RESUMO
The clinical heterogeneity of idiopathic nephrotic syndrome in childhood may reflect different mechanisms of disease that are as yet unclear. Here, we evaluated the association between an atypical presence of IgM on the surface of T cells (T-cell IgM) and the response to steroid therapy in a total of 153 pediatric patients with idiopathic nephrotic syndrome in different phases of disease. At disease onset, T-cell IgM median levels were significantly elevated and predictive of risk of relapse in 47 patients. They were also significantly increased comparing 58 steroid-dependent to 8 infrequently relapsing and 14 frequently relapsing patients, especially during relapse, whereas they were within the normal range in 7 genetic steroid-resistant patients. T-cell IgM in vivo was not affected by the amount of total circulating IgM, nor by concomitant acute infections or oral immunosuppression. However, it was affected by rituximab treatment in 21 steroid-dependent patients. By in vitro experiments, elevated T-cell IgM was not influenced by total circulating IgM levels or by the presence of other circulating factors, and there was no distinctive antigen-specificity or atypical IgM polymerization. Rather, we found that increased T-cell IgM correlates with reduced IgM sialylation, which influences T-cell response to steroid inhibition and T-cell production of podocyte-damaging factors. Thus, the atypical presence of IgM on the surface of T cells may predispose a subset of steroid-sensitive pediatric patients with idiopathic nephrotic syndrome to a poor response to steroid therapy since disease onset.
Assuntos
Glucocorticoides/farmacologia , Imunoglobulina M/metabolismo , Síndrome Nefrótica/tratamento farmacológico , Linfócitos T/imunologia , Adolescente , Criança , Pré-Escolar , Resistência a Medicamentos/genética , Quimioterapia Combinada/métodos , Feminino , Seguimentos , Glucocorticoides/uso terapêutico , Humanos , Imunoglobulina M/análise , Imunoglobulina M/imunologia , Lactente , Masculino , Síndrome Nefrótica/sangue , Síndrome Nefrótica/genética , Síndrome Nefrótica/imunologia , Podócitos , Estudos Prospectivos , Recidiva , Rituximab/farmacologia , Rituximab/uso terapêutico , Ácidos Siálicos/metabolismo , Linfócitos T/metabolismo , Resultado do TratamentoRESUMO
The involvement of the glomerulus in the pathogenesis of cystinosis, caused by loss-of-function mutations in cystinosin (CTNS, 17p13), is a matter of controversy. Although patients with cystinosis demonstrate glomerular lesions and high-molecular-weight proteinuria starting from an early age, a mouse model of cystinosis develops only signs of proximal tubular dysfunction. Here we studied podocyte damage in patients with cystinosis by analyzing urinary podocyte excretion and by in vitro studies of podocytes deficient in cystinosin. Urine from patients with cystinosis presented a significantly higher amount of podocytes compared with controls. In culture, cystinotic podocytes accumulated cystine compatible with cystinosin deficiency. The expression of podocyte specific genes CD2AP, podocalyxin, and synaptopodin and of the WT1 protein was evident in all cell lines. Conditionally immortalized podocyte lines of 2 patients with different CTNS mutations had altered cytoskeleton, impaired cell adhesion sites, and increased individual cell motility. Moreover, these cells showed enhanced phosphorylation of both Akt1 and Akt2 (isoforms of protein kinase B). Inhibition of Akt by a specific inhibitor (Akti inhibitor 1/2) resulted in normalization of the hypermotile phenotype. Thus, our study extends the list of genetic disorders causing podocyte damage and provides the evidence of altered cell signaling cascades resulting in impaired cell adhesion and enhanced cell motility in cystinosis.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Movimento Celular , Cistinose/metabolismo , Podócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adolescente , Sistemas de Transporte de Aminoácidos Neutros/deficiência , Sistemas de Transporte de Aminoácidos Neutros/genética , Estudos de Casos e Controles , Adesão Celular , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Criança , Pré-Escolar , Cistinose/genética , Cistinose/patologia , Cistinose/urina , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Regulação para Baixo , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Mutação , Fenótipo , Fosforilação , Podócitos/efeitos dos fármacos , Podócitos/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Transdução de Sinais , Urina/citologia , Proteínas WT1/genética , Proteínas WT1/metabolismo , Adulto JovemRESUMO
