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Mutations of the ADAR1 gene encoding an RNA deaminase cause severe diseases associated with chronic activation of type I interferon (IFN) responses, including Aicardi-Goutières syndrome and bilateral striatal necrosis1-3. The IFN-inducible p150 isoform of ADAR1 contains a Zα domain that recognizes RNA with an alternative left-handed double-helix structure, termed Z-RNA4,5. Hemizygous ADAR1 mutations in the Zα domain cause type I IFN-mediated pathologies in humans2,3 and mice6-8; however, it remains unclear how the interaction of ADAR1 with Z-RNA prevents IFN activation. Here we show that Z-DNA-binding protein 1 (ZBP1), the only other protein in mammals known to harbour Zα domains9, promotes type I IFN activation and fatal pathology in mice with impaired ADAR1 function. ZBP1 deficiency or mutation of its Zα domains reduced the expression of IFN-stimulated genes and largely prevented early postnatal lethality in mice with hemizygous expression of ADAR1 with mutated Zα domain (Adar1mZα/- mice). Adar1mZα/- mice showed upregulation and impaired editing of endogenous retroelement-derived complementary RNA reads, which represent a likely source of Z-RNAs activating ZBP1. Notably, ZBP1 promoted IFN activation and severe pathology in Adar1mZα/- mice in a manner independent of RIPK1, RIPK3, MLKL-mediated necroptosis and caspase-8-dependent apoptosis, suggesting a novel mechanism of action. Thus, ADAR1 prevents endogenous Z-RNA-dependent activation of pathogenic type I IFN responses by ZBP1, suggesting that ZBP1 could contribute to type I interferonopathies caused by ADAR1 mutations.
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Adenosina Desaminase , Interferon Tipo I , Proteínas de Ligação a RNA , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Apoptose , Caspase 8/metabolismo , Interferon Tipo I/antagonistas & inibidores , Interferon Tipo I/imunologia , Camundongos , Mutação , Necroptose , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Organ fibrosis is a shared endpoint of many diseases, yet underlying mechanisms are not well understood. Several pathways governed by the primary cilium, a sensory antenna present on most vertebrate cells, have been linked with fibrosis. Ciliopathies usually start early in life and represent a considerable disease burden. We performed massively parallel sequencing by using cohorts of genetically unsolved individuals with unexplained liver and kidney failure and correlated this with clinical, imaging, and histopathological analyses. Mechanistic studies were conducted with a vertebrate model and primary cells. We detected bi-allelic deleterious variants in TULP3, encoding a critical adaptor protein for ciliary trafficking, in a total of 15 mostly adult individuals, originating from eight unrelated families, with progressive degenerative liver fibrosis, fibrocystic kidney disease, and hypertrophic cardiomyopathy with atypical fibrotic patterns on histopathology. We recapitulated the human phenotype in adult zebrafish and confirmed disruption of critical ciliary cargo composition in several primary cell lines derived from affected individuals. Further, we show interaction between TULP3 and the nuclear deacetylase SIRT1, with roles in DNA damage repair and fibrosis, and report increased DNA damage ex vivo. Transcriptomic studies demonstrated upregulation of profibrotic pathways with gene clusters for hypertrophic cardiomyopathy and WNT and TGF-ß signaling. These findings identify variants in TULP3 as a monogenic cause for progressive degenerative disease of major organs in which affected individuals benefit from early detection and improved clinical management. Elucidation of mechanisms crucial for DNA damage repair and tissue maintenance will guide novel therapeutic avenues for this and similar genetic and non-genomic diseases.
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Cardiomiopatia Hipertrófica , Cílios , Adulto , Animais , Cardiomiopatia Hipertrófica/metabolismo , Criança , Cílios/genética , Cílios/metabolismo , Fibrose , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Rim , Fígado , Mutação/genética , Peixe-Zebra/genéticaRESUMO
BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder characterized by a life-threatening cytokine storm and immunopathology. Familial HLH type 3 (FHL3) accounts for approximately 30% of all inborn HLH cases worldwide. It is caused by mutations in the UNC13D gene that result in impaired degranulation of cytotoxic vesicles and hence compromised T-cell- and natural killer-cell-mediated killing. Current treatment protocols, including allogeneic hematopoietic stem cell (HSC) transplantation, still show high mortality. OBJECTIVE: We sought to develop and evaluate a curative genome editing strategy in the preclinical FHL3 Jinx mouse model. Jinx mice harbor a cryptic splice donor site in Unc13d intron 26 and develop clinical symptoms of human FHL3 upon infection with lymphocytic choriomeningitis virus (LCMV). METHODS: We employed clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technology to delete the disease-causing mutation in HSCs and transplanted Unc13d-edited stem cells into busulfan-conditioned Jinx recipient mice. Safety studies included extensive genotyping and chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing (CAST-Seq)-based off-target analyses. Cure from HLH predisposition was assessed by LCMV infection. RESULTS: Hematopoietic cells isolated from transplanted mice revealed efficient gene editing (>95%), polyclonality of the T-cell receptor repertoire, and neither signs of off-target effects nor leukemogenesis. Unc13d transcription levels of edited and wild-type cells were comparable. While LCMV challenge resulted in acute HLH in Jinx mice transplanted with mock-edited HSCs, Jinx mice grafted with Unc13d-edited cells showed rapid virus clearance and protection from HLH. CONCLUSIONS: Our study demonstrates that transplantation of CRISPR-Cas edited HSCs supports the development of a functional polyclonal T-cell response in the absence of genotoxicity-associated clonal outgrowth.
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Linfo-Histiocitose Hemofagocítica , Humanos , Camundongos , Animais , Linfo-Histiocitose Hemofagocítica/genética , Linfo-Histiocitose Hemofagocítica/terapia , Linfo-Histiocitose Hemofagocítica/diagnóstico , Linfócitos T , Edição de Genes , Mutação , Vírus da Coriomeningite Linfocítica , Células-Tronco Hematopoéticas , Proteínas de Membrana/genéticaRESUMO
State-of-the-art mass spectrometers combined with modern bioinformatics algorithms for peptide-to-spectrum matching (PSM) with robust statistical scoring allow for more variable features (i.e., post-translational modifications) being reliably identified from (tandem-) mass spectrometry data, often without the need for biochemical enrichment. Semi-specific proteome searches, that enforce a theoretical enzymatic digestion to solely the N- or C-terminal end, allow to identify of native protein termini or those arising from endogenous proteolytic activity (also referred to as "neo-N-termini" analysis or "N-terminomics"). Nevertheless, deriving biological meaning from these search outputs can be challenging in terms of data mining and analysis. Thus, we introduce TermineR, a data analysis approach for the (1) annotation of peptides according to their enzymatic cleavage specificity and known protein processing features, (2) differential abundance and enrichment analysis of N-terminal sequence patterns, and (3) visualization of neo-N-termini location. We illustrate the use of TermineR by applying it to tandem mass tag (TMT)-based proteomics data of a mouse model of polycystic kidney disease, and assess the semi-specific searches for biological interpretation of cleavage events and the variable contribution of proteolytic products to general protein abundance. The TermineR approach and example data are available as an R package at https://github.com/MiguelCos/TermineR.
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SIGNIFICANCE STATEMENT: Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates and complex morphology, such as podocytes. To improve our understanding on how disturbances of these trafficking pathways are linked to podocyte depletion and slit diaphragm (SD) injury, the authors explored the role of the small GTPase Rab7, which is linked to endosomal, lysosomal, and autophagic pathways, using as model systems mice and Drosophila with podocyte-specific or nephrocyte-specific loss of Rab7, and a human podocyte cell line depleted for Rab7. Their findings point to maturation and fusion events during endolysosomal and autophagic maturation as key processes for podocyte homeostasis and function and identify altered lysosomal pH values as a putative novel mechanism for podocytopathies. BACKGROUND: Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates, such as podocytes. How disturbances within these trafficking pathways may act as factors in proteinuric glomerular diseases is poorly understood. METHODS: To explore how disturbances in trafficking pathways may act as factors in proteinuric glomerular diseases, we focused on Rab7, a highly conserved GTPase that controls the homeostasis of late endolysosomal and autophagic processes. We generated mouse and Drosophila in vivo models lacking Rab7 exclusively in podocytes or nephrocytes, and performed histologic and ultrastructural analyses. To further investigate Rab7 function on lysosomal and autophagic structures, we used immortalized human cell lines depleted for Rab7. RESULTS: Depletion of Rab7 in mice, Drosophila , and immortalized human cell lines resulted in an accumulation of diverse vesicular structures resembling multivesicular bodies, autophagosomes, and autoendolysosomes. Mice lacking Rab7 developed a severe and lethal renal phenotype with early-onset proteinuria and global or focal segmental glomerulosclerosis, accompanied by an altered distribution of slit diaphragm proteins. Remarkably, structures resembling multivesicular bodies began forming within 2 weeks after birth, prior to the glomerular injuries. In Drosophila nephrocytes, Rab7 knockdown resulted in the accumulation of vesicles and reduced slit diaphragms. In vitro , Rab7 knockout led to similar enlarged vesicles and altered lysosomal pH values, accompanied by an accumulation of lysosomal marker proteins. CONCLUSIONS: Disruption within the final common pathway of endocytic and autophagic processes may be a novel and insufficiently understood mechanism regulating podocyte health and disease.
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Glomérulos Renais , Podócitos , Animais , Camundongos , Humanos , Glomérulos Renais/patologia , Podócitos/metabolismo , Endossomos , Drosophila , Rim , MamíferosRESUMO
PURPOSE: About 15% of patients with common variable immunodeficiency (CVID) develop a small intestinal enteropathy, which resembles celiac disease with regard to histopathology but evolves from a distinct, poorly defined pathogenesis that has been linked in some cases to chronic norovirus (NV) infection. Interferon-driven inflammation is a prominent feature of CVID enteropathy, but it remains unknown how NV infection may contribute. METHODS: Duodenal biopsies of CVID patients, stratified according to the presence of villous atrophy (VA), IgA plasma cells (PCs), and chronic NV infection, were investigated by flow cytometry, multi-epitope-ligand cartography, bulk RNA-sequencing, and RT-qPCR of genes of interest. RESULTS: VA development was connected to the lack of intestinal (IgA+) PC, a T helper 1/T helper 17 cell imbalance, and increased recruitment of granzyme+CD8+ T cells and pro-inflammatory macrophages to the affected site. A mixed interferon type I/III and II signature occurred already in the absence of histopathological changes and increased with the severity of the disease and in the absence of (IgA+) PCs. Chronic NV infection exacerbated this signature when compared to stage-matched NV-negative samples. CONCLUSIONS: Our study suggests that increased IFN signaling and T-cell cytotoxicity are present already in mild and are aggravated in severe stages (VA) of CVID enteropathy. NV infection preempts local high IFN-driven inflammation, usually only seen in VA, at milder disease stages. Thus, revealing the impact of different drivers of the pathological mixed IFN type I/III and II signature may allow for more targeted treatment strategies in CVID enteropathy and supports the goal of viral elimination.
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Infecções por Caliciviridae , Imunodeficiência de Variável Comum , Norovirus , Humanos , Atrofia/complicações , Atrofia/patologia , Infecções por Caliciviridae/imunologia , Linfócitos T CD8-Positivos , Imunodeficiência de Variável Comum/complicações , Imunodeficiência de Variável Comum/imunologia , Imunoglobulina A , Inflamação/complicações , Interferons , Norovirus/fisiologiaRESUMO
BACKGROUND & AIMS: Exhaustion of CD8 T cells has been suggested to inform different clinical outcomes in Crohn's disease, but detailed analyses are lacking. This study aimed to identify the role of exhaustion on a single-cell level and identify relevant CD8 T cell populations in Crohn's disease. METHODS: Blood and intestinal tissue from 58 patients with Crohn's disease (active disease or remission) were assessed for CD8 T cell expression of exhaustion markers and their cytokine profile by highly multiplexed flow and mass cytometry. Key disease-associated subsets were sorted and analyzed by RNA sequencing. CD39 inhibition assays were performed in vitro. RESULTS: Activated CD39+ and CD39+PD-1+ CD8 T cell subsets expressing multiple exhaustion markers were enriched at low frequency in active Crohn's disease. Their cytokine production capacity was inversely linked to the Harvey-Bradshaw Index. Subset-level protein and transcriptome profiling revealed co-existence of effector and exhaustion programs in CD39+ and CD39+ PD-1+CD8 T cells, with CD39+ cells likely originating from the intestine. CD39 enzymatic activity controlled T cell cytokine production. Importantly, transcriptional exhaustion signatures were enriched in remission in CD39-expressing subsets with up-regulation of TOX. Subset-level transcriptomics revealed a CD39-related gene module that is associated with the clinical course. CONCLUSIONS: These data showed a role for the exhaustion of peripheral CD39-expressing CD8 T cell subsets in Crohn's disease. Their low frequency illustrated the utility of single-cell cytometry methods for identification of relevant immune populations. Importantly, the link of their exhaustion status to the clinical activity and their specific gene signatures have implications for exhaustion-based personalized medicine approaches.
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Apirase , Linfócitos T CD8-Positivos , Doença de Crohn , Apirase/sangue , Apirase/genética , Apirase/imunologia , Biomarcadores/sangue , Linfócitos T CD8-Positivos/imunologia , Doença de Crohn/sangue , Doença de Crohn/genética , Doença de Crohn/imunologia , Citocinas/imunologia , Humanos , Prognóstico , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Subpopulações de Linfócitos TRESUMO
BACKGROUND: The cell-matrix adhesion between podocytes and the glomerular basement membrane is essential for the integrity of the kidney's filtration barrier. Despite increasing knowledge about the complexity of integrin adhesion complexes, an understanding of the regulation of these protein complexes in glomerular disease remains elusive. METHODS: We mapped the in vivo composition of the podocyte integrin adhesome. In addition, we analyzed conditional knockout mice targeting a gene (Parva) that encodes an actin-binding protein (α-parvin), and murine disease models. To evaluate podocytes in vivo, we used super-resolution microscopy, electron microscopy, multiplex immunofluorescence microscopy, and RNA sequencing. We performed functional analysis of CRISPR/Cas9-generated PARVA single knockout podocytes and PARVA and PARVB double knockout podocytes in three- and two-dimensional cultures using specific extracellular matrix ligands and micropatterns. RESULTS: We found that PARVA is essential to prevent podocyte foot process effacement, detachment from the glomerular basement membrane, and the development of FSGS. Through the use of in vitro and in vivo models, we identified an inherent PARVB-dependent compensatory module at podocyte integrin adhesion complexes, sustaining efficient mechanical linkage at the filtration barrier. Sequential genetic deletion of PARVA and PARVB induces a switch in structure and composition of integrin adhesion complexes. This redistribution of these complexes translates into a loss of the ventral actin cytoskeleton, decreased adhesion capacity, impaired mechanical resistance, and dysfunctional extracellular matrix assembly. CONCLUSIONS: The findings reveal adaptive mechanisms of podocyte integrin adhesion complexes, providing a conceptual framework for therapeutic strategies to prevent podocyte detachment in glomerular disease.
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Barreira de Filtração Glomerular , Proteínas dos Microfilamentos , Podócitos , Animais , Barreira de Filtração Glomerular/metabolismo , Integrinas/metabolismo , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Podócitos/metabolismoRESUMO
BACKGROUND: Previous research demonstrated that small Rho GTPases, modulators of the actin cytoskeleton, are drivers of podocyte foot-process effacement in glomerular diseases, such as FSGS. However, a comprehensive understanding of the regulatory networks of small Rho GTPases in podocytes is lacking. METHODS: We conducted an analysis of podocyte transcriptome and proteome datasets for Rho GTPases; mapped in vivo, podocyte-specific Rho GTPase affinity networks; and examined conditional knockout mice and murine disease models targeting Srgap1. To evaluate podocyte foot-process morphology, we used super-resolution microscopy and electron microscopy; in situ proximity ligation assays were used to determine the subcellular localization of the small GTPase-activating protein SRGAP1. We performed functional analysis of CRISPR/Cas9-generated SRGAP1 knockout podocytes in two-dimensional and three-dimensional cultures and quantitative interaction proteomics. RESULTS: We demonstrated SRGAP1 localization to podocyte foot processes in vivo and to cellular protrusions in vitro. Srgap1fl/fl*Six2Cre but not Srgap1fl/fl*hNPHS2Cre knockout mice developed an FSGS-like phenotype at adulthood. Podocyte-specific deletion of Srgap1 by hNPHS2Cre resulted in increased susceptibility to doxorubicin-induced nephropathy. Detailed analysis demonstrated significant effacement of podocyte foot processes. Furthermore, SRGAP1-knockout podocytes showed excessive protrusion formation and disinhibition of the small Rho GTPase machinery in vitro. Evaluation of a SRGAP1-dependent interactome revealed the involvement of SRGAP1 with protrusive and contractile actin networks. Analysis of glomerular biopsy specimens translated these findings toward human disease by displaying a pronounced redistribution of SRGAP1 in FSGS. CONCLUSIONS: SRGAP1, a podocyte-specific RhoGAP, controls podocyte foot-process architecture by limiting the activity of protrusive, branched actin networks. Therefore, elucidating the complex regulatory small Rho GTPase affinity network points to novel targets for potentially precise intervention in glomerular diseases.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Podócitos/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Actomiosina/metabolismo , Animais , Extensões da Superfície Celular/metabolismo , Extensões da Superfície Celular/ultraestrutura , Células Cultivadas , Modelos Animais de Doenças , Feminino , Proteínas Ativadoras de GTPase/deficiência , Proteínas Ativadoras de GTPase/genética , Glomerulosclerose Segmentar e Focal/etiologia , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Integrinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Síndrome Nefrótica/etiologia , Síndrome Nefrótica/metabolismo , Síndrome Nefrótica/patologia , Podócitos/ultraestrutura , Mapeamento de Interação de Proteínas , Proteoma , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , TranscriptomaRESUMO
Vascular endothelial growth factor A (VEGFA) secretion from podocytes is crucial for maintaining endothelial integrity within the glomerular filtration barrier. However, until now, the molecular mechanisms underlying podocyte secretory function remained unclear. Through podocyte-specific deletion of BECLIN1 (ATG6 or Becn1), a key protein in autophagy initiation, we identified a major role for this molecule in anterograde Golgi trafficking. The Becn1-deficient podocytes displayed aberrant vesicle formation in the trans-Golgi network (TGN), leading to dramatic vesicle accumulation and complex disrupted patterns of intracellular vesicle trafficking and membrane dynamics. Phenotypically, podocyte-specific deletion of Becn1 resulted in early-onset glomerulosclerosis, which rapidly progressed and dramatically reduced mouse life span. Further, in vivo and in vitro studies clearly showed that VEGFA secretion, and thereby endothelial integrity, greatly depended on BECLIN1 availability and function. Being the first to demonstrate the importance of a secretory pathway for podocyte integrity and function, we identified BECLIN1 as a key component in this complex cellular process. Functionally, by promoting VEGFA secretion, a specific secretory pathway emerged as an essential component for the podocyte-endothelial crosstalk that maintains the glomerular filtration barrier.
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Podócitos , Animais , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Barreira de Filtração Glomerular/metabolismo , Camundongos , Podócitos/metabolismo , Via Secretória , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
INTRODUCTION AND OBJECTIVE: Thermal injuries associated with Holmium laser lithotripsy of the urinary tract are an underestimated problem in stone therapy. Surgical precision relies exclusively on visual target identification when applying laser energy for stone disintegration. This study evaluates a laser system that enables target identification automatically during bladder stone lithotripsy, URS, and PCNL in a porcine animal model. METHODS: Holmium laser lithotripsy was performed on two domestic pigs by an experienced endourology surgeon in vivo. Human stone fragments (4-6 mm) were inserted in both ureters, renal pelvises, and bladders. Ho:YAG laser lithotripsy was conducted as a two-arm comparison study, evaluating the target identification system against common lithotripsy. We assessed the ureters' lesions according to PULS and the other locations descriptively. Post-mortem nephroureterectomy and cystectomy specimens were examined by a pathologist. RESULTS: The sufficient disintegration of stone samples was achieved in both setups. Endoscopic examination revealed numerous lesions in the urinary tract after the commercial Holmium laser system. The extent of lesions with the feedback system was semi-quantitatively and qualitatively lower. The energy applied was significantly less, with a mean reduction of more than 30% (URS 27.1%, PCNL 52.2%, bladder stone lithotripsy 17.1%). Pathology examination revealed only superficial lesions in both animals. There was no evidence of organ perforation in either study arm. CONCLUSIONS: Our study provides proof-of-concept for a laser system enabling automatic real-time target identification during lithotripsy on human urinary stones. Further studies in humans are necessary, and to objectively quantify this new system's advantages, investigations involving a large number of cases are mandatory.
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Litotripsia a Laser/métodos , Ureteroscopia , Cálculos da Bexiga Urinária/terapia , Animais , Feminino , SuínosRESUMO
Podocyte maintenance and stress resistance are exquisitely based on high basal rates of autophagy making these cells a unique model to unravel mechanisms of autophagy regulation. Polyamines have key cellular functions such as proliferation, nucleic acid biosynthesis and autophagy. Here we test whether endogenous spermidine signaling is a driver of basal and dynamic autophagy in podocytes by using genetic and pharmacologic approaches to interfere with different steps of polyamine metabolism. Translational studies revealed altered spermidine signaling in focal segmental glomerulosclerosis in vivo and in vitro. Exogenous spermidine supplementation emerged as new treatment strategy by successfully activating autophagy in vivo via inhibition of EP300, a protein with an essential role in controlling cell growth, cell division and prompting cells to differentiate to take on specialized functions. Surprisingly, gas chromatography-mass spectroscopy based untargeted metabolomics of wild type and autophagy deficient primary podocytes revealed a positive feedback mechanism whereby autophagy itself maintains polyamine metabolism and spermidine synthesis. The transcription factor MAFB acted as an upstream regulator of polyamine metabolism. Thus, our data highlight a novel positive feedback loop of autophagy and spermidine signaling allowing maintenance of high basal levels of autophagy as a key mechanism to sustain the filtration barrier. Hence, spermidine supplementation may emerge as a new therapeutic to restore autophagy in glomerular disease.
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Glomerulosclerose Segmentar e Focal , Podócitos , Espermidina , Autofagia , Proliferação de Células , Humanos , Espermidina/metabolismoRESUMO
Podocytes form the outer part of the glomerular filter, where they have to withstand enormous transcapillary filtration forces driving glomerular filtration. Detachment of podocytes from the glomerular basement membrane precedes most glomerular diseases. However, little is known about the regulation of podocyte adhesion in vivo. Thus, we systematically screened for podocyte-specific focal adhesome (FA) components, using genetic reporter models in combination with iTRAQ-based mass spectrometry. This approach led to the identification of FERM domain protein EPB41L5 as a highly enriched podocyte-specific FA component in vivo. Genetic deletion of Epb41l5 resulted in severe proteinuria, detachment of podocytes, and development of focal segmental glomerulosclerosis. Remarkably, by binding and recruiting the RhoGEF ARGHEF18 to the leading edge, EPB41L5 directly controls actomyosin contractility and subsequent maturation of focal adhesions, cell spreading, and migration. Furthermore, EPB41L5 controls matrix-dependent outside-in signaling by regulating the focal adhesome composition. Thus, by linking extracellular matrix sensing and signaling, focal adhesion maturation, and actomyosin activation EPB41L5 ensures the mechanical stability required for podocytes at the kidney filtration barrier. Finally, a diminution of EPB41L5-dependent signaling programs appears to be a common theme of podocyte disease, and therefore offers unexpected interventional therapeutic strategies to prevent podocyte loss and kidney disease progression.
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Actomiosina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Adesões Focais/metabolismo , Proteínas de Membrana/metabolismo , Podócitos/metabolismo , Animais , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Feminino , Adesões Focais/patologia , Técnicas de Inativação de Genes , Glomerulosclerose Segmentar e Focal/etiologia , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Síndrome Nefrótica/etiologia , Síndrome Nefrótica/metabolismo , Síndrome Nefrótica/patologia , Podócitos/patologia , Gravidez , Proteômica , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Renal oncocytomas (ROs) are benign epithelial tumors of the kidney whereas chromophobe renal cell carcinoma (chRCCs) are malignant renal tumors. The latter constitute 5-7% of renal neoplasias. ROs and chRCCs show pronounced molecular and histological similarities, which renders their differentiation demanding. We aimed for the differential proteome profiling of ROs and early-stage chRCCs in order to better understand distinguishing protein patterns. METHODS: We employed formalin-fixed, paraffin-embedded samples (six RO cases, six chRCC cases) together with isotopic triplex dimethylation and a pooled reference standard to enable cohort-wide quantitative comparison. For lysosomal-associated membrane protein 1 (LAMP1) and integrin alpha-V (ITGAV) we performed corroborative immunohistochemistry (IHC) in an extended cohort of 42 RO cases and 31 chRCC cases. RESULTS: At 1% false discovery rate, we identified > 3900 proteins, of which > 2400 proteins were consistently quantified in at least four RO and four chRCC cases. The proteomic expression profiling discriminated ROs and chRCCs and highlighted established features such as accumulation of mitochondrial proteins in ROs together with emphasizing the accumulation of endo-lysosomal proteins in chRCCs. In line with the proteomic data, IHC showed enrichment of LAMP1 in chRCC and of ITGAV in RO. CONCLUSION: We present one of the first differential proteome profiling studies on ROs and chRCCs and highlight differential abundance of LAMP1 and ITGAV in these renal tumors.
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Podocytes exhibit a unique cytoskeletal architecture that is fundamentally linked to their function in maintaining the kidney filtration barrier. The cytoskeleton regulates podocyte shape, structure, stability, slit diaphragm insertion, adhesion, plasticity, and dynamic response to environmental stimuli. Genetic mutations demonstrate that even slight impairment of the podocyte cytoskeletal apparatus results in proteinuria and glomerular disease. Moreover, mechanisms underpinning all acquired glomerular pathologies converge on disruption of the cytoskeleton, suggesting that this subcellular structure could be targeted for therapeutic purposes. This review summarizes our current understanding of the function of the cytoskeleton in podocytes and the associated implications for pathophysiology.
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Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , Podócitos/fisiologia , Citoesqueleto/genética , Feminino , Humanos , MasculinoRESUMO
The mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin and its analogs are being increasingly used in solid-organ transplantation. A commonly reported side effect is male subfertility to infertility, yet the precise mechanisms of mTOR interference with male fertility remain obscure. With the use of a conditional mouse genetic approach we demonstrate that deficiency of mTORC1 in the epithelial derivatives of the Wolffian duct is sufficient to cause male infertility. Analysis of spermatozoa from Raptor fl/fl*KspCre mice revealed an overall decreased motility pattern. Both epididymis and seminal vesicles displayed extensive organ regression with increasing age. Histologic and ultrastructural analyses demonstrated increased amounts of destroyed and absorbed spermatozoa in different segments of the epididymis. Mechanistically, genetic and pharmacologic mTORC1 inhibition was associated with an impaired cellular metabolism and a disturbed protein secretion of epididymal epithelial cells. Collectively, our data highlight the role of mTORC1 to preserve the function of the epididymis, ductus deferens, and the seminal vesicles. We thus reveal unexpected new insights into the frequently observed mTORC1 inhibitor side effect of male infertility in transplant recipients.
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Proliferação de Células/efeitos dos fármacos , Fertilidade/efeitos dos fármacos , Complexos Multiproteicos/efeitos dos fármacos , Glândulas Seminais/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/efeitos dos fármacos , Animais , Masculino , Mamíferos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos Transgênicos , Fosforilação , Glândulas Seminais/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Congenital anomalies of the kidney and urinary tract (CAKUT) account for 40-50% of chronic kidney disease that manifests in the first two decades of life. Thus far, 31 monogenic causes of isolated CAKUT have been described, explaining ~12% of cases. To identify additional CAKUT-causing genes, we performed whole-exome sequencing followed by a genetic burden analysis in 26 genetically unsolved families with CAKUT. We identified two heterozygous mutations in SRGAP1 in 2 unrelated families. SRGAP1 is a small GTPase-activating protein in the SLIT2-ROBO2 signaling pathway, which is essential for development of the metanephric kidney. We then examined the pathway-derived candidate gene SLIT2 for mutations in cohort of 749 individuals with CAKUT and we identified 3 unrelated individuals with heterozygous mutations. The clinical phenotypes of individuals with mutations in SLIT2 or SRGAP1 were cystic dysplastic kidneys, unilateral renal agenesis, and duplicated collecting system. We show that SRGAP1 is expressed in early mouse nephrogenic mesenchyme and that it is coexpressed with ROBO2 in SIX2-positive nephron progenitor cells of the cap mesenchyme in developing rat kidney. We demonstrate that the newly identified mutations in SRGAP1 lead to an augmented inhibition of RAC1 in cultured human embryonic kidney cells and that the SLIT2 mutations compromise the ability of the SLIT2 ligand to inhibit cell migration. Thus, we report on two novel candidate genes for causing monogenic isolated CAKUT in humans.
Assuntos
Proteínas Ativadoras de GTPase , Peptídeos e Proteínas de Sinalização Intercelular , Mutação , Proteínas do Tecido Nervoso , Receptores Imunológicos , Transdução de Sinais/genética , Anormalidades Urogenitais , Refluxo Vesicoureteral , Animais , Exoma , Proteínas Ativadoras de GTPase/biossíntese , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mesoderma/metabolismo , Camundongos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/metabolismo , Ratos , Receptores Imunológicos/biossíntese , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Fatores de Risco , Anormalidades Urogenitais/embriologia , Anormalidades Urogenitais/genética , Refluxo Vesicoureteral/embriologia , Refluxo Vesicoureteral/genéticaRESUMO
Precise positioning of the highly complex interdigitating podocyte foot processes is critical to form the normal glomerular filtration barrier, but the molecular programs driving this process are unknown. The protein atypical protein kinase C (aPKC)--a component of the Par complex, which localizes to tight junctions and interacts with slit diaphragm proteins--may play a role. Here, we found that the combined deletion of the aPKCλ/ι and aPKCζ isoforms in podocytes associated with incorrectly positioned centrosomes and Golgi apparatus and mislocalized molecules of the slit diaphragm. Furthermore, aPKC-deficient podocytes failed to form the normal network of foot processes, leading to defective glomerular maturation with incomplete capillary formation and mesangiolysis. Our results suggest that aPKC isoforms orchestrate the formation of the podocyte processes essential for normal glomerular development and kidney function. Defective aPKC signaling results in a dramatically simplified glomerular architecture, causing severe proteinuria and perinatal death.
Assuntos
Isoenzimas/metabolismo , Podócitos/enzimologia , Podócitos/patologia , Proteína Quinase C/metabolismo , Proteinúria/fisiopatologia , Animais , Capilares/metabolismo , Capilares/patologia , Capilares/ultraestrutura , Diferenciação Celular/fisiologia , Centrossomo/enzimologia , Centrossomo/patologia , Centrossomo/ultraestrutura , Feminino , Complexo de Golgi/enzimologia , Complexo de Golgi/patologia , Complexo de Golgi/ultraestrutura , Isoenzimas/genética , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/citologia , Glomérulos Renais/enzimologia , Masculino , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Podócitos/ultraestrutura , Proteína Quinase C/genética , Proteinúria/metabolismo , Proteinúria/patologia , Transdução de Sinais/fisiologia , Junções Íntimas/enzimologia , Junções Íntimas/patologia , Junções Íntimas/ultraestruturaRESUMO
Alteration of cortical actin structures is the common final pathway leading to podocyte foot process effacement and proteinuria. The molecular mechanisms that safeguard podocyte foot process architecture and maintain the three-dimensional actin network remain elusive. Here, we demonstrate that neuronal Wiskott-Aldrich syndrome protein (N-WASP), which promotes actin nucleation, is required to stabilize podocyte foot processes. Mice lacking N-WASP specifically in podocytes were born with normal kidney function but developed significant proteinuria 3 weeks after birth, suggesting an important role for N-WASP in maintaining foot processes. In addition, inducing deletion of N-WASP in adult mice resulted in severe proteinuria and kidney failure. Electron microscopy showed an accumulation of electron-dense patches of actin and strikingly altered morphology of podocyte foot processes. Although basic actin-based processes such as cell migration were not affected, primary cultures of N-WASP-deficient podocytes revealed significant impairment of dynamic actin reorganization events, including the formation of circular dorsal ruffles. Taken together, our findings suggest that N-WASP-mediated actin nucleation of branched microfilament networks is specifically required for the maintenance of foot processes, presumably sustaining the mechanical resistance of the filtration barrier.
Assuntos
Podócitos/fisiologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/fisiologia , Citoesqueleto de Actina/química , Actinas/química , Animais , Células Cultivadas , Camundongos , Camundongos Knockout , Podócitos/química , Podócitos/ultraestrutura , Proteína Neuronal da Síndrome de Wiskott-Aldrich/análiseRESUMO
The molecular mechanisms that maintain podocytes and consequently, the integrity of the glomerular filtration barrier are incompletely understood. Here, we show that the class III phosphoinositide 3-kinase vacuolar protein sorting 34 (Vps34) plays a central role in modulating endocytic pathways, maintaining podocyte homeostasis. In mice, podocyte-specific conditional knockout of Vps34 led to early proteinuria, glomerular scarring, and death within 3-9 weeks of age. Vps34-deficient podocytes exhibited substantial vacuolization and foot process effacement. Although the formation of autophagosomes and autophagic flux were impaired, comparisons between podocyte-specific Vps34-deficient mice, autophagy-deficient mice, and doubly deficient mice suggested that defective autophagy was not primarily responsible for the severe phenotype caused by the loss of Vps34. In fact, Rab5-positive endosomal compartments, endocytosis, and fluid-phase uptake were severely disrupted in Vps34-deficient podocytes. Vps34 deficiency in nephrocytes, the podocyte-like cells of Drosophila melanogaster, resulted in a block between Rab5- and Rab7-positive endosomal compartments. In summary, these data identify Vps34 as a major regulator of endolysosomal pathways in podocytes and underline the fundamental roles of endocytosis and fluid-phase uptake for the maintenance of the glomerular filtration barrier.