells. The FFDD skin lesions arise along the sites of fusion of the maxillary and mandibular prominences early in facial development, and Cyp26c1 was expressed exactly along the fusion line for these facial prominences in the first branchial arch in mice. Sequencing of four additional, unrelated Type IV FFDD patients and eight Type II or III TWIST2-negative FFDD patients revealed that three of the Type IV patients were homozygous for the duplication, whereas none of the Type II or III patients had CYP26C1 mutations. The seven base pairs duplication was present in 0.3% of healthy controls and 0.3% of patients with other birth defects. These findings suggest that the phenotypic manifestations of FFDD Type IV can be non-penetrant or underascertained. Thus, FFDD Type IV results from the loss of function mutations in CYP26C1.