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Focal and segmental glomerulosclerosis (FSGS) is a severe form of idiopathic nephrotic syndrome (INS), a glomerulopathy of presumably immune origin that is attributed to extrarenal pathogenic circulating factors. The recurrence of FSGS (rFSGS) after transplant occurs in 30% to 50% of cases. The direct analysis of patient plasma proteome has scarcely been addressed to date, mainly due to the methodological difficulties associated with plasma complexity and dynamic range. In this study, first, we compared different methods of plasma preparation, second, we compared the plasma proteomes of rFSGS and controls using two preparation methods, and third, we analyzed the early proximal signaling events in podocytes subjected to patient plasma, through a combination of phosphoproteomics and lipid-raft proteomics (raftomics). By combining immunodepletion and high pH fractionation, we performed a differential proteomic analysis of soluble plasma proteins and of extracellular vesicles (EV) obtained from healthy controls, non-INS patient controls, and rFSGS patients (n = 4). In both the soluble- and the EV-protein sets from the rFSGS patients, we found a statistically significant increase in a cluster of proteins involved in neutrophil degranulation. A group of lipid-binding proteins, generally associated with lipoproteins, was found to be decreased in the soluble set from the rFSGS patients. In addition, three amino acid transporters involved in mTORC1 activation were found to be significantly increased in the EV from the rFSGS. Next, we incubated human podocytes for 30 min with 10% plasma from both groups of patients. The phosphoproteomics and raftomics of the podocytes revealed profound differences in the proteins involved in the mTOR pathway, in autophagy, and in cytoskeleton organization. We analyzed the correlation between the abundance of plasma and plasma-regulated podocyte proteins. The observed changes highlight some of the mechanisms involved in FSGS recurrence and could be used as specific early markers of circulating-factor activity in podocytes.
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Tailor-made and designed micro- and nanocarriers can bring significant benefits over their traditional macroscopic counterparts in drug delivery applications. For the successful loading and subsequent release of bioactive compounds, carriers should present a high loading capacity, trigger release mechanisms, biodegradability and biocompatibility. Hydrophobic drug molecules can accumulate in fat tissues, resulting in drawbacks for the patient's recovery. To address these issues, we propose to combine the advantageous features of both host molecules (cyclodextrin) and calcium carbonate (CaCO3) particles in order to load hydrophobic chemicals. Herein, hybrid cyclodextrin-CaCO3 micro- to nano-particles have been fabricated by combining Na2CO3 solution and CaCl2 solution in the presence of an additive, namely poly (vinylsulfonic acid) (PVSA) or glycerol (gly). By investigating experimental parameters and keeping the Na2CO3 and CaCl2 concentrations constant (0.33 M), we have evidenced that the PVSA or gly concentration and mixing time have a direct impact on the final cyclodextrine-CaCO3 particle size. Indeed, by increasing the concentration of PVSA (5 mM to 30 mM) or gly (0.7 mM to 4 mM) or the reaction time (from 10 min to 4 h), particles with a size of 200 nm could be reached. Interestingly, the vaterite or calcite form could also be selected, according to the experimental conditions. We hypothesised that the incorporation of PVSA or gly into the precipitation reaction might reduce the nucleation rate by sequestering Ca2+. The obtained particles have been found to keep their crystal structure and surface charge after storage in aqueous media for at least 6 months. In the context of improving the therapeutic benefit of hydrophobic drugs, the developed particles were used to load the hydrophobic drug tocopherol acetate. The resulting particles are biocompatible and highly stable in a physiological environment (pH 7.4, 0.15 M NaCl). A selective release of the cargo is observed in acidic media (pH lower than 5).
