RESUMO
Stabilisation of fragile oligonucleotides, typically small interfering RNA (siRNA), is one of the most critical issues for oligonucleotide therapeutics. Many previous studies encapsulated oligonucleotides into ~100-nm nanoparticles. However, such nanoparticles inevitably accumulate in liver and spleen. Further, some intractable cancers, e.g., tumours in pancreas and brain, have inherent barrier characteristics preventing the penetration of such nanoparticles into tumour microenvironments. Herein, we report an alternative approach to cancer-targeted oligonucleotide delivery using a Y-shaped block catiomer (YBC) with precisely regulated chain length. Notably, the number of positive charges in YBC is adjusted to match that of negative charges in each oligonucleotide strand (i.e., 20). The YBC rendezvouses with a single oligonucleotide in the bloodstream to generate a dynamic ion-pair, termed unit polyion complex (uPIC). Owing to both significant longevity in the bloodstream and appreciably small size (~18 nm), the uPIC efficiently delivers oligonucleotides into pancreatic tumour and brain tumour models, exerting significant antitumour activity.
Assuntos
Antineoplásicos/metabolismo , Neoplasias Encefálicas/terapia , Regulação Neoplásica da Expressão Gênica , Nanoestruturas/química , Oligonucleotídeos/genética , Neoplasias Pancreáticas/terapia , RNA Interferente Pequeno/genética , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Carbocianinas/química , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Portadores de Fármacos/síntese química , Portadores de Fármacos/farmacocinética , Corantes Fluorescentes/química , Humanos , Injeções Intravenosas , Masculino , Camundongos , Nanoestruturas/administração & dosagem , Oligonucleotídeos/síntese química , Oligonucleotídeos/metabolismo , Oligonucleotídeos/farmacocinética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidade , Polietilenoglicóis/química , Polilisina/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacocinética , Eletricidade Estática , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto , Quinase 1 Polo-LikeRESUMO
Although techniques for cell-specific gene expression via viral transfer have advanced, many challenges (e.g., viral vector design, transduction of genes into specific target cells) still remain. We investigated a novel, simple methodology for using adenovirus transfer to target specific cells of the kidney tubules for the expression of exogenous proteins. We selected genes encoding sodium-dependent phosphate transporter type 2a (NPT2a) in the proximal tubule, sodium-potassium-2-chloride cotransporter (NKCC2) in the thick ascending limb of Henle (TALH), and aquaporin 2 (AQP2) in the collecting duct. The promoters of the three genes were linked to a GFP-coding fragment, the final constructs were then incorporated into an adenovirus vector, and this was then used to generate gene-manipulated viruses. After flushing circulating blood, viruses were directly injected into the renal arteries of rats and were allowed to site-specifically expression in tubule cells, and rats were then euthanized to obtain kidney tissues for immunohistochemistry. Double staining with adenovirus-derived EGFP and endogenous proteins were examined to verify orthotopic expression, i.e. "adenovirus driven NPT2a-EGFP and endogenous NHE3 protein", "adenovirus driven NKCC2-EGFP and endogenous NKCC2 protein" and "adenovirus driven AQP2-EGFP and endogenous AQP2 protein". Owing to a lack of finding good working anti-NPT2a antibody, an antibody against a different protein (sodium-hydrogen exchanger 3 or NHE3) that is also specifically expressed in the proximal tubule was used. Kidney structures were well-preserved, and other organ tissues did not show EGFP staining. Our gene transfer method is easier than using genetically engineered animals, and it confers the advantage of allowing the manipulation of gene transfer after birth. This is the first method to successfully target gene expression to specific cells in the kidney tubules. This study may serve as the first step for safe and effective gene therapy in the kidney tubule diseases.
