RESUMEN
BACKGROUND: Minimal change disease and primary focal segmental glomerulosclerosis in adults, along with idiopathic nephrotic syndrome in children, are immune-mediated podocytopathies that lead to nephrotic syndrome. Autoantibodies targeting nephrin have been found in patients with minimal change disease, but their clinical and pathophysiological roles are unclear. METHODS: We conducted a multicenter study to analyze antinephrin autoantibodies in adults with glomerular diseases, including minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, IgA nephropathy, antineutrophil cytoplasmic antibody-associated glomerulonephritis, and lupus nephritis, as well as in children with idiopathic nephrotic syndrome and in controls. We also created an experimental mouse model through active immunization with recombinant murine nephrin. RESULTS: The study included 539 patients (357 adults and 182 children) and 117 controls. Among the adults, antinephrin autoantibodies were found in 46 of the 105 patients (44%) with minimal change disease, 7 of 74 (9%) with primary focal segmental glomerulosclerosis, and only in rare cases among the patients with other conditions. Of the 182 children with idiopathic nephrotic syndrome, 94 (52%) had detectable antinephrin autoantibodies. In the subgroup of patients with active minimal change disease or idiopathic nephrotic syndrome who were not receiving immunosuppressive treatment, the prevalence of antinephrin autoantibodies was as high as 69% and 90%, respectively. At study inclusion and during follow-up, antinephrin autoantibody levels were correlated with disease activity. Experimental immunization induced a nephrotic syndrome, a minimal change disease-like phenotype, IgG localization to the podocyte slit diaphragm, nephrin phosphorylation, and severe cytoskeletal changes in mice. CONCLUSIONS: In this study, circulating antinephrin autoantibodies were common in patients with minimal change disease or idiopathic nephrotic syndrome and appeared to be markers of disease activity. Their binding at the slit diaphragm induced podocyte dysfunction and nephrotic syndrome, which highlights their pathophysiological significance. (Funded by Deutsche Forschungsgemeinschaft and others.).
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Autoanticuerpos , Proteínas de la Membrana , Síndrome Nefrótico , Podocitos , Proteínas de la Membrana/inmunología , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Humanos , Animales , Ratones , Niño , Podocitos/inmunología , Adulto , Síndrome Nefrótico/inmunología , Masculino , Femenino , Persona de Mediana Edad , Modelos Animales de Enfermedad , Adolescente , Nefrosis Lipoidea/inmunología , Preescolar , Glomeruloesclerosis Focal y Segmentaria/inmunología , Adulto Joven , AncianoRESUMEN
The mechanistic target of rapamycin (mTOR) forms two distinct intracellular multiprotein complexes that control a multitude of intracellular processes linked to metabolism, proliferation, actin cytoskeleton, and survival. Recent studies have identified the importance of these complexes for transport regulation of ions and nutrients along the entire nephron. First reports could link altered activity of these complexes to certain disease entities, i.e. diabetic nephropathy, acute kidney injury or hyperkalemia.
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Sirolimus , Serina-Treonina Quinasas TOR , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Complejos MultiproteicosRESUMEN
Polycystic kidney disease (PKD) and other renal ciliopathies are characterized by cysts, inflammation, and fibrosis. Cilia function as signaling centers, but a molecular link to inflammation in the kidney has not been established. Here, we show that cilia in renal epithelia activate chemokine signaling to recruit inflammatory cells. We identify a complex of the ciliary kinase LKB1 and several ciliopathy-related proteins including NPHP1 and PKD1. At homeostasis, this ciliary module suppresses expression of the chemokine CCL2 in tubular epithelial cells. Deletion of LKB1 or PKD1 in mouse renal tubules elevates CCL2 expression in a cell-autonomous manner and results in peritubular accumulation of CCR2+ mononuclear phagocytes, promoting a ciliopathy phenotype. Our findings establish an epithelial organelle, the cilium, as a gatekeeper of tissue immune cell numbers. This represents an unexpected disease mechanism for renal ciliopathies and establishes a new model for how epithelial cells regulate immune cells to affect tissue homeostasis.