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Bacteria resistance to antibiotics has emerged as a major health problem. Developing new antibacterial systems is then of major interest. In this sense, we present biocapsules presenting inherent antibacterial capacity. The self-assembly of charged biopolymer, namely diethylaminoethyl-dextran hydrochloride (dex+) and dextran sulfate (dex-), were done on calcium carbonate microparticles, used as a template. Zeta potential measurements have shown the successful alternate adsorption of these biopolymers and related charge reversal upon the multilayer film construction onto the particles surface. The shape of the capsules was characterized by scanning electron microscopy (SEM). These particles were tested against bacteria resistant to antibiotics, namely kanamycin-resistant Escherichia coli. An inhibitory effect of the particles was observed during bacterial growth in liquid medium, i.e. in the range of 10 % for (dex+/dex-)n coated CaCO3 materials and of 50% for (dex+/dex-)n capsules. These findings evidence the high potential of capsules to act as antimicrobial agents in future and in treatments against infections.
Assuntos
AntibacterianosRESUMO
BACKGROUND: The Wilms tumor 1 suppressor gene, WT1, is expressed throughout life in podocytes and is essential for their function. Downregulation of WT1 has been reported in podocyte diseases but the underlying mechanisms remain unclear. Podocyte injury is the hallmark of idiopathic nephrotic syndrome (INS), the most frequent glomerular disease in children and young adults. An increase in the abundance of Cmaf-inducing protein (CMIP) has been found to alter podocyte function, but it is not known whether CMIP affects WT1 expression. METHODS: Transcriptional and post-transcriptional regulation of WT1in the presence of CMIP was studied using transient transfection, mouse models, and siRNA handling. RESULTS: We showed that overproduction of CMIP in the podocyte was consistently associated with a downregulation of WT1 according to two mechanisms. We found that CMIP prevented the NF-kB-mediated transcriptional activation of WT1. We demonstrated that CMIP interacts directly with WT1 through its leucine-rich repeat domain. Overexpression of CMIP in the M15 cell line induced a downregulation of WT1, which was prevented by lactacystin, a potent proteasome inhibitor. We showed that CMIP exhibits an E3 ligase activity and targets WT1 to proteasome degradation. Intravenous injection of Cmip-siRNA specifically prevented the repression of Wt1 in lipopolysaccharides-induced proteinuria in mice. CONCLUSIONS: These data suggest that CMIP is a repressor of WT1 and might be a critical player in the pathophysiology of some podocyte diseases. Because WT1 is required for podocyte integrity, CMIP could be considered a therapeutic target in podocyte diseases.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Síndrome Nefrótica/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas WT1/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Regulação para Baixo/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/metabolismo , Síndrome Nefrótica/metabolismo , Podócitos/citologia , Podócitos/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Ligação Proteica , Proteinúria/patologia , Proteinúria/prevenção & controle , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/metabolismo , Ativação Transcricional , Proteínas WT1/genéticaRESUMO
Escherichia coli is one of the most common commensal aerobic bacteria in the gut microbiota of humans (and other mammals). Nevertheless, if left free to proliferate, it can induce a large range of diseases from diarrhoea to extra-intestinal diseases. In recent years, this bacterium had become increasingly resistant to antibiotics. It is therefore essential to implement new approaches able to maintain both bacterial viability and to control their proliferation. In this context, we developed a process to encapsulate Escherichia coli in polymer shells. We took advantage of the fact that this bacterium has a negatively charged surface and modified it via a layer-by-layer process, i.e. with oppositely charged polyelectrolyte pairs (namely chitosan as the polycation and alginate or dextran sulfate as polyanion). We successfully demonstrate the controlled coating of the bacterial surface via zeta potential measurement, the viability of the encapsulated bacteria and a delay in growth due to the multilayer coating. This delay was dependent on the number of polyelectrolyte layers.
Assuntos
Quitosana , Escherichia coli , Alginatos , Animais , Antibacterianos/farmacologia , Humanos , Viabilidade MicrobianaRESUMO
The analysis of T cell lipid raft proteome is challenging due to the highly dynamic nature of rafts and the hydrophobic character of raft-resident proteins. We explored an innovative strategy for bottom-up lipid raftomics based on suspension-trapping (S-Trap) sample preparation. Mouse T cells were prepared from splenocytes by negative immunoselection, and rafts were isolated by a detergent-free method and OptiPrep gradient ultracentrifugation. Microdomains enriched in flotillin-1, LAT, and cholesterol were subjected to proteomic analysis through an optimized protocol based on S-Trap and high pH fractionation, followed by nano-LC-MS/MS. Using this method, we identified 2,680 proteins in the raft-rich fraction and established a database of 894 T cell raft proteins. We then performed a differential analysis on the raft-rich fraction from nonstimulated versus anti-CD3/CD28 T cell receptor (TCR)-stimulated T cells. Our results revealed 42 proteins present in one condition and absent in the other. For the first time, we performed a proteomic analysis on rafts from ex vivo T cells obtained from individual mice, before and after TCR activation. This work demonstrates that the proposed method utilizing an S-Trap-based approach for sample preparation increases the specificity and sensitivity of lipid raftomics.
Assuntos
Lipídeos/análise , Proteoma/análise , Linfócitos T/química , Animais , Células Cultivadas , Cromatografia Líquida , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Espectrometria de Massas em TandemRESUMO
Upon their interaction with cognate antigen, T cells integrate different extracellular and intracellular signals involving basal and induced protein-protein interactions, as well as the binding of proteins to lipids, which can lead to either cell activation or inhibition. Here, we show that the selective T cell expression of CMIP, a new adapter protein, by targeted transgenesis drives T cells toward a naïve phenotype. We found that CMIP inhibits activation of the Src kinases Fyn and Lck after CD3/CD28 costimulation and the subsequent localization of Fyn and Lck to LRs. Video microscopy analysis showed that CMIP blocks the recruitment of LAT and the lipid raft marker cholera toxin B at the site of TCR engagement. Proteomic analysis identified several protein clusters differentially modulated by CMIP and, notably, Cofilin-1, which is inactivated in CMIP-expressing T cells. Moreover, transgenic T cells exhibited the downregulation of GM3 synthase, a key enzyme involved in the biosynthesis of gangliosides. These results suggest that CMIP negatively impacts proximal signaling and cytoskeletal rearrangement and defines a new mechanism for the negative regulation of T cells that could be a therapeutic target.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antígenos CD28/metabolismo , Complexo CD3/metabolismo , Polaridade Celular , Citocinas/metabolismo , Ativação Enzimática , Glicoesfingolipídeos/metabolismo , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Microdomínios da Membrana/metabolismo , Camundongos Transgênicos , Fenótipo , Proteômica , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Quinases da Família src/metabolismoRESUMO
Most of the drug molecules are partially insoluble in aqueous solution and then may accumulate in fat tissues hampering efficient therapy. Innovative drug delivery strategies have emerged in industry or academia over the last decades, however preserving the activity of the encapsulated drug, having high drug loading capacity and controlling drug release kinetics, are still challenging. In this context, we explored the preparation of new nanocarriers, namely nanocapsules, via a templating method, and using polysaccharides exhibiting biological functions. Cationic poly(cyclodextrin) (P(CD+)) and alginate (alg-) were initially self-assembled layer-by-layer on colloidal gold nanoparticles. Removal of gold nanoparticles was then induced thorough cyanide-assisted hydrolysis, enabling the recovery of nanocapsules. A hydrophobic drug known to allow the mutation of genes inside cells, namely 4-hydroxy-tamoxifen, was loaded within the nanocapsules' shell via inclusion with the cyclodextrin cavities. The so-designed nanomaterials were incubated with immortalized podocytes to investigate i) their incorporation inside cells and ii) their efficiency for in vitro 4-hydroxy-tamoxifen-induced CreERT2 recombination. This work undoubtedly highlights a proof-of-concept for drug delivery using polysaccharides-based capsules with host properties.
Assuntos
Alginatos/química , Ciclodextrinas/química , Sistemas de Liberação de Medicamentos , Nanocápsulas/química , Podócitos/metabolismo , Tamoxifeno/farmacologia , Adsorção , Animais , Cátions , Coloides/química , Ouro/química , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Camundongos , Nanocápsulas/ultraestrutura , Podócitos/efeitos dos fármacos , Polieletrólitos/química , Eletricidade Estática , Água/químicaRESUMO
Lupus glomerulopathies are classified into various histological patterns, which probably result from different pathophysiological origins. Podocyte injury can be demonstrated in lupus nephritis but its clinical relevance is far little appreciated and is often masked by proliferative lesions and inflammatory cell infiltrations. Two patterns of podocyte lesions may be considered, either occurring in the context of renal inflammation or reflecting podocyte dysfunction in non-proliferative and non-inflammatory glomerulopathies. This distinction remains elusive since no reliable biomarker discriminates between both entities. CMIP was recently found induced in some glomerular disease but its expression in different lupus nephritis classes has not been investigated. Twenty-four adult patients with lupus nephritis, including non-proliferative (n = 11) and proliferative (n = 13) glomerulopathies were analyzed. Clinical, biological and immunological data were compared with immunomorphological findings. We analyzed by quantitative and qualitative methods the expression of CMIP in different histological classes. We found CMIP abundance selectively increased in podocytes in class II and class V glomerulopathies, while in proliferative forms (class III and class IV), CMIP was rarely detected. CMIP was not expressed in cellular crescents, endothelial cells or mesangial cells. CMIP colocalized with some subsets of B and T cells within glomerular or interstitial mononuclear cell infiltrates but never with macrophages. Hematuria is rarely present in lupus glomerulopathies expressing CMIP. There was no correlation between classical immunological markers and CMIP expression. Thus, CMIP induction in lupus nephritis seems restricted to non-proliferative glomerulopathies and may define a specific pattern of podocyte injury.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação da Expressão Gênica , Nefrite Lúpica/metabolismo , Nefrite Lúpica/patologia , Podócitos/metabolismo , Adulto , Feminino , Humanos , Glomérulos Renais/patologia , Nefrite Lúpica/terapia , Masculino , FenótipoRESUMO
Minimal-change nephrotic syndrome (MCNS) is an immune-mediated glomerular disease. We have analyzed the modifications on T-cell subsets in twenty-three patients who were highly steroid/calcineurin inhibitor and/or mycophenolate mofetil-dependent for frequently relapsing nephrotic syndrome (FRNS) and who were enrolled in a multicenter, double-blind, randomized, placebo vs Rituximab-controlled trial. Patients with FRNS entered the trial at remission and were randomly assigned to receive either Rituximab or placebo. In both groups, patient blood samples were analyzed at inclusion and then monthly until six months post-perfusion. Disclosure of patient's allocation code occurred in relapse or at the end of the trial. All patients under placebo displaying relapse were subsequently treated with Rituximab. Despite the significant decrease of immunosuppressive drugs, remission was maintained in all patients included in the Rituximab group, except one (n = 9/10). On the other hand, relapses occurred within a few weeks (means ≈ 7.3 weeks) in all patients receiving placebo (n = 13). At inclusion, before rituximab therapy, the frequency of different T-cell subsets were highly similar in both groups, except for CD8+ and invariant TCRVα24 T-cell subsets, which were significantly increased in patients of the Placebo group ((p = 0,0414 and p = 0.0428, respectively). Despite the significant decrease of immunosuppressive drugs, remission was maintained in all patients included in the Rituximab group (n = 10), except one. Relapses were associated with a significant decrease in CD4+CD25highFoxP3high Tregulatory cells (p = 0.0005) and IL2 expression (p = 0.0032), while CMIP abundance was significantly increased (p = 0.03). Remissions after Rituximab therapy were associated in both groups with significant decrease in the frequency of CD4+CD45RO+CXCR5+, invariant natural killer T-cells (INKT) and CD4-CD8- (double-negative, DN) T-cells expressing the invariant Vα24 chain (DN-TCR Vα24) T-cells, suggesting that MCNS involves a disorder of innate and adaptive immune response, which can be stabilized by Rituximab treatment.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células T Matadoras Naturais/imunologia , Nefrose Lipoide/tratamento farmacológico , Rituximab/uso terapêutico , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Imunidade Adaptativa , Adolescente , Antígenos CD20/imunologia , Criança , Pré-Escolar , Método Duplo-Cego , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Imunidade Inata , Masculino , Placebos , Receptores de Antígenos de Linfócitos T/metabolismo , Resultado do TratamentoRESUMO