Assuntos
Adenoviridae/genética , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Regiões Promotoras Genéticas/genética , Transdução Genética/métodos , Expressão Gênica , Vetores Genéticos/genética , Células HEK293 , Humanos , SegurançaRESUMO
Polyion complexes (b-PICs) are prepared by mixing single- or double-stranded oligo RNA (aniomer) with poly(ethylene glycol)-b-poly(L-lysine) (PEG-PLL) (block catiomer) to clarify the effect of aniomer chain rigidity on association behaviors at varying concentrations. Here, a 21-mer single-stranded RNA (ssRNA) (persistence length: 1.0 nm) and a 21-mer double-stranded RNA (small interfering RNA, siRNA) (persistence length: 62 nm) are compared. Both oligo RNAs form a minimal charge-neutralized ionomer pair with a single PEG-PLL chain, termed unit b-PIC (uPIC), at low concentrations (<≈ 0.01 mg mL(-1)). Above the critical association concentration (≈ 0.01 mg mL(-1)), ssRNA b-PICs form secondary associates, PIC micelles, with sizes up to 30-70 nm, while no such multimolecular assembly is observed for siRNA b-PICs. The entropy gain associated with the formation of a segregated PIC phase in the multimolecular PIC micelles may not be large enough for rigid siRNA strands to compensate with appreciably high steric repulsion derived from PEG chains. Chain rigidity appears to be a critical parameter in polyion complex association.
Assuntos
Lisina/análogos & derivados , Polietilenoglicóis/química , RNA de Cadeia Dupla/química , RNA Interferente Pequeno/química , Entropia , Concentração de Íons de Hidrogênio , Lisina/química , Micelas , Eletricidade EstáticaRESUMO
Low-density lipoprotein apheresis (LDL-A) has been shown to reduce proteinuria in a subgroup of nephrotic syndrome patients refractory to immunosuppressive therapy. Factors influencing the efficacy of LDL-A in nephrotic syndrome are completely unknown. Using a proteomics approach, we aimed to identify biological markers that predict the response to LDL-A in patients with steroid-resistant nephrotic syndrome (SRNS). Identification of plasma proteins bound to the dextran-sulfate column at the first session of LDL-A was determined by mass spectrometry. To investigate biological factors associated with the response to LDL-A, we compared profiles of column-bound proteins between responders (defined by more than 50% reduction of proteinuria after the treatment) and non-responders by 2-dimensional gel electrophoresis (2-DE) coupled to mass spectrometry in seven patients with SRNS. Evaluation of proteins adsorbed to LDL-A column in patients with SRNS revealed the identity of 62 proteins, which included apolipoproteins, complement components, and serum amyloid P-component (SAP). Comparative analysis of the column-bound proteins between responders and non-responders by 2-DE demonstrated that apolipoprotein E (APOE) and SAP levels were increased in non-responders as compared with responders. These results were confirmed by western blotting. Moreover, serum levels of APOE and SAP were significantly higher in the non-responder group than in the responder group by ELISA. Our data provide comprehensive analysis of proteins adsorbed by LDL-A in SRNS, and demonstrate that the serum levels of APOE and SAP may be used to predict the response to LDL-A in these patients.
Assuntos
Remoção de Componentes Sanguíneos/métodos , LDL-Colesterol/sangue , Síndrome Nefrótica/terapia , Proteômica/métodos , Adulto , Idoso , Apolipoproteínas E/sangue , Proteínas Sanguíneas/metabolismo , Sulfato de Dextrana/química , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Feminino , Glucocorticoides/uso terapêutico , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Componente Amiloide P Sérico/metabolismoRESUMO
Efficient systems for delivery of small interfering RNA (siRNA) are required for clinical application of RNA interference (RNAi) in cancer therapy. Herein, we developed a safe and efficient nanocarrier comprising poly(ethylene glycol)-block-charge-conversional polymer (PEG-CCP)/calcium phosphate (CaP) hybrid micelles for systemic delivery of siRNA and studied their efficacy in spontaneous bioluminescent pancreatic tumors from transgenic mice. PEG-CCP was engineered to provide the siRNA-loaded hybrid micelles with enhanced colloidal stability and biocompatibility due to the PEG capsule and with endosome-disrupting functionality due to the acidic pH-responsive CCP segment where the polyanionic structure could be converted to polycationic structure at acidic pH through cis-aconitic amide cleavage. The resulting hybrid micelles were confirmed to have a diameter of <50nm, with a narrow size distribution. Intravenously injected hybrid micelles significantly reduced the luciferase-based luminescent signal from the spontaneous pancreatic tumors in an siRNA sequence-specific manner. The gene silencing activity of the hybrid micelles correlated with their preferential tumor accumulation, as indicated by fluorescence imaging and histological analysis. Moreover, there were no significant changes in hematological parameters in mice treated with the hybrid micelles. These results demonstrate the great potential of the hybrid micelles as siRNA carriers for RNAi-based cancer therapy.