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Quimiocina CCL2/metabolismo , Cilios/patología , Enfermedades Renales Quísticas/congénito , Riñón Poliquístico Autosómico Dominante/patología , Proteína Quinasa C/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Quinasas Activadas por AMP , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Proteínas del Citoesqueleto , Perros , Células Epiteliales/metabolismo , Femenino , Células HEK293 , Humanos , Enfermedades Renales Quísticas/patología , Túbulos Renales/citología , Túbulos Renales/patología , Macrófagos/metabolismo , Células de Riñón Canino Madin Darby , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis/fisiología , Riñón Poliquístico Autosómico Dominante/genética , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Pez CebraRESUMEN
Anemia is a common complication of chronic kidney disease, affecting the quality of life of patients. Among various factors, such as iron and erythropoietin deficiency, reduced red blood cell (RBC) lifespan has been implicated in the pathogenesis of anemia. However, mechanistic data on in vivo RBC dysfunction in kidney disease are lacking. Herein, we describe the development of chronic kidney disease-associated anemia in mice with proteinuric kidney disease resulting from either administration of doxorubicin or an inducible podocin deficiency. In both experimental models, anemia manifested at day 10 and progressed at day 30 despite increased circulating erythropoietin levels and erythropoiesis in the bone marrow and spleen. Circulating RBCs in both mouse models displayed altered morphology and diminished osmotic-sensitive deformability together with increased phosphatidylserine externalization on the outer plasma membrane, a hallmark of RBC death. Fluorescence-labelling of RBCs at day 20 of mice with doxorubicin-induced kidney disease revealed premature clearance from the circulation. Metabolomic analyses of RBCs from both mouse models demonstrated temporal changes in redox recycling pathways and Lands' cycle, a membrane lipid remodeling process. Anemic patients with proteinuric kidney disease had an increased proportion of circulating phosphatidylserine-positive RBCs. Thus, our observations suggest that reduced RBC lifespan, mediated by altered RBC metabolism, reduced RBC deformability, and enhanced cell death contribute to the development of anemia in proteinuric kidney disease.
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Anemia , Insuficiencia Renal Crónica , Anemia/inducido químicamente , Animales , Eritrocitos , Humanos , Longevidad , Ratones , Calidad de Vida , Insuficiencia Renal Crónica/complicacionesRESUMEN
Crescentic glomerulonephritis represents a group of kidney diseases characterized by rapid loss of kidney function and the formation of glomerular crescents. While the role of the immune system has been extensively studied in relation to the development of crescents, recent findings show that parietal epithelial cells play a key role in the pathophysiology of crescent formation, even in the absence of immune modulation. This review highlights our current understanding of parietal epithelial cell biology and the reported physiological and pathological roles that these cells play in glomerular lesion formation, especially in the context of crescentic glomerulonephritis.
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Células Epiteliales/patología , Glomerulonefritis/patología , Glomérulos Renales/patología , Animales , HumanosRESUMEN
End-stage renal disease is a major global health burden. The only definitive treatment existing is renal transplantation. Worldwide, the demand for donated kidneys by far exceeds the supply. A novel technique for organ preservation, normothermic machine perfusion (NMP), now promises to increase the potential pool of available organs by extending the spectrum of donors and reducing the incidence of graft failure. First studies in humans and large animals are being performed with promising results, but refinement of the technique, buffer, and machines involved is labor-intensive and expensive. To our knowledge, this is the first report of a small animal model of NMP and subsequent transplantation.