The WT1 (Wilm's tumor suppressor) gene is expressed throughout life in podocytes and is essential for the functional integrity of the glomerular filtration barrier. We have previously shown that CMIP (C-Maf inducing protein) is overproduced in podocyte diseases and alters intracellular signaling. Here we isolated the proximal region of the human CMIP promoter and showed by chromatin immunoprecipitation assays and electrophoretic-mobility shift that Wilm's tumor protein (WT1) bound to 2 WT1 response elements, located at positions -290/-274 and -57/-41 relative to transcription start site. Unlike the human CMIP gene, only one Wt1 response element was identified in the mouse Cmip proximal promoter located at position -217/-206. Luciferase reporter assays indicated that WT1 dose-dependently inhibited the transcriptional induction of the CMIP promoter. Transfection of decoy oligonucleotides mimicking the WT1 response elements prevented the inhibition of WT1 on CMIP promoter activity. Furthermore, WT1 silencing promoted Cmip expression. In line with these findings, the abundance of Cmip was early and significantly increased at the transcript and protein level in podocytes displaying a primary defect in Wt1, including Denys-Drash syndrome and Frasier syndrome. Thus, WT1 is a major repressor of the CMIP gene in physiological situations, while conditional deletion of CMIP in the developing kidney did not affect the development of mature glomeruli.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Podócitos/metabolismo , Proteínas WT1/metabolismo , Animais , Sequência de Bases , Síndrome de Denys-Drash/metabolismo , Feminino , Síndrome de Frasier/metabolismo , Regulação da Expressão Gênica , Humanos , Rim/embriologia , Masculino , Camundongos , Regiões Promotoras GenéticasRESUMO
Idiopathic change nephrotic syndrome (INS), the most frequent glomerular disease in children and young adults, is characterized by heavy proteinuria and a relapsing remitting course. Although the mechanisms underlying the pathophysiology of proteinuria remain unclear, clinical and experimental observations suggest that lymphocyte and podocyte disturbances are two sides of the disease. The current hypothesis suggests that immune cells release a putative factor, which alters podocyte function resulting in nephrotic proteinuria. Besides T-cell abnormalities, recent evidence of B-cell depletion efficacy in sustained remissions added a new challenge in understanding the immunological mechanisms of INS. In this review, we discuss recent insights related to podocyte disorders occurring in INS and their relevance in human diseases.
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Linfócitos B , Nefropatias , Podócitos , Proteinúria , Linfócitos T , Adulto , Linfócitos B/imunologia , Linfócitos B/patologia , Criança , Pré-Escolar , Humanos , Nefropatias/imunologia , Nefropatias/patologia , Podócitos/imunologia , Podócitos/patologia , Proteinúria/imunologia , Proteinúria/patologia , Síndrome , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Renal toxicity constitutes a dose-limiting side effect of anticancer therapies targeting vascular endothelial growth factor (VEGF). In order to study this further, we followed up 29 patients receiving this treatment, who experienced proteinuria, hypertension, and/or renal insufficiency. Eight developed minimal change nephropathy/focal segmental glomerulopathy (MCN/FSG)-like lesions and 13 developed thrombotic microangiopathy (TMA). Patients receiving receptor tyrosine kinase inhibitors (RTKIs) mainly developed MCN/FSG-like lesions, whereas TMA complicated anti-VEGF therapy. There were no mutations in factor H, factor I, or membrane cofactor protein of the complement alternative pathway, while plasma ADAMTS13 activity persisted and anti-ADAMTS13 antibodies were undetectable in patients with TMA. Glomerular VEGF expression was undetectable in TMA and decreased in MCN/FSG. Glomeruli from patients with TMA displayed a high abundance of RelA in endothelial cells and in the podocyte nuclei, but c-mip was not detected. Conversely, MCN/FSG-like lesions exhibited a high abundance of c-mip, whereas RelA was scarcely detected. RelA binds in vivo to the c-mip promoter and prevents its transcriptional activation, whereas RelA knockdown releases c-mip activation. The RTKI sorafenib inhibited RelA activity, which then promoted c-mip expression. Thus, our results suggest that c-mip and RelA define two distinct types of renal damage associated with VEGF-targeted therapies.