Assuntos
Nanopartículas/administração & dosagem , Neoplasias Pancreáticas/genética , RNA Interferente Pequeno/administração & dosagem , Animais , Fosfatos de Cálcio/química , Linhagem Celular Tumoral , Inativação Gênica , Luciferases/genética , Masculino , Camundongos , Camundongos Transgênicos , Micelas , Nanopartículas/química , Neoplasias Pancreáticas/metabolismo , Polietilenoglicóis/química , RNA Interferente Pequeno/farmacocinética , Distribuição TecidualRESUMO
OBJECTIVE: Autosomal dominant hypocalcemia (ADH) is a congenital isolated hypoparathyroidism caused by activating mutations in the calcium-sensing receptor (CASR) gene. The clinical features of ADH are heterogeneous; some patients are asymptomatic, and others show severe hypocalcemia with Bartter's syndrome. We therefore recruited 12 patients with ADH to clarify the determinants of their clinical presentation. DESIGN AND METHODS: We studied two sporadic and 10 familial cases of ADH. Serum concentrations of calcium, intact PTH, and magnesium (Mg(2+)) were measured in each patient. Fractional excretion of Mg (FE(Mg)) was calculated in spot urine samples. A nuclear factor of activated T cells luciferase assay was used to analyze the responsiveness of each mutant CaSR to extracellular Ca(2+). RESULTS: Genomic analysis revealed five known activating mutations and a novel mutation, E481K, in the CASR. Patients with the A843E, C131W, or F788C mutation showed hypomagnesemia with elevated FE(Mg). Intact PTH in these patients was consistently near the detection limit. In contrast, patients with the P221L, K47N, or E481K mutation exhibited normal Mg(2+) levels. In these patients, intact PTH increased in response to low calcium, and their maximum intact PTH exceeded the lower limit of the reference range. Functional analysis showed an association between the disease severity and the in vitro activity of the mutant CaSR. CONCLUSIONS: The functional activity of mutant CaSR determines the serum Mg(2+) level, renal Mg(2+) handling, and intact PTH in patients with ADH. The presence of hypomagnesemia with elevated FE(Mg) may indicate the diagnosis of ADH among patients with PTH-deficient hypoparathyroidism.
Assuntos
Hipercalciúria/diagnóstico , Hipocalcemia/diagnóstico , Hipoparatireoidismo/congênito , Mutação , Receptores de Detecção de Cálcio/genética , Adulto , Cálcio/sangue , Criança , Feminino , Humanos , Hipercalciúria/genética , Hipercalciúria/metabolismo , Hipocalcemia/genética , Hipocalcemia/metabolismo , Hipoparatireoidismo/diagnóstico , Hipoparatireoidismo/genética , Hipoparatireoidismo/metabolismo , Magnésio/sangue , Masculino , Pessoa de Meia-Idade , Hormônio Paratireóideo/sangue , Fenótipo , Receptores de Detecção de Cálcio/metabolismoRESUMO
For systemic small interfering RNA (siRNA) delivery to tumor mass, a multifunctional polyion complex micelle was constructed with a block copolymer bearing a targeting ligand and a micelle-stabilizing moiety as well as an endosome-disrupting cationic unit. The block copolymer was comprised of poly(ethylene glycol) (PEG) and a polyaspartamide derivative with flanking cationic tetraethylenepentamine (TEP) moiety (PAsp(TEP)), in which the distal ends of PEG and PAsp(TEP) were further installed with cyclic RGD (cRGD) peptide ligand and cholesteryl (Chol) moiety, respectively. The resulting polymer was confirmed to form siRNA-loaded micelle with a diameter of sub 50 nm and a narrow size distribution. In the stability assays with fluorescently labeled siRNA, the terminal Chol moiety significantly suppressed both the rapid dissociation of the micelles in the serum-containing medium and their rapid elimination from the bloodstream, presumably due to its hydrophobic interactions in the micellar core. Moreover, the targeting cRGD ligand, associated with the stabilizing moiety, significantly enhanced the accumulation of siRNA-loaded micelle in a subcutaneous lung (A549) tumor, compared to a non-targeted control, after systemic administration. Ultimately, significant tumor growth inhibition was achieved by systemic administration of the targeted/stabilized micelle incorporating polo-like kinase 1 (Plk1) siRNA with negligible liver toxicity, consistent with the significant sequence-specific gene silencing of Plk1 in the tumor tissue. These results demonstrated the therapeutic potential of cRGD-PEG-PAsp(TEP)-Chol/siRNA micelle for systemic siRNA delivery toward cancer therapy.