Idiopathic focal segmental glomerulosclerosis (FSGS) is a progressive and proteinuric kidney disease that starts with podocyte injury. Podocytes cover the external side of the glomerular capillary by a complex web of primary and secondary ramifications. Similar to dendritic spines of neuronal cells, podocyte processes rely on a dynamic actin-based cytoskeletal architecture to maintain shape and function. Brain-derived neurotrophic factor (BDNF) is a pleiotropic neurotrophin that binds to the tropomyosin-related kinase B receptor (TrkB) and has crucial roles in neuron maturation, survival, and activity. In neuronal cultures, exogenously added BDNF increases the number and size of dendritic spines. In animal models, BDNF administration is beneficial in both central and peripheral nervous system disorders. Here we show that BDNF has a TrkB-dependent trophic activity on podocyte cell processes; by affecting microRNA-134 and microRNA-132 signalling, BDNF up-regulates Limk1 translation and phosphorylation, and increases cofilin phosphorylation, which results in actin polymerization. Importantly, BDNF effectively repairs podocyte damage in vitro, and contrasts proteinuria and glomerular lesions in in vivo models of FSGS, opening a potential new perspective to the treatment of podocyte disorders.
Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Actinas/metabolismo , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Glomerulosclerose Segmentar e Focal/prevenção & controle , MicroRNAs/metabolismo , Podócitos/efeitos dos fármacos , Células 3T3 , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/patologia , Fatores de Despolimerização de Actina/metabolismo , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Glomerulosclerose Segmentar e Focal/induzido quimicamente , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Larva/efeitos dos fármacos , Larva/metabolismo , Quinases Lim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/genética , Fosforilação , Podócitos/metabolismo , Podócitos/patologia , Polimerização , Proteinúria/metabolismo , Proteinúria/prevenção & controle , Interferência de RNA , Receptor trkB/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Peixe-ZebraRESUMO
Nephrin is an immunoglobulin-like adhesion molecule first discovered as a major component of the podocyte slit diaphragm, where its integrity is essential to the function of the glomerular filtration barrier. Outside the kidney, nephrin has been shown in other restricted locations, most notably in the central nervous system (CNS) of embryonic and newborn rodents. With the aim of better characterizing nephrin expression and its role in the CNS of adult rodents, we studied its expression pattern and possible binding partners in CNS tissues and cultured neuronal cells and compared these data to those obtained in control renal tissues and podocyte cell cultures. Our results show that, besides a number of locations already found in embryos and newborns, endogenous nephrin in adult rodent CNS extends to the pons and corpus callosum and is expressed by granule cells and Purkinje cells of the cerebellum, with a characteristic alternating expression pattern. In primary neuronal cells we find nephrin expression close to synaptic proteins and demonstrate that nephrin co-immunoprecipitates with Fyn kinase, glutamate receptors and the scaffolding molecule PSD95, an assembly that is reminiscent of those made by synaptic adhesion molecules. This role seems to be confirmed by our findings of impaired maturation and reduced glutamate exocytosis occurring in Neuro2A cells upon nephrin silencing. Of note, we disclose that the very same nephrin interactions occur in renal glomeruli and cultured podocytes, supporting our hypothesis that podocytes organize and use similar molecular intercellular signalling modules to those used by neuronal cells.