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Trasplante de Riñón/métodos , Modelos Animales , Preservación de Órganos/métodos , Perfusión/instrumentación , Recolección de Tejidos y Órganos/métodos , Animales , Humanos , Riñón/cirugía , Fallo Renal Crónico/cirugía , Nefrectomía/métodos , Preservación de Órganos/instrumentación , Ratas , Ratas Sprague-Dawley , Proyectos de Investigación , Recolección de Tejidos y Órganos/instrumentaciónRESUMEN
BACKGROUND: Understanding podocyte-specific responses to injury at a systems level is difficult because injury leads to podocyte loss or an increase of extracellular matrix, altering glomerular cellular composition. Finding a window into early podocyte injury might help identify molecular pathways involved in the podocyte stress response. METHODS: We developed an approach to apply proteome analysis to very small samples of purified podocyte fractions. To examine podocytes in early disease states in FSGS mouse models, we used podocyte fractions isolated from individual mice after chemical induction of glomerular disease (with Doxorubicin or LPS). We also applied single-glomerular proteome analysis to tissue from patients with FSGS. RESULTS: Transcriptome and proteome analysis of glomeruli from patients with FSGS revealed an underrepresentation of podocyte-specific genes and proteins in late-stage disease. Proteome analysis of purified podocyte fractions from FSGS mouse models showed an early stress response that includes perturbations of metabolic, mechanical, and proteostasis proteins. Additional analysis revealed a high correlation between the amount of proteinuria and expression levels of the mechanosensor protein Filamin-B. Increased expression of Filamin-B in podocytes in biopsy samples from patients with FSGS, in single glomeruli from proteinuric rats, and in podocytes undergoing mechanical stress suggests that this protein has a role in detrimental stress responses. In Drosophila, nephrocytes with reduced filamin homolog Cher displayed altered filtration capacity, but exhibited no change in slit diaphragm structure. CONCLUSIONS: We identified conserved mechanisms of the podocyte stress response through ultrasensitive proteome analysis of human glomerular FSGS tissue and purified native mouse podocytes during early disease stages. This approach enables systematic comparisons of large-scale proteomics data and phenotype-to-protein correlation.
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Filaminas/genética , Regulación de la Expresión Génica , Glomeruloesclerosis Focal y Segmentaria/patología , Proteómica/métodos , Estrés Fisiológico/genética , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Glomeruloesclerosis Focal y Segmentaria/genética , Humanos , Ratones , Podocitos/metabolismo , Proteinuria/genética , Proteinuria/fisiopatología , Distribución Aleatoria , RatasRESUMEN
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 del Citoesqueleto/metabolismo , Adhesiones Focales/metabolismo , Proteínas de la Membrana/metabolismo , Podocitos/metabolismo , Animales , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/genética , Femenino , Adhesiones Focales/patología , Técnicas de Inactivación de Genes , Glomeruloesclerosis Focal y Segmentaria/etiología , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomeruloesclerosis Focal y Segmentaria/patología , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Síndrome Nefrótico/etiología , Síndrome Nefrótico/metabolismo , Síndrome Nefrótico/patología , Podocitos/patología , Embarazo , Proteómica , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Nephron number is a major determinant of long-term renal function and cardiovascular risk. Observational studies suggest that maternal nutritional and metabolic factors during gestation contribute to the high variability of nephron endowment. However, the underlying molecular mechanisms have been unclear. METHODS: We used mouse models, including DNA methyltransferase (Dnmt1, Dnmt3a, and Dnmt3b) knockout mice, optical projection tomography, three-dimensional reconstructions of the nephrogenic niche, and transcriptome and DNA methylation analysis to characterize the role of DNA methylation for kidney development. RESULTS: We demonstrate that DNA hypomethylation is a key feature of nutritional kidney growth restriction in vitro and in vivo, and that DNA methyltransferases Dnmt1 and Dnmt3a are highly enriched in the nephrogenic zone of the developing kidneys. Deletion of Dnmt1 in nephron progenitor cells (in contrast to deletion of Dnmt3a or Dnm3b) mimics nutritional models of kidney growth restriction and results in a substantial reduction of nephron number as well as renal hypoplasia at birth. In Dnmt1-deficient mice, optical projection tomography and three-dimensional reconstructions uncovered a significant reduction of stem cell niches and progenitor cells. RNA sequencing analysis revealed that global DNA hypomethylation interferes in the progenitor cell regulatory network, leading to downregulation of genes crucial for initiation of nephrogenesis, Wt1 and its target Wnt4. Derepression of germline genes, protocadherins, Rhox genes, and endogenous retroviral elements resulted in the upregulation of IFN targets and inhibitors of cell cycle progression. CONCLUSIONS: These findings establish DNA methylation as a key regulatory event of prenatal renal programming, which possibly represents a fundamental link between maternal nutritional factors during gestation and reduced nephron number.
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ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Riñón/embriología , Organogénesis/genética , Células Madre/citología , Animales , Diferenciación Celular/genética , Células Cultivadas , Metilación de ADN , Regulación del Desarrollo de la Expresión Génica/genética , Inmunohistoquímica , Ratones , Ratones Noqueados , Nefronas/citología , Nefronas/fisiología , Ratas , Ratas Wistar , Sensibilidad y Especificidad , Células Madre/fisiología , ADN Metiltransferasa 3BRESUMEN
In immune-mediated glomerular diseases like crescentic glomerulonephritis (cGN), inflammatory CD4+ T cells accumulate within the tubulointerstitial compartment in close contact to proximal and distal tubular epithelial cells and drive renal inflammation and tissue damage. However, whether renal epithelial cell populations play a role in the pathogenesis of cGN by modulating CD4+ T cell responses is less clear. In the present study, we aimed to investigate the potential of renal epithelial cells to function as antigen-presenting cells, thereby stimulating CD4+ T cell responses. Using a FACS-based protocol that allowed comparative analysis of cortical epithelial cell populations, we showed that particularly proximal tubular epithelial cells (PTECs) express molecules linked with antigen-presenting cell function, including major histocompatibility complex class II (MHCII), CD74, CD80, and CD86 in homeostasis and nephrotoxic nephritis, a murine model of cGN. Protein expression was visualized at the PTEC single cell level by imaging flow cytometry. Interestingly, we found inflammation-dependent regulation of epithelium-expressed CD74, CD80, and CD86, whereas MHCII expression was not altered. Antigen-specific stimulation of CD4+ T cells by PTECs in vitro supported CD4+ T cell survival and induced CD4+ T cell activation, proliferation, and inflammatory cytokine production. In patients with antineutrophil cytoplasmic antibody-associated glomerulonephritis, MHCII and CD74 were expressed by both proximal and distal tubules, whereas CD86 was predominantly expressed by proximal tubules. Thus, particularly PTECs have the potential to induce an inflammatory phenotype in CD4+ T cells in vitro, which might also play a role in the pathology of immune-mediated kidney disease.
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Células Presentadoras de Antígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Células Epiteliales/inmunología , Glomerulonefritis/inmunología , Túbulos Renales Proximales/inmunología , Activación de Linfocitos , Comunicación Paracrina , Animales , Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Anticuerpos Anticitoplasma de Neutrófilos/metabolismo , Células Presentadoras de Antígenos/metabolismo , Células Presentadoras de Antígenos/patología , Antígenos de Diferenciación de Linfocitos B/inmunología , Antígenos de Diferenciación de Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Glomerulonefritis/metabolismo , Glomerulonefritis/patología , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Masculino , Ratones Endogámicos C57BL , Fenotipo , Transducción de SeñalAsunto(s)
Adenina/análogos & derivados , Glomeruloesclerosis Focal y Segmentaria , Péptidos y Proteínas de Señalización Intracelular , Leucemia Linfocítica Crónica de Células B , Proteínas de la Membrana , Síndrome Nefrótico , Piperidinas , Humanos , Glomeruloesclerosis Focal y Segmentaria/inducido químicamente , Glomeruloesclerosis Focal y Segmentaria/tratamiento farmacológico , Glomeruloesclerosis Focal y Segmentaria/genética , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Mutación , Síndrome Nefrótico/genéticaRESUMEN
Podocyte injury is an early event in diabetic kidney disease and is a hallmark of glomerulopathy. MicroRNA-146a (miR-146a) is highly expressed in many cell types under homeostatic conditions, and plays an important anti-inflammatory role in myeloid cells. However, its role in podocytes is unclear. Here, we show that miR-146a expression levels decrease in the glomeruli of patients with type 2 diabetes (T2D), which correlates with increased albuminuria and glomerular damage. miR-146a levels are also significantly reduced in the glomeruli of albuminuric BTBR ob/ob mice, indicating its significant role in maintaining podocyte health. