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1.
Kidney Int ; 96(2): 270-272, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31331463

RESUMO

The ability of proximal tubule cells to internalize filtered proteins over a broad concentration range is essential for maintaining a protein-free urine but also renders these cells uniquely susceptible to cytotoxic damage. Morace et al. find that knockout of globotriaosylceramide synthase, an enzyme required for production of Gb3 and other members of the globo series of glycosphingolipids, impairs endocytic uptake of filtered proteins and preserves kidney function in mouse models of acute kidney injury.

3.
J. bras. nefrol ; 40(4): 333-338, Out.-Dec. 2018. tab, graf
Artigo em Inglês | LILACS-Express | ID: biblio-984593

RESUMO

ABSTRACT Introduction: Fabry disease (FD) is a disorder caused by mutations in the gene encoding for lysosomal enzyme α-galactosidase A (α-GAL). Reduced α-GAL activity leads to progressive accumulation of globotriaosylceramide (Gb3), also known as CD77. The recent report of increased expression of CD77 in blood cells of patients with FD indicated that this molecule can be used as a potential marker for monitoring enzyme replacement therapy (ERT). Objective: The purpose of this study was to evaluate the CD77 levels throughout ERT in FD patients (V269M mutation). Methods: We evaluated the fluctuations in PBMC (peripheral blood mononuclear cell) membrane CD77 expression in FD patients undergoing ERT and correlated these levels with those observed in different cell types. Results: A greater CD77 expression was found in phagocytes of patients compared to controls at baseline. Interestingly, the variability in CD77 levels is larger in patients at baseline (340 - 1619 MIF) and after 12 months of ERT (240 - 530 MIF) compared with the control group (131 - 331 MFI). Furthermore, by analyzing the levels of CD77 in phagocytes from patients throughout ERT, we found a constant decrease in CD77 levels. Conclusion: The increased CD77 levels in the phagocytes of Fabry carriers together with the decrease in CD77 levels throughout ERT suggest that measuring CD77 levels in phagocytes is a promising tool for monitoring the response to ERT in FD.


RESUMO Introdução: A doença de Fabry (DF) é um distúrbio causado por mutações no gene que codifica a enzima lisossômica α-galactosidase A (α-GAL). A redução da atividade de α-GAL leva ao acúmulo progressivo de globotriaosilceramida (Gb3), também conhecida como CD77. O recente relato de aumento da expressão de CD77 em células sanguíneas de pacientes com DF indicou que essa molécula pode ser utilizada como um potencial marcador para o monitoramento da terapia de reposição enzimática (TRE). Objetivo: O objetivo deste estudo foi avaliar os níveis de CD77 ao longo da TRE em pacientes com DF (mutação V269M). Métodos: Foram avaliadas as flutuações na expressão de CD77 nas membranas das CMSP (células mononucleares do sangue periférico) em pacientes com DF submetidos à TRE e correlacionados com aqueles observados em diferentes tipos de células. Resultados: Uma maior expressão de CD77 foi encontrada em fagócitos de pacientes em comparação aos controles no início do estudo. Curiosamente, a variabilidade nos níveis de CD77 é maior em pacientes no início do estudo (340 - 1619 MIF) e após 12 meses de TRE (240 - 530 MIF) em comparação com o grupo controle (131 - 331 MFI). Além disso, analisando os níveis de CD77 em fagócitos de pacientes ao longo da TRE, encontramos uma diminuição constante nos níveis de CD77. Conclusão: O aumento nos níveis de CD77 nos fagócitos de portadores de Fabry, juntamente com a diminuição nos níveis de CD77 ao longo da TRE, sugerem que medir os níveis de CD77 nos fagócitos é uma ferramenta promissora para monitorar a resposta à TRE na DF.