Assuntos
Encéfalo/metabolismo , Proteínas de Membrana/biossíntese , Receptores de Glutamato/metabolismo , Animais , Células Cultivadas , Córtex Cerebral/metabolismo , Glomérulos Renais/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias , Neurônios/metabolismo , Podócitos/metabolismo , Proteínas Tirosina Quinases , Células de Purkinje/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Quinases da Família src/metabolismoRESUMO
BACKGROUND/AIMS: The relative contribution of genetic factors and dietary patterns to glomerular damage in healthy individuals and prediabetic conditions is currently unclear. All Rab3A knockout (KO) mice spontaneously develop macroalbuminuria, but only male mice exhibit a glucose-intolerant phenotype, thus making the model suitable to examine the impact of a diet on preexisting podocyte damage. METHODS: Male and female Rab3A KO and wild-type (WT) mice were chronically fed a high-glucose diet (HGD). Biochemical tests, histology and immunohistochemistry were periodically performed whilst primary podocytes served for in vitro analyses. RESULTS: Chronic administration of an HGD did not induce de novo alterations in WT kidneys but caused progressive worsening of podocyte and glomerular damage in both male and female Rab3A KO. Though glomerular lesions, reminiscent of human diabetic nephropathy, were more severe in male mice, overt proteinuria and renal damage were also evident in female mice. The in vitro analysis of Rab3A WT and KO podocytes revealed diminished actin plasticity in the cell processes of KO podocytes. Furthermore, a modest increase in glucose concentration induced profound cytoskeletal changes only in Rab3A KO cells. CONCLUSIONS: Our data show that chronic administration of an HGD to Rab3A KO mice that have a genetic defect that impairs podocyte actin plasticity results in increased podocyte damage and leads to overt proteinuria. If the same diet is given to male Rab3A KO animals, with additionally altered glucose homeostasis, this results in renal lesions similar to those of human diabetic nephropathy.
Assuntos
Dieta , Glucose/administração & dosagem , Glomérulos Renais/patologia , Proteinúria/patologia , Proteína rab3A de Ligação ao GTP/deficiência , Actinas/metabolismo , Animais , Células Cultivadas , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Feminino , Glucose/toxicidade , Humanos , Imuno-Histoquímica , Glomérulos Renais/metabolismo , Glomérulos Renais/ultraestrutura , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Microscopia Eletrônica , Microscopia de Fluorescência , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Podócitos/patologia , Proteinúria/etiologia , Proteinúria/genética , Fatores Sexuais , Proteína rab3A de Ligação ao GTP/genéticaRESUMO
Podocytes possess the complete machinery for glutamatergic signaling, raising the possibility that neuron-like signaling contributes to glomerular function. To test this, we studied mice and cells lacking Rab3A, a small GTPase that regulates glutamate exocytosis. In addition, we blocked the glutamate ionotropic N-methyl-d-aspartate receptor (NMDAR) with specific antagonists. In mice, the absence of Rab3A and blockade of NMDAR both associated with an increased urinary albumin/creatinine ratio. In humans, NMDAR blockade, obtained by addition of ketamine to general anesthesia, also had an albuminuric effect. In vitro, Rab3A-null podocytes displayed a dysregulated release of glutamate with higher rates of spontaneous exocytosis, explained by a reduction in Rab3A effectors resulting in freedom of vesicles from the actin cytoskeleton. In addition, NMDAR antagonism led to profound cytoskeletal remodeling and redistribution of nephrin in cultured podocytes; the addition of the agonist NMDA reversed these changes. In summary, these results suggest that glutamatergic signaling driven by podocytes contributes to the integrity of the glomerular filtration barrier and that derangements in this signaling may lead to proteinuric renal diseases.
Assuntos
Exocitose/fisiologia , Taxa de Filtração Glomerular/fisiologia , Ácido Glutâmico/metabolismo , Podócitos/metabolismo , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Citoesqueleto/metabolismo , Maleato de Dizocilpina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Ketamina/análogos & derivados , Ketamina/farmacologia , Nefropatias/metabolismo , Nefropatias/fisiopatologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Podócitos/citologia , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Proteína rab3A de Ligação ao GTP/genética , Proteína rab3A de Ligação ao GTP/metabolismoRESUMO
Synaptopodin is the founding member of a novel class of proline-rich actin-associated proteins highly expressed in telencephalic dendrites and renal podocytes. Synaptopodin-deficient (synpo(-/-)) mice lack the dendritic spine apparatus and display impaired activity-dependent long-term synaptic plasticity. In contrast, the ultrastructure of podocytes in synpo(-/-) mice is normal. Here we show that synpo(-/-) mice display impaired recovery from protamine sulfate-induced podocyte foot process (FP) effacement and LPS-induced nephrotic syndrome. Similarly, synpo(-/-) podocytes show impaired actin filament reformation in vitro. We further demonstrate that synaptopodin exists in 3 isoforms, neuronal Synpo-short (685 AA), renal Synpo-long (903 AA), and Synpo-T (181 AA). The C terminus of Synpo-long is identical to that of Synpo-T. All 3 isoforms specifically interact with alpha-actinin and elongate alpha-actinin-induced actin filaments. synpo(-/-) mice lack Synpo-short and Synpo-long expression but show an upregulation of Synpo-T protein expression in podocytes, though not in the brain. Gene silencing of Synpo-T abrogates stress-fiber formation in synpo(-/-) podocytes, demonstrating that Synpo-T serves as a backup for Synpo-long in synpo(-/-) podocytes. In concert, synaptopodin regulates the actin-bundling activity of alpha-actinin in highly dynamic cell compartments, such as podocyte FPs and the dendritic spine apparatus.
Assuntos
Actinina/metabolismo , Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Encéfalo/metabolismo , Rim/metabolismo , Camundongos , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Microscopia Eletrônica , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Kidney podocytes and their slit diaphragms form the final barrier to urinary protein loss. This explains why podocyte injury is typically associated with nephrotic syndrome. The present study uncovered an unanticipated novel role for costimulatory molecule B7-1 in podocytes as an inducible modifier of glomerular permselectivity. B7-1 in podocytes was found in genetic, drug-induced, immune-mediated, and bacterial toxin-induced experimental kidney diseases with nephrotic syndrome. The clinical significance of our results is underscored by the observation that podocyte expression of B7-1 correlated with the severity of human lupus nephritis. In vivo, exposure to low-dose LPS rapidly upregulates B7-1 in podocytes of WT and SCID mice, leading to nephrotic-range proteinuria. Mice lacking B7-1 are protected from LPS-induced nephrotic syndrome, suggesting a link between podocyte B7-1 expression and proteinuria. LPS signaling through toll-like receptor-4 reorganized the podocyte actin cytoskeleton in vitro, and activation of B7-1 in cultured podocytes led to reorganization of vital slit diaphragm proteins. In summary, upregulation of B7-1 in podocytes may contribute to the pathogenesis of proteinuria by disrupting the glomerular filter and provides a novel molecular target to tackle proteinuric kidney diseases. Our findings suggest a novel function for B7-1 in danger signaling by nonimmune cells.
Assuntos
Antígeno B7-1/biossíntese , Rim/imunologia , Síndrome Nefrótica/imunologia , Actinas/metabolismo , Animais , Antígeno B7-1/genética , Sequência de Bases , DNA/genética , Humanos , Técnicas In Vitro , Integrina alfa3/genética , Integrina alfa3/metabolismo , Rim/patologia , Lipopolissacarídeos/toxicidade , Nefrite Lúpica/etiologia , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Knockout , Camundongos SCID , Síndrome Nefrótica/etiologia , Síndrome Nefrótica/patologia , Proteínas/genética , Proteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like , Receptores Toll-LikeRESUMO
Although patients with chronic renal failure are increasing worldwide, many aspects of kidney biology remain to be elucidated. Recent research has uncovered several molecular properties of the glomerular filtration barrier, in which podocytes, highly differentiated, ramified cells that enwrap the glomerular basement membrane, have been reported to be mainly responsible for filter's selectivity. We previously described that podocytes express Rab3A, a GTPase restricted to cell types that are capable of highly regulated exocytosis, such as neuronal cells. Here, we first demonstrate by a proteomic study that Rab3A in podocytes coimmmunoprecipitates with molecules once thought to be synapse specific. We then show that podocytes possess structures resembling synaptic vesicles, which contain glutamate, coexpress Rab3A and synaptotagmin 1, and undergo spontaneous and stimulated exocytosis and recycling, with glutamate release. Finally, from the results of a cDNA microarray study, we describe the presence of a series of neuron- and synapse-specific molecules in normal human glomeruli and confirm the glomerular protein expression of both metabotropic and ionotropic glutamate receptors. These data point toward a synaptic-like mechanism of communication among glomerular cells, which perfectly fits with the molecular composition of the glomerular filter and puts in perspective several previous observations, proposing a different working hypothesis for understanding glomerular signaling dynamics.