Assuntos
Inibidores da Angiogênese/efeitos adversos , Proteínas de Transporte/metabolismo , Nefropatias/induzido quimicamente , Glomérulos Renais/efeitos dos fármacos , Niacinamida/análogos & derivados , Compostos de Fenilureia/efeitos adversos , Inibidores de Proteínas Quinases/efeitos adversos , Fator de Transcrição RelA/metabolismo , Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Idoso , Animais , Sequência de Bases , Sítios de Ligação , Biomarcadores/metabolismo , Proteínas de Transporte/genética , Estudos de Casos e Controles , Linhagem Celular , Feminino , Regulação da Expressão Gênica , Glomerulosclerose Segmentar e Focal/induzido quimicamente , Glomerulosclerose Segmentar e Focal/diagnóstico , Glomerulosclerose Segmentar e Focal/enzimologia , Humanos , Hipertensão/induzido quimicamente , Hipertensão/diagnóstico , Hipertensão/enzimologia , Nefropatias/diagnóstico , Nefropatias/enzimologia , Glomérulos Renais/enzimologia , Glomérulos Renais/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Dados de Sequência Molecular , Nefrose Lipoide/induzido quimicamente , Nefrose Lipoide/diagnóstico , Nefrose Lipoide/enzimologia , Niacinamida/efeitos adversos , Valor Preditivo dos Testes , Regiões Promotoras Genéticas , Proteinúria/induzido quimicamente , Proteinúria/diagnóstico , Proteinúria/enzimologia , Insuficiência Renal/induzido quimicamente , Insuficiência Renal/diagnóstico , Insuficiência Renal/enzimologia , Sorafenibe , Microangiopatias Trombóticas/induzido quimicamente , Microangiopatias Trombóticas/diagnóstico , Microangiopatias Trombóticas/enzimologia , Fator de Transcrição RelA/deficiência , Fator de Transcrição RelA/genética , Transcrição Gênica , Transfecção , Fatores de Crescimento do Endotélio Vascular/metabolismo , Adulto JovemRESUMO
Membranous nephropathy is a glomerular disease typified by a nephrotic syndrome without infiltration of inflammatory cells or proliferation of resident cells. Although the cause of the disease is unknown, the primary pathology involves the generation of autoantibodies against antigen targets on the surface of podocytes. The mechanisms of nephrotic proteinuria, which reflect a profound podocyte dysfunction, remain unclear. We previously found a new gene, c-mip (c-maf-inducing protein), that was associated with the pathophysiology of idiopathic nephrotic syndrome. Here we found that c-mip was not detected in the glomeruli of rats with passive-type Heymann nephritis given a single dose of anti-megalin polyclonal antibody, yet immune complexes were readily present, but without triggering of proteinuria. Rats reinjected with anti-megalin develop heavy proteinuria a few days later, concomitant with c-mip overproduction in podocytes. This overexpression was associated with the downregulation of synaptopodin in patients with membranous nephropathy, rats with passive Heymann nephritis, and c-mip transgenic mice, while the abundance of death-associated protein kinase and integrin-linked kinase was increased. Cyclosporine treatment significantly reduced proteinuria in rats with passive Heymann nephritis, concomitant with downregulation of c-mip in podocytes. Thus, c-mip has an active role in the podocyte disorders of membranous nephropathy.
Assuntos
Proteínas de Transporte/fisiologia , Glomerulonefrite Membranosa/patologia , Podócitos/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Proteínas Reguladoras de Apoptose/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Ciclosporina/uso terapêutico , Proteínas Quinases Associadas com Morte Celular , Glomerulonefrite Membranosa/tratamento farmacológico , Humanos , Podócitos/patologia , Proteínas Serina-Treonina Quinases/fisiologia , Regulação para CimaRESUMO
The mechanisms of podocyte disorders in cases of idiopathic nephrotic syndrome (INS) are complex and remain incompletely elucidated. The abnormal regulation of NF-κB may play a key role in the pathophysiology of these podocyte diseases, but at present, NF-κB has not been thoroughly investigated. In this study, we report that induction of c-mip in podocytes of patients with INS is associated with a down-regulation of RelA, a potent antiapoptotic factor that belongs to the NF-κB family. Overexpression of c-mip in differentiated podocytes promotes apoptosis by inducing caspase-3 activity and up-regulating the proapoptotic protein Bax, whereas the overall levels of the antiapoptotic protein Bcl-2 was concomitantly decreased. The associated overexpression of RelA prevented the proapoptotic effects of c-mip. In addition, the targeted induction of c-mip in podocytes in vivo inhibited the expression of the RelA protein and increased the Bax/Bcl-2 ratio. The expression of both c-mip and active caspase-3 increased in focal and segmental glomerulosclerosis biopsies, and both proteins displayed a close spatial relationship. These results suggest that alterations in NF-κB activity might result from the up-regulation of c-mip and are likely to contribute to podocyte disorders in cases of INS.
Assuntos
Apoptose/fisiologia , Proteínas de Transporte/fisiologia , NF-kappa B/metabolismo , Síndrome Nefrótica/metabolismo , Podócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Animais , Proteínas de Transporte/biossíntese , Caspase 3/metabolismo , Linhagem Celular , Regulação para Baixo/fisiologia , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Síndrome Nefrótica/patologia , Podócitos/patologia , Fator de Transcrição RelA/biossíntese , Fator de Transcrição RelA/genética , Regulação para Cima/fisiologiaRESUMO
Immune mechanisms underlying the pathophysiology of idiopathic nephrotic syndrome, the most frequent glomerular disease in children, are believed to involve a systemic disorder of T cell function and cell mediated immunity. How these perturbations take place remains unclear. We report here that NFRKB, a member of the chromatin remodeling complex, is upregulated in MCNS relapse, mainly in CD4+T cells and B cells and undergo post-translational modifications including sumoylation. We showed that NFRKB was highly expressed in nuclear compartment during the relapse, while it was restricted to cytoplasm in remission. NFRKB induced the activation of AP1 signaling pathway by upregulating the expression of c-jun. We showed that NFRKB promotes hypomethylation of genomic DNA, suggesting its implication in regulation of gene expression by enhancing the binding of transcription factors through chromatin remodeling. These results suggest for the first time that NFRKB may be involved in the disorders of transcriptional regulation commonly observed in MCNS relapse.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Nefrose Lipoide/genética , Adolescente , Adulto , Clonagem Molecular , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica/fisiologia , Predisposição Genética para Doença , Células HEK293 , Humanos , Células Jurkat , Pessoa de Meia-Idade , Nefrose Lipoide/metabolismo , Nefrose Lipoide/patologia , Processamento de Proteína Pós-Traducional , Recidiva , Transcrição Gênica/genética , Regulação para Cima/genética , Adulto JovemRESUMO
Idiopathic nephrotic syndrome is the most frequent glomerular disease in children. The mechanisms underlying its pathophysiology have been investigated by genetic, cellular and molecular approaches. While genetic analyses have provided new insights into disease pathogenesis through the discovery of several podocyte genes mutated in distinct forms of inherited nephrotic syndrome, the molecular bases of minimal change nephrotic syndrome and focal and segmental glomerulosclerosis with relapse remain unclear. The immune system seems to play a critical role in the active phase of this disease through disturbances involving several cell subsets, mainly T cells. The innate immune system may also contribute to the immune disorders. In this review, we discuss recent insights from the molecular and immunological findings and their significance in the context of the clinical course of the disease.
Assuntos
Nefrose Lipoide/imunologia , Nefrose Lipoide/fisiopatologia , Citocinas/fisiologia , Glomerulosclerose Segmentar e Focal/imunologia , Glomerulosclerose Segmentar e Focal/fisiopatologia , Humanos , Imunidade Inata/fisiologia , Podócitos/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Idiopathic nephrotic syndrome comprises several podocyte diseases of unknown origin that affect the glomerular podocyte, which controls the permeability of the filtration barrier in the kidney to proteins. It is characterized by the daily loss of more than 3 g of protein in urine and the lack of inflammatory lesions or cell infiltration. We found that the abundance of c-mip (c-maf inducing protein) was increased in the podocytes of patients with various acquired idiopathic nephrotic syndromes in which the podocyte is the main target of injury. Mice engineered to have excessive c-mip in podocytes developed proteinuria without morphological alterations, inflammatory lesions, or cell infiltration. Excessive c-mip blocked podocyte signaling by preventing the interaction of the slit diaphragm transmembrane protein nephrin with the tyrosine kinase Fyn, thereby decreasing phosphorylation of nephrin in vitro and in vivo. Moreover, c-mip inhibited interactions between Fyn and the cytoskeletal regulator N-WASP (neural Wiskott-Aldrich syndrome protein) and between the adaptor protein Nck and nephrin, potentially accounting for cytoskeletal disorganization and the effacement of foot processes seen in idiopathic nephrotic syndromes. The intravenous injection of small interfering RNA targeting c-mip prevented lipopolysaccharide-induced proteinuria in mice. Together, these results identify c-mip as a key component in the molecular pathogenesis of acquired podocyte diseases.
Assuntos
Proteínas de Transporte/fisiologia , Podócitos/fisiologia , Proteinúria/fisiopatologia , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Fosforilação , Podócitos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Interferência de RNA , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
It is currently considered that idiopathic minimal change nephrotic syndrome is an immune-mediated glomerular disease. Its association with classical Hodgkin lymphoma minimal change nephrotic syndrome (cHL-MCNS) suggests a molecular link, which remains to be elucidated. We analyzed the expression of cmaf inducing protein (c-mip) in lymphomatous tissues and kidney biopsy samples of patients with cHL-MCNS (n = 8) and in lymphomatous tissues of patients with isolated cHL (n = 9). Because c-mip affects the regulatory loop involving Fyn, we investigated possible structural defects in this signaling pathway, using laser capture microdissection, reverse transcription polymerase chain reaction, and Western blotting. We found that c-mip was selectively expressed in Hodgkin and Reed-Sternberg (HRS) cells and podocytes of patients with cHL-MCNS but is undetectable in patients with isolated cHL. We demonstrated that c-mip was specifically involved in the negative regulation of early proximal signaling through its interaction with phosphoprotein associated with glycosphingolipid-enriched microdomains and Fyn. We showed that the up-regulation of c-mip in cHL-MCNS was associated with a possible Fyn defect in HRS cells and podocytes. Moreover, we showed that c-mip was up-regulated in Fyn-deficient podocytes. c-mip may be a useful marker of cHL-MCNS and its induction reflects the dysregulation of proximal signaling.
Assuntos
Proteínas de Transporte/metabolismo , Doença de Hodgkin/complicações , Nefrose Lipoide/complicações , Podócitos/metabolismo , Células de Reed-Sternberg/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Western Blotting , Proteína Tirosina Quinase CSK , Estudos de Casos e Controles , Células Cultivadas , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Doença de Hodgkin/metabolismo , Humanos , Hibridização In Situ , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Microdissecção , Nefrose Lipoide/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima , Quinases da Família srcRESUMO
In naive T cells, Lck exerts a negative control on the ERK/MAPK pathway. We show that c-mip (c-maf inducing protein) interacts with the p85 subunit of PI3 kinase and inactivates Lck, which results in Erk1/2 and p38 MAPK activation. This effect is not enough to activate AP1 given the inability of ERK to migrate into the nucleus and to transactivate its target genes. We demonstrate that c-mip interacts with Dip1 and upregulates DAPK, which blocks the nuclear translocation of ERK1/2. This dual effect of c-mip is unique and might represent a potential mechanism to prevent the development of an immune response.