4.
Wellcome Open Res ; 3: 46, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29900417

RESUMO

Tatton-Brown-Rahman syndrome (TBRS; OMIM 615879), also known as the DNMT3A-overgrowth syndrome, is an overgrowth intellectual disability syndrome first described in 2014 with a report of 13 individuals with constitutive heterozygous DNMT3A variants. Here we have undertaken a detailed clinical study of 55 individuals with de novoDNMT3A variants, including the 13 previously reported individuals. An intellectual disability and overgrowth were reported in >80% of individuals with TBRS and were designated major clinical associations. Additional frequent clinical associations (reported in 20-80% individuals) included an evolving facial appearance with low-set, heavy, horizontal eyebrows and prominent upper central incisors; joint hypermobility (74%); obesity (weight ³2SD, 67%); hypotonia (54%); behavioural/psychiatric issues (most frequently autistic spectrum disorder, 51%); kyphoscoliosis (33%) and afebrile seizures (22%). One individual was diagnosed with acute myeloid leukaemia in teenage years. Based upon the results from this study, we present our current management for individuals with TBRS.

5.
J Bras Nefrol ; 2018 Jun 04.
Artigo em Inglês, Português | MEDLINE | ID: mdl-29927462

RESUMO

INTRODUCTION: Fabry disease (FD) is a disorder caused by mutations in the gene encoding for lysosomal enzyme α-galactosidase A (α-GAL). Reduced α-GAL activity leads to progressive accumulation of globotriaosylceramide (Gb3), also known as CD77. The recent report of increased expression of CD77 in blood cells of patients with FD indicated that this molecule can be used as a potential marker for monitoring enzyme replacement therapy (ERT). OBJECTIVE: The purpose of this study was to evaluate the CD77 levels throughout ERT in FD patients (V269M mutation). METHODS: We evaluated the fluctuations in PBMC (peripheral blood mononuclear cell) membrane CD77 expression in FD patients undergoing ERT and correlated these levels with those observed in different cell types. RESULTS: A greater CD77 expression was found in phagocytes of patients compared to controls at baseline. Interestingly, the variability in CD77 levels is larger in patients at baseline (340 - 1619 MIF) and after 12 months of ERT (240 - 530 MIF) compared with the control group (131 - 331 MFI). Furthermore, by analyzing the levels of CD77 in phagocytes from patients throughout ERT, we found a constant decrease in CD77 levels. CONCLUSION: The increased CD77 levels in the phagocytes of Fabry carriers together with the decrease in CD77 levels throughout ERT suggest that measuring CD77 levels in phagocytes is a promising tool for monitoring the response to ERT in FD.

6.
Am J Physiol Renal Physiol ; 311(5): F1015-F1024, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27681560

RESUMO

Fabry nephropathy is a major cause of morbidity and premature death in patients with Fabry disease (FD), a rare X-linked lysosomal storage disorder. Gb3, the main substrate of α-galactosidase A (α-Gal A), progressively accumulates within cells in a variety of tissues. Establishment of cell models has been useful as a tool for testing hypotheses of disease pathogenesis. We applied CRISPR/Cas9 genome editing technology to the GLA gene to develop human kidney cell models of FD in human immortalized podocytes, which are the main affected renal cell type. Our podocytes lack detectable α-Gal A activity and have increased levels of Gb3. To explore different pathways that could have distinct patterns of activation under conditions of α-gal A deficiency, we used a high-throughput antibody array to perform phosphorylation profiling of CRISPR/Cas9-edited and control podocytes. Changes in both total protein levels and in phosphorylation status per site were observed. Analysis of our candidate proteins suggests that multiple signaling pathways are impaired in FD.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Doença de Fabry/metabolismo , Rim/metabolismo , Podócitos/metabolismo , alfa-Galactosidase/metabolismo , Linhagem Celular , Doença de Fabry/genética , Doença de Fabry/patologia , Humanos , Rim/patologia , Podócitos/patologia , Transdução de Sinais/fisiologia , alfa-Galactosidase/genética
7.
J Bras Nefrol ; 38(1): 49-53, 2016 Mar.
Artigo em Inglês, Português | MEDLINE | ID: mdl-27049364

RESUMO

INTRODUCTION: Fabry disease is a lysosomal storage disorder due to abnormalities in the GLA gene (Xq22). Such changes result in the reduction/absence of activity of the lysosome enzyme α-GAL, whose function is to metabolize globotriaosylceramide (Gb3). Renal disease is a major clinical outcome of the accumulation of Gb3. Podocyte injury is thought to be a major contributor to the progressive loss of the renal function and may be found altered even before the onset of microalbuminuria. OBJECTIVE: The aim of this study was to quantify the urinary excretion of podocytes in Fabry disease patients (V269M, n = 14) and healthy controls (n = 40), and to correlate podocyturia with the variables gender, age, time of therapy and albumin: creatinine ratio (ACR). METHODS: Urinary podocytes were stained using immunofluorescence to podocalyxin and DAPi. The number of podocalyxin-positive cells was quantified and the average number was taken (normal range 0-0.6 podocytes/mL). RESULTS: The average number of podocytes in the urine of Fabry disease patients was significantly higher than in healthy controls (p < 0.0001). We observed a positive correlation between podocyturia and ACR (p = 0.004; (r2 = 0.6417). We found no correlation between podocyturia and gender, age or duration of therapy. CONCLUSION: Podocyturia is an important parameter in the assessment of renal disease in general, and it may serve as an additional early tool for monitoring Fabry disease nephropathy even before changes in ACR are seen. This may prove to be a useful tool to assess disease progression in patients expected to have a more aggressive phenotype.


Assuntos
Doença de Fabry/fisiopatologia , Podócitos/patologia , Adulto , Idoso , Albuminúria , Estudos de Casos e Controles , Criança , Creatinina/urina , Progressão da Doença , Doença de Fabry/urina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Urinálise , Adulto Jovem
8.
J. bras. nefrol ; 38(1): 49-53, jan.-mar. 2016. tab, graf
Artigo em Português | LILACS-Express | ID: lil-777499

RESUMO

Resumo Introdução: A doença de Fabry (DF) é uma desordem lisossômica ligada ao cromossomo X ocasionada por mutações no gene que codifica a enzima lisossômica α-galactosidase A (α-GAL). A redução ou ausência da atividade dessa enzima leva ao acúmulo progressivo de gb3. A doença renal é uma importante consequência clínica da acumulação de Gb3. Podócito é o tipo celular mais afetado na doença renal, que mostra apenas uma resposta parcial à Terapia de Reposição Enzimática. Além disso, a disfunção podocitária é a principal contribuinte para a perda progressiva da função renal e pode ser encontrada alterada mesmo antes do início da microalbuminúria. Assim, a podocitúria na DF pode ser uma ferramenta importante para prever a doença renal. Objetivo: O objetivo deste estudo foi quantificar a excreção urinária de podócitos em pacientes com DF (V269M, n = 14) e controles saudáveis (n = 40), e relacioná-las com as variáveis sexo, idade, tempo de terapia e a razão albumina: creatinina (AUC). Métodos: Podócitos urinários foram identificados utilizando imunofluorescência para podocalixina e DAPI. O número de células podocalixina positivo foi contado e o número médio foi utilizado (faixa normal 0-0.6 podócitos/mL). Resultados: O número médio de podócitos na urina de pacientes com DF foi significativamente maior do que os controles saudáveis (p < 0.0001). Observou-se uma correlação positiva entre podocitúria e AUC (p = 0.004; r2 = 0.6417). Conclusão: A podocitúria pode ser uma ferramenta adicional para avaliar a progressão da doença renal em pacientes que se espera que tenha um fenótipo mais agressivo.