Assuntos
Podócitos/citologia , Podócitos/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Células Cultivadas , Endocitose/fisiologia , Exocitose/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ácido Glutâmico/metabolismo , Humanos , Camundongos , Podócitos/efeitos dos fármacos , Venenos de Aranha/farmacologia , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismo , Proteína rab3A de Ligação ao GTP/metabolismoAssuntos
Barreira de Filtração Glomerular/fisiologia , Proteínas de Membrana/fisiologia , Transdução de Sinais/fisiologia , Animais , Adesão Celular , Linhagem Celular , Humanos , Proteínas de Membrana/química , Neurônios/fisiologia , Neurônios/ultraestrutura , Podócitos/metabolismo , Podócitos/fisiologia , Podócitos/ultraestrutura , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Sinapses/fisiologiaRESUMO
Proteinuria is a common symptom of glomerular diseases and is due to leakage of proteins from the glomerular filtration barrier, a three-layer structure composed by two post-mitotic highly specialized and interdependent cell populations, i.e. glomerular endothelial cells and podocytes, and the basement membrane in between. Despite enormous progresses made in the last years, pathogenesis of proteinuria remains to be completely uncovered. Studies in the field could largely benefit from an in vitro model of the glomerular filter, but such a system has proved difficult to realize. Here we describe a method to obtain and utilize a three-dimensional podocyte-endothelial co-culture which can be largely adopted by the scientific community because it does not rely on special instruments nor on the synthesis of devoted biomaterials. The device is composed by a porous membrane coated on both sides with type IV collagen. Adhesion of podocytes on the upper side of the membrane has to be preceded by VEGF-induced maturation of endothelial cells on the lower side. The co-culture can be assembled with podocyte cell lines as well as with primary podocytes, extending the use to cells derived from transgenic mice. An albumin permeability assay has been extensively validated and applied as functional readout, enabling rapid drug testing. Additionally, the bottom of the well can be populated with a third cell type, which multiplies the possibilities of analyzing more complex glomerular intercellular signaling events. In conclusion, the ease of assembly and versatility of use are the major advantages of this three-dimensional model of the glomerular filtration barrier over existing methods. The possibility to run a functional test that reliably measures albumin permeability makes the device a valid companion in several research applications ranging from drug screening to intercellular signaling studies.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Células Endoteliais , Podócitos , Albuminas/metabolismo , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Colágeno Tipo I/farmacologia , Colágeno Tipo IV/farmacologia , Dexametasona/farmacologia , Doxorrubicina/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Ácido Glutâmico/farmacologia , Humanos , Glomérulos Renais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Receptores de Glutamato/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Progresses in podocyte biology have been strictly connected with genetic advances; the identification of genes mutated in familial and sporadic forms of nephrotic syndrome has been followed by functional studies of the encoded proteins, revealing numerous properties of the cell. The molecules uncovered so far belong to three main categories: a) proteins located at the slit diaphragm, the intercellular junction which laterally connects podocyte processes and is responsible for selectivity of the glomerular filter, b) molecules involved in regulation of actin dynamics, which are essential for the maintenance of podocyte structure and function, and c) molecules belonging to intracellular organelles, such as mitochondria and lysosomes, which are central players in podocyte metabolism. Considering the key role of the podocyte in health and disease of the glomerular filter, better knowledge of this cell is a pre-requisite for developing targeted therapies of glomerular diseases.
Assuntos
Síndrome Nefrótica/etiologia , Podócitos/fisiologia , Animais , Citoesqueleto , Humanos , Lisossomos , Proteínas de Membrana/fisiologia , Mitocôndrias , Síndrome Nefrótica/genética , Podócitos/ultraestruturaRESUMO
Podocytes are postmitotic renal glomerular cells with multiple ramifications that extend from the cell body. Processes departing from a podocyte interdigitate with corresponding projections from neighboring cells and form an intricate web that enwraps the glomerular capillary completely. Podocyte processes are interconnected by the slit diaphragm, an adhesion junction mostly formed by Ig-like molecules, cadherins/protocadherins, ephrin/eph, and neurexin molecules organized in an assembly that resembles synaptic junctions. Podocyte failure is primarily or secondarily implicated in all forms of proteinuric glomerular diseases, as confirmed by the morphological changes of their elaborate cell architecture detectable by electron microscopy. Importantly, mutations of podocyte proteins are responsible for the most severe forms of congenital nephrotic syndrome. In the last 15 years, progressive technological advances have aided the study of podocyte biology and pathology, confirming the relevance of podocyte molecules and signaling pathways for the function of the glomerular filter. This review will examine the most important and newest discoveries in the field, which is rapidly evolving, hopefully leading to a detailed knowledge of this fascinating cell and to the development of specific therapeutic options for proteinuric diseases.
Assuntos
Podócitos/fisiologia , Proteinúria/patologia , Animais , Humanos , Nefropatias/metabolismo , Nefropatias/patologia , Glomérulos Renais/citologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Camundongos , Modelos Biológicos , Podócitos/citologia , Podócitos/patologia , Podócitos/ultraestrutura , Proteinúria/metabolismo , Transdução de SinaisRESUMO
The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line. The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features. After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules. Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.
Assuntos
Técnicas de Cultura de Células/métodos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteócitos/citologia , Osteócitos/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Meios de Cultura , Camundongos , Camundongos TransgênicosRESUMO
The morphology of healthy podocyte foot processes is necessary for maintaining the characteristics of the kidney filtration barrier. In most forms of glomerular disease, abnormal filter barrier function results when podocytes undergo foot process spreading and retraction by remodeling their cytoskeletal architecture and intercellular junctions during a process known as effacement. The cell adhesion protein nephrin is necessary for establishing the morphology of the kidney podocyte in development by transducing from the specialized podocyte intercellular junction phosphorylation-mediated signals that regulate cytoskeletal dynamics. The present studies extend our understanding of nephrin function by showing that nephrin activation in cultured podocytes induced actin dynamics necessary for lamellipodial protrusion. This process required a PI3K-, Cas-, and Crk1/2-dependent signaling mechanism distinct from the previously described nephrin-Nck1/2 pathway necessary for assembly and polymerization of actin filaments. Our present findings also support the hypothesis that mechanisms governing lamellipodial protrusion in culture are similar to those used in vivo during foot process effacement in a subset of glomerular diseases. In mice, podocyte-specific deletion of Crk1/2 prevented foot process effacement in one model of podocyte injury and attenuated foot process effacement and associated proteinuria in a delayed fashion in a second model. In humans, focal adhesion kinase and Cas phosphorylation - markers of focal adhesion complex-mediated Crk-dependent signaling - was induced in minimal change disease and membranous nephropathy, but not focal segmental glomerulosclerosis. Together, these observations suggest that activation of a Cas-Crk1/2-dependent complex is necessary for foot process effacement observed in distinct subsets of human glomerular diseases.