RESUMO
The development of nanocarriers that selectively release guest molecules on sensing a particular biological signal is being actively pursued in nanomedicine for diagnostic and therapeutic purposes. Here we report a protein-based nanocarrier that opens in the presence of intracellular adenosine-5'-triphosphate (ATP). The nanocarrier consists of multiple barrel-shaped chaperonin units assembled through coordination with Mg(2+) into a tubular structure that protects guest molecules against biological degradation. When its surface is functionalized with a boronic acid derivative, the nanocarrier is able to enter cells. The hydrolysis of intracellular ATP into adenosine-5'-diphosphate (ADP) induces conformational changes of the chaperonin units, which in turns generate a mechanical force that leads to the disassembly of the tube and release of the guests. This scission occurs with a sigmoidal dependence on ATP concentration, which means that the nanocarrier can differentiate biological environments in terms of the concentration of ATP for selective guest release. Furthermore, biodistribution tests reveal preferential accumulation of the nanocarriers in a tumour tissue.
Assuntos
Trifosfato de Adenosina/metabolismo , Nanoestruturas , Robótica , Proteínas de Fluorescência Verde/química , Hidrólise , Magnésio/metabolismo , Microscopia Eletrônica de Transmissão , Conformação ProteicaRESUMO
Multifunctional delivery systems of small interfering RNA (siRNA) are needed to overcome the intrinsic biological barriers toward efficient gene silencing in the cell cytoplasm. In this report, a smart multilayered assembly (SMA) was fabricated by a layer-by-layer method with polyionic materials. The SMA was designed to feature a siRNA-loaded core, a transiently core-stabilizing silica interlayer, an endosome-disrupting polycation interlayer, and a biocompatible poly(ethylene glycol) (PEG) shell with reductive environment-responsive detachability. The SMA was confirmed to be approximately 160 nm in size with narrow distribution and spherical morphology by DLS and TEM analyses. The PEG detachability of the SMA based on disulfide cleavage was also confirmed by the increase in both ζ-potential and size due to the exposure of the polycation interlayer and the compromised colloidal stability. The silica interlayer rendered the SMA highly tolerant to dissociation induced by anionic lipids, while after 24 h dialysis siRNA release from the SMA was clearly observed, presumably due to gradual dissolution of the silica interlayer based on the equilibrium shift to silicate ions. The entrapment ratio of siRNA delivered by the SMA within the endosome was significantly lower than that by nondisulfide control (NDC) without PEG detachability, suggesting the improved endosomal escape of SMA with the exposed, endosome-disrupting interlayer after PEG detachment. SMAs induced significantly higher gene silencing efficiency in various cultured cells, compared to NDC, without associated cytotoxicity. The systemic administration of SMAs for subcutaneous tumor-bearing mice achieved significant endogenous gene silencing in tumor tissue without hematological toxicity.
Assuntos
Preparações de Ação Retardada/química , Endossomos/química , Endossomos/fisiologia , Nanocápsulas/química , Neoplasias Experimentais/genética , RNA Interferente Pequeno/genética , Transfecção/métodos , Animais , Cristalização/métodos , Preparações de Ação Retardada/administração & dosagem , Substâncias Macromoleculares/química , Teste de Materiais , Camundongos , Conformação Molecular , Nanocápsulas/ultraestrutura , Tamanho da Partícula , Polietilenoglicóis/química , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , Propriedades de SuperfícieRESUMO
Development of an efficient in vivo delivery vehicle of small interfering RNA (siRNA) is the key challenge for successful siRNA-based therapies. In this study, toward systemic delivery of siRNA to solid tumors, a smart polymer/calcium phosphate (CaP)/siRNA hybrid nanoparticle was prepared to feature biocompatibility, reversible stability and endosomal escape functionality using a pH sensitive block copolymer of poly(ethylene glycol) and charge-conversional polymer (PEG-CCP), of which anionic functional groups could be converted to cationic groups in an endosomal acidic condition for facilitated endosomal escape. Nanoparticles were confirmed to be approximately 100nm in size, narrowly dispersed and spherical. Also, the nanoparticle was highly tolerable in medium containing serum, while releasing the entrapped siRNA in a cytoplasm-mimicking ionic condition, presumably based on the equilibrium between CaP complexes and calcium ions. Further, the nanoparticle showed high gene silencing efficiency in cultured pancreatic cancer cells (BxPC3) without associated cytotoxicity. Ultimately, systemic administration of the nanoparticles carrying vascular endothelium growth factor (VEGF) siRNA led to the significant reduction in the subcutaneous BxPC3 tumor growth, well consistent with the enhanced accumulation of siRNA and the significant VEGF gene silencing (~68%) in the tumor. Thus, the hybrid nanoparticle was demonstrated to be a promising formulation toward siRNA-based cancer therapies.
Assuntos
Portadores de Fármacos/administração & dosagem , Neoplasias Pancreáticas/terapia , RNA Interferente Pequeno/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Fosfatos de Cálcio/administração & dosagem , Linhagem Celular Tumoral , Feminino , Inativação Gênica , Humanos , Camundongos , Camundongos Nus , Nanopartículas/administração & dosagem , Neoplasias Pancreáticas/patologia , Polietilenoglicóis/administração & dosagem , RNA Mensageiro/metabolismoRESUMO
We describe the development and application of intravital confocal micro-videography to visualize entrance, distribution, and clearance of drugs within various tissues and organs. We use a Nikon A1R confocal laser scanning microscope system attached to an upright ECLIPSE FN1. The Nikon A1R allows simultaneous four channel acquisition and speed of 30 frames per second while maintaining high resolution of 512 × 512 scanned points. The key techniques of our intravital imaging are (1) to present a flat and perpendicular surface to the objective lens, and (2) to expose the subject with little or no bleeding to facilitate optical access to multiple tissues and organs, and (3) to isolate the subject from the body movement without compressing the blood vessels, and (4) to insert a tail vein catheter for timed injection without moving the subject. Ear lobe dermis tissue was accessible without surgery. Liver, kidney, and subcutaneous tumor were accessed following exteriorization through skin incision. In order to image initial extravasations of compounds into tissue following intravenous injection, movie acquisition was initialized prior to drug administration. Our technique can serve as a powerful tool for investigating biological mechanisms and functions of intravenously injected drugs, with both spatial and temporal resolution.
RESUMO
Bartter's syndrome was reported in 1962, and Gitelman's syndrome, which is subtype of Bartter's syndrome was described later. These syndromes are characterized by hypokalemia, hypochloremic metabolic alkalosis, normal to low blood pressure, although they show hyperreninemia, and hyperaldosteronemia. The cause of these diseases have been unexplained for a long time. Recently however, from 1996 to 2002, several causes have identified. Bartter's syndrome can occur due to a loss of function mutation in NKCC2, ROMK, CLC-Kb and barttin, or a gain of function mutation of calcium-sensing receptor. Gitelman's syndrome can occur due to a loss of function mutation in NCC. Different causes need different treatment and have different prognosis. In fact, we cannot examine all DNA sequences in regular hospitals. So it is our goal to make a clinical diagnostic standard to appropriate treatment.
Assuntos
Síndrome de Bartter , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/fisiologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Síndrome de Bartter/genética , Síndrome de Bartter/fisiopatologia , Síndrome de Bartter/terapia , Canais de Cloreto/genética , Canais de Cloreto/fisiologia , Colecalciferol/uso terapêutico , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Eletrólitos/administração & dosagem , Hidratação , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Mutação , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Receptores de Detecção de Cálcio/genética , Receptores de Detecção de Cálcio/fisiologia , Simportadores de Cloreto de Sódio/genética , Simportadores de Cloreto de Sódio/fisiologia , Simportadores de Cloreto de Sódio-Potássio/genética , Simportadores de Cloreto de Sódio-Potássio/fisiologia , Membro 1 da Família 12 de Carreador de SolutoRESUMO
Autosomal dominant hypocalcemia (ADH) caused by activating mutations of calcium-sensing receptor (CaSR) is characterized by hypocalcemia with inappropriately low concentration of PTH and relative hypercalciuria. Active vitamin D treatment often leads to nephrolithiasis and renal impairment in patients with ADH. However, differential diagnosis between ADH and idiopathic hypoparathyroidism is sometimes very difficult. Here, we report a mutation of CaSR and its functional property found in three generations of a Japanese family. The proband developed seizures at 7 days of age. His mother and elder sister were discovered to have hypoparathyroidism by family survey, but his father was normocalcemic. His grandfather developed heart failure and was found to have hypoparathyroidism. All affected members had been treated with active vitamin D3 and bilateral nephrolithiasis were detected in three of them. DNA sequencing revealed that all affected patients had a heterozygous mutation in CaSR gene that causes proline to leucine substitution at codon 221 (P221L). In vitro functional analysis of the mutant CaSR by measuring inositol 1,4,5-trisphosphate production in response to changes of extracellular Ca indicated that this mutation is an activating one and responsible for ADH in this family. Therefore, careful monitoring of urinary Ca excretion before and during treatment of PTH-deficient hypoparathyroidism is very important, and screening of CaSR mutation should be considered in patients with relative hypercalciuria or with a family history of hypocalcemia.
Assuntos
Saúde da Família , Hipocalcemia/genética , Mutação Puntual , Receptores de Detecção de Cálcio/genética , Adulto , Pré-Escolar , Análise Mutacional de DNA , Feminino , Genes Dominantes , Humanos , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Mutagênese Sítio-Dirigida , Linhagem , Receptores de Detecção de Cálcio/metabolismoRESUMO
The thiazolidinedione (TZD) class of antidiabetic drugs has been shown to inhibit the formation of bone-resorbing osteoclasts in vitro and to decrease bone resorption markers in vivo. These drugs also inhibit the expression of leptin in adipocytes. Less leptin can be associated with higher bone mass, based on analyses of mice deficient in leptin action. Effects of 1-year treatment with troglitazone, a member of the TZDs, on bone mineral density (BMD) and bone metabolism were examined in 25 Japanese type 2 diabetic patients. Glucose metabolism was improved, whereas body mass index and percent body fat did not change throughout the study. The percent change of BMD was negatively correlated with that of serum leptin, whereas it was not associated with changes of bone metabolic markers, type I collagen N-telopeptide (NTx), bone alkaline phosphatase (ALP), body mass index, or HbA1c. Serum leptin decreased in 68% of subjects (responders) after 1-month treatment and was consistently lower than the basal level throughout the treatment. Percent changes of BMD were significantly higher in the responders than in the nonresponders and in nondiabetic subjects at 6 and 12 months. NTx and bone ALP decreased at 1 month but increased thereafter in either group of patients. Thus, it is suggested that the decrease in serum leptin with no reduction in body fat mass by troglitazone is associated with preventing bone loss in type 2 diabetic patients. Hence, TZDs may have an advantage for diabetic patients who have risk factors for osteoporosis.
Assuntos
Cromanos/uso terapêutico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Leptina/sangue , Osteoporose/prevenção & controle , Tiazolidinedionas/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Fosfatase Alcalina/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Densidade Óssea , Colágeno/efeitos dos fármacos , Colágeno/metabolismo , Colágeno Tipo I , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Feminino , Hemoglobinas Glicadas/efeitos dos fármacos , Humanos , Vértebras Lombares/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Osteoporose/sangue , Osteoporose/etiologia , Peptídeos/efeitos dos fármacos , Peptídeos/metabolismo , Estudos Prospectivos , TroglitazonaRESUMO
Cytokines in interleukin (IL)-11 subfamily participate in the regulation of bone cell proliferation and differentiation. We report here positive effects of IL-11 on osteoblasts and bone formation. Overexpression of human IL-11 gene in transgenic mice resulted in the stimulation of bone formation to increase cortical thickness and strength of long bones, and in the prevention of cortical bone loss with advancing age. Bone resorption and osteoclastogenesis were not affected in IL-11 transgenic mice. In experiments in vitro, IL-11 stimulated transcription of the target gene for bone morphogenetic protein (BMP) via STAT3, leading to osteoblastic differentiation in the presence of BMP-2, but inhibited adipogenesis in bone marrow stromal cells. These results indicate that IL-11 is a stimulatory factor for osteoblastogenesis and bone formation to conserve cortical bone, possibly by enhancing BMP actions in bone. IL-11 may be a new therapeutic target for senile osteoporosis.
Assuntos
Envelhecimento/patologia , Desenvolvimento Ósseo/efeitos dos fármacos , Reabsorção Óssea/prevenção & controle , Interleucina-11/farmacologia , Tiazolidinedionas , Animais , Sequência de Bases , Células da Medula Óssea/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/fisiologia , Células Cultivadas , Cromanos/farmacologia , Primers do DNA , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Recombinantes/farmacologia , Tiazóis/farmacologia , Transcrição Gênica/efeitos dos fármacos , TroglitazonaRESUMO
Bartter's syndrome is a heterogeneous disorder characterised by deficient renal reabsorption of sodium and chloride, and hypokalaemic metabolic alkalosis with hyper-reninaemia and hyperaldosteronaemia. Mutations in several ion transporters and channels have been associated with the pathogenesis of Bartter's syndrome. We describe two hypocalcaemic patients with deficient parathyroid hormone secretion who also showed characteristics of Bartter's syndrome. We found activating mutations of the gene for the calcium-sensing receptor (CASR) in both patients. Activation of this calcium-sensing receptor inhibits the activity of a renal outer-medullary potassium channel that is mutated in type 2 Bartter's syndrome. We therefore suggest that some activating mutations of CASR could provide new mechanisms for the development of Bartter's syndrome.
Assuntos
Síndrome de Bartter/genética , Receptores de Superfície Celular/genética , Adulto , Síndrome de Bartter/sangue , Síndrome de Bartter/complicações , Feminino , Humanos , Hipocalcemia/complicações , Hipoparatireoidismo/complicações , Masculino , Mutação , Hormônio Paratireóideo/deficiência , Receptores de Detecção de CálcioRESUMO
Calcium-sensing receptor (CaSR) plays an essential role in regulating secretion of parathyroid hormone. After the identification of CaSR, some cases of familial hypocalciuric hypercalcemia (FHH) were shown to have heterozygous inactivating mutations of CaSR. However, linkage study showed that there are additional two genetic loci for FHH in addition to the chromosomal location of CaSR gene. Furthermore, one family with heterozygous inactivating mutation of CaSR was shown to exhibit hypercalcemia with hypercalciuria. Therefore, heterozygous inactivating mutation of CaSR is not synonymous with FHH. In addition, patients with neonatal severe hyperparathyroidism were shown to have homozygous or compound heterozygous inactivating mutations of CaSR.