Abstract Introduction: Fabry disease is a lysosomal storage disorder due to abnormalities in the GLA gene (Xq22). Such changes result in the reduction/absence of activity of the lysosome enzyme α-GAL, whose function is to metabolize globotriaosylceramide (Gb3). Renal disease is a major clinical outcome of the accumulation of Gb3. Podocyte injury is thought to be a major contributor to the progressive loss of the renal function and may be found altered even before the onset of microalbuminuria. Objective: The aim of this study was to quantify the urinary excretion of podocytes in Fabry disease patients (V269M, n = 14) and healthy controls (n = 40), and to correlate podocyturia with the variables gender, age, time of therapy and albumin: creatinine ratio (ACR). Methods: Urinary podocytes were stained using immunofluorescence to podocalyxin and DAPi. The number of podocalyxin-positive cells was quantified and the average number was taken (normal range 0-0.6 podocytes/mL). Results: The average number of podocytes in the urine of Fabry disease patients was significantly higher than in healthy controls (p < 0.0001). We observed a positive correlation between podocyturia and ACR (p = 0.004; (r2 = 0.6417). We found no correlation between podocyturia and gender, age or duration of therapy. Conclusion: Podocyturia is an important parameter in the assessment of renal disease in general, and it may serve as an additional early tool for monitoring Fabry disease nephropathy even before changes in ACR are seen. This may prove to be a useful tool to assess disease progression in patients expected to have a more aggressive phenotype.

9.
FEBS J ; 282(23): 4466-79, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26299746

RESUMO

Basement membranes (BMs) are thin sheets of extracellular matrix that outline epithelia, muscle fibers, blood vessels and peripheral nerves. The current view of BM structure and functions is based mainly on transmission electron microscopy imaging, in vitro protein binding assays, and phenotype analysis of human patients, mutant mice and invertebrata. Recently, MS-based protein analysis, biomechanical testing and cell adhesion assays with in vivo derived BMs have led to new and unexpected insights. Proteomic analysis combined with ultrastructural studies showed that many BMs undergo compositional and structural changes with advancing age. Atomic force microscopy measurements in combination with phenotype analysis have revealed an altered mechanical stiffness that correlates with specific BM pathologies in mutant mice and human patients. Atomic force microscopy-based height measurements strongly suggest that BMs are more than two-fold thicker than previously estimated, providing greater freedom for modelling the large protein polymers within BMs. In addition, data gathered using BMs extracted from mutant mice showed that laminin has a crucial role in BM stability. Finally, recent evidence demonstrate that BMs are bi-functionally organized, leading to the proposition that BM-sidedness contributes to the alternating epithelial and stromal tissue arrangements that are found in all metazoan species. We propose that BMs are ancient structures with tissue-organizing functions and were essential in the evolution of metazoan species.


Assuntos
Membrana Basal/química , Membrana Basal/metabolismo , Animais , Membrana Basal/ultraestrutura , Humanos , Microscopia de Força Atômica , Proteômica
10.
PLoS One ; 9(1): e86425, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24466086

RESUMO

Epithelial cells elaborate specialized domains that have distinct protein and lipid compositions, including the apical and basolateral surfaces and primary cilia. Maintaining the identity of these domains is required for proper cell function, and requires the efficient and selective SNARE-mediated fusion of vesicles containing newly synthesized and recycling proteins with the proper target membrane. Multiple pathways exist to deliver newly synthesized proteins to the apical surface of kidney cells, and the post-Golgi SNAREs, or VAMPs, involved in these distinct pathways have not been identified. VAMP7 has been implicated in apical protein delivery in other cell types, and we hypothesized that this SNARE would have differential effects on the trafficking of apical proteins known to take distinct routes to the apical surface in kidney cells. VAMP7 expressed in polarized Madin Darby canine kidney cells colocalized primarily with LAMP2-positive compartments, and siRNA-mediated knockdown modulated lysosome size, consistent with the known function of VAMP7 in lysosomal delivery. Surprisingly, VAMP7 knockdown had no effect on apical delivery of numerous cargoes tested, but did decrease the length and frequency of primary cilia. Additionally, VAMP7 knockdown disrupted cystogenesis in cells grown in a three-dimensional basement membrane matrix. The effects of VAMP7 depletion on ciliogenesis and cystogenesis are not directly linked to the disruption of lysosomal function, as cilia lengths and cyst morphology were unaffected in an MDCK lysosomal storage disorder model. Together, our data suggest that VAMP7 plays an essential role in ciliogenesis and lumen formation. To our knowledge, this is the first study implicating an R-SNARE in ciliogenesis and cystogenesis.


Assuntos
Cílios/metabolismo , Células Epiteliais/metabolismo , Rim/metabolismo , Proteínas R-SNARE/metabolismo , Animais , Linhagem Celular , Expressão Gênica , Técnicas de Silenciamento de Genes , Lisossomos/metabolismo , Isoformas de Proteínas , Transporte Proteico , Proteínas R-SNARE/genética , Ratos
11.
Mol Genet Metab ; 111(2): 184-92, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24215843

RESUMO

Accumulation of globotriaosylceramide (Gb3) and other neutral glycosphingolipids with galactosyl residues is the hallmark of Fabry disease, a lysosomal storage disorder caused by deficiency of the enzyme alpha-galactosidase A (α-gal A). These lipids are incorporated into the plasma membrane and intracellular membranes, with a preference for lipid rafts. Disruption of raft mediated cell processes is implicated in the pathogenesis of several human diseases, but little is known about the effects of the accumulation of glycosphingolipids on raft dynamics in the context of Fabry disease. Using siRNA technology, we have generated a polarized renal epithelial cell model of Fabry disease in Madin-Darby canine kidney cells. These cells present increased levels of Gb3 and enlarged lysosomes, and progressively accumulate zebra bodies. The polarized delivery of both raft-associated and raft-independent proteins was unaffected by α-gal A knockdown, suggesting that accumulation of Gb3 does not disrupt biosynthetic trafficking pathways. To assess the effect of α-gal A silencing on lipid raft dynamics, we employed number and brightness (N&B) analysis to measure the oligomeric status and mobility of the model glycosylphosphatidylinositol (GPI)-anchored protein GFP-GPI. We observed a significant increase in the oligomeric size of antibody-induced clusters of GFP-GPI at the plasma membrane of α-gal A silenced cells compared with control cells. Our results suggest that the interaction of GFP-GPI with lipid rafts may be altered in the presence of accumulated Gb3. The implications of our results with respect to the pathogenesis of Fabry disease are discussed.


Assuntos
Proteínas de Fluorescência Verde/metabolismo , Microdomínios da Membrana/metabolismo , Modelos Biológicos , alfa-Galactosidase/metabolismo , Animais , Cães , Doença de Fabry/enzimologia , Doença de Fabry/patologia , Expressão Gênica , Glicosilfosfatidilinositóis/metabolismo , Proteínas de Fluorescência Verde/genética , Humanos , Rim/enzimologia , Rim/patologia , Lisossomos/enzimologia , Lisossomos/patologia , Células Madin Darby de Rim Canino , Microdomínios da Membrana/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Triexosilceramidas/biossíntese , alfa-Galactosidase/antagonistas & inibidores , alfa-Galactosidase/genética
12.
Gene ; 536(1): 118-22, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24334114

RESUMO

This study investigated the potential relationship between the expression levels of lysosome-associated membrane proteins (LAMP) 1 and 2 and responses to enzyme replacement therapy (ERT) in the members of a single family with Fabry disease (FD). LAMP levels were assessed by flow cytometry in leukocytes from 17 FD patients who received an eight-month course of ERT course and 101 healthy individuals. We found that phagocytic cells from the FD patients had higher expression levels of both LAMP-1 and LAMP-2, relative to the levels in phagocytes from the healthy controls (p=0.001). Furthermore, the LAMP-1 and LAMP-2 levels in phagocytes from the FD carriers continuously decreased with ERT administration to reach levels similar to those in healthy controls. We suggest that LAMP-1 and LAMP-2 could be used as additional markers with which to assess ERT effectiveness in FD.


Assuntos
Doença de Fabry/genética , Proteína 2 de Membrana Associada ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Adolescente , Adulto , Idoso , Biomarcadores Farmacológicos/análise , Estudos de Casos e Controles , Terapia de Reposição de Enzimas , Doença de Fabry/tratamento farmacológico , Feminino , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Linhagem , Adulto Jovem , alfa-Galactosidase/uso terapêutico
13.
Mutat Res ; 752(1-2): 1-7, 2013 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-23376767

RESUMO

Acrylamide (AA) is a probable human carcinogen generated in carbohydrate-rich foodstuffs upon heating. Glycidamide (GA), formed via epoxidation, presumably mediated by cytochrome P450 2E1, is considered to be the active metabolite that plays a central role in the genotoxicity of AA. The aim of this work was to evaluate the cytogenetic damage induced by AA and GA in cultured human lymphocytes by use of the sister chromatid exchange (SCE) assay. Furthermore, this report addresses the role of individual genetic polymorphisms in key genes involved in detoxification and DNA-repair pathways (BER, NER, HRR and NHEJ) on the induction of SCE by GA. While AA induced the number of SCE/metaphase only slightly, especially for the highest concentration tested (2000µM), GA markedly induced SCEs in a concentration-dependent manner up to concentrations of 750µM, leading to an increase in SCEs of up to about 10-fold compared with controls. By combining DNA damage in GA-treated lymphocytes and data on polymorphisms, associations between the induction of SCEs with GSTP1 (Ile105Val) and GSTA2 (Glu210Ala) genotypes are suggested.


Assuntos
Acrilamida/toxicidade , Dano ao DNA , Reparo do DNA , Compostos de Epóxi/toxicidade , Mutagênicos/toxicidade , Polimorfismo Genético , Troca de Cromátide Irmã , Glutationa S-Transferase pi/genética , Glutationa Transferase/genética , Humanos , Inativação Metabólica/genética , Isoenzimas/genética , Linfócitos/efeitos dos fármacos
14.
J Cell Sci ; 125(Pt 22): 5546-54, 2012 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-22976307

RESUMO

Kidney function requires the appropriate distribution of membrane proteins between the apical and basolateral surfaces along the kidney tubule. Further, the absolute amount of a protein at the cell surface versus intracellular compartments must be attuned to specific physiological needs. Endolyn (CD164) is a transmembrane protein that is expressed at the brush border and in apical endosomes of the proximal convoluted tubule and in lysosomes of more distal segments of the kidney. Endolyn has been shown to regulate CXCR4 signaling in hematopoietic precursor cells and myoblasts; however, little is known about endolyn function in the adult or developing kidney. Here we identify endolyn as a gene important for zebrafish pronephric kidney function. Zebrafish endolyn lacks the N-terminal mucin-like domain of the mammalian protein, but is otherwise highly conserved. Using in situ hybridization we show that endolyn is expressed early during development in zebrafish brain, eye, gut and pronephric kidney. Embryos injected with a translation-inhibiting morpholino oligonucleotide targeted against endolyn developed pericardial edema, hydrocephaly and body curvature. The pronephric kidney appeared normal morphologically, but clearance of fluorescent dextran injected into the common cardinal vein was delayed, consistent with a defect in the regulation of water balance in morphant embryos. Heterologous expression of rat endolyn rescued the morphant phenotypes. Interestingly, rescue experiments using mutant rat endolyn constructs revealed that both apical sorting and endocytic/lysosomal targeting motifs are required for normal pronephric kidney function. This suggests that both polarized targeting and postendocytic trafficking of endolyn are essential for the protein's proper function in mammalian kidney.


Assuntos
Polaridade Celular , Endocitose , Endolina/metabolismo , Rim/embriologia , Rim/metabolismo , Pronefro/embriologia , Peixe-Zebra/embriologia , Envelhecimento/metabolismo , Animais , Polaridade Celular/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Endocitose/efeitos dos fármacos , Endolina/química , Técnicas de Silenciamento de Genes , Rim/anatomia & histologia , Rim/citologia , Células Madin Darby de Rim Canino , Mamíferos/embriologia , Mamíferos/metabolismo , Morfolinos/farmacologia , Especificidade de Órgãos , Pronefro/metabolismo , Estrutura Terciária de Proteína , Ratos , Relação Estrutura-Atividade , Peixe-Zebra/metabolismo
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