Assuntos
Nefropatias/patologia , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Podócitos/metabolismo , Proteínas Proto-Oncogênicas c-crk/metabolismo , Adolescente , Adulto , Idoso , Animais , Linhagem Celular , Proteína Substrato Associada a Crk/genética , Proteína Substrato Associada a Crk/metabolismo , Feminino , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Nefropatias/metabolismo , Glomérulos Renais/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/metabolismo , Podócitos/ultraestrutura , Proteínas Proto-Oncogênicas c-crk/genética , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Adulto JovemRESUMO
Adducins are cytoskeletal actin-binding proteins (alpha, beta, gamma) that function as heterodimers and heterotetramers and are encoded by distinct genes. Experimental and clinical evidence implicates alpha- and beta-adducin variants in hypertension and renal dysfunction. Here, we have addressed the role of alpha- and beta-adducin on glomerular function and disease using beta-adducin null mice, congenic substrains for alpha- and beta-adducin from the Milan hypertensive (MHS) and Milan normotensive (MNS) rats and patients with IgA nephropathy. Targeted deletion of beta-adducin in mice reduced urinary protein excretion, preceded by an increase of podocyte protein expression (phospho-nephrin, synaptopodin, alpha-actinin, ZO-1, Fyn). The introgression of polymorphic MHS beta-adducin locus into MNS (Add2, 529R) rats was associated with an early reduction of podocyte protein expression (nephrin, synaptopodin, alpha-actinin, ZO-1, podocin, Fyn), followed by severe glomerular and interstitial lesions and increased urinary protein excretion. These alterations were markedly attenuated when the polymorphic MHS alpha-adducin locus was also present (Add1, 316Y). In patients with IgA nephropathy, the rate of decline of renal function over time was associated to polymorphic beta-adducin (ADD2, 1797T, rs4984) with a significant interaction with alpha-adducin (ADD1, 460W, rs4961). These findings suggest that adducin genetic variants participate in the development of glomerular lesions by modulating the expression of specific podocyte proteins.
Assuntos
Proteínas de Ligação a Calmodulina/genética , Glomerulonefrite por IGA/genética , Podócitos , Polimorfismo Genético , Proteinúria/genética , Animais , Proteínas de Ligação a Calmodulina/metabolismo , Glomerulonefrite por IGA/patologia , Humanos , Rim/metabolismo , Rim/patologia , Testes de Função Renal , Camundongos , Podócitos/metabolismo , Podócitos/patologia , Ratos , RoedoresRESUMO
BACKGROUND: In recent studies performed on cultured cells and experimental nephropathies, it has been hypothesized that tubular epithelial cells (TEC), via epithelial-mesenchymal transformation (EMT), can become collagen-producing cells. According to this theory, they should proceed through several activating steps, such as proliferation and phenotype changes, to eventually synthesize extracellular matrix (ECM). METHODS: To evaluate whether EMT operates in human TECs, 133 renal biopsies of different renal diseases were studied, analyzing by immunohistochemistry and in situ hybridization the possible expression of markers of proliferation (PCNA, Mib-1), cellular phenotype (vimentin, alpha-SMA, cytokeratin, ZO-1) and ECM production (prolyl 4-hydroxylase, HSP47, interstitial collagens). RESULTS: Independently of histological diagnosis, variable degrees of TEC positivity for PCNA (2.7 +/- 2.4 cells/field) and Mib-1 (1.9 +/- 2.3) were present. TECs expressing vimentin (1.4 +/- 4.7) and alpha-smooth muscle actin (alpha-SMA; 0.04 +/- 0.4) also were detected. It was possible to observe loss of epithelial antigens from 8 to 10% of the tubular cross sections. Moreover, TECs were stained by prolyl 4-hydroxylase (3.6 +/- 4.3), heat shock protein-47 (HSP47; 2.9 +/- 5.4), collagen type I (0.2 +/- 2.7) and type III (0.3 +/- 2.0). Collagen types I and III mRNAs were found in 0.8 to 1.4 cells/field. The number of TEC with EMT features were associated with serum creatinine and the degree of interstitial damage (P< or = 0.03), and even considering the 45 cases with mild interstitial lesions, the tubular expression of all markers remained strictly associated with renal function (P< or = 0.01). CONCLUSIONS: Our results suggest that, via transition to a mesenchymal phenotype, TEC can produce ECM proteins in human disease and directly intervene in the fibrotic processes. Moreover, the association of EMT features with serum creatinine supports the value of these markers in the assessment of disease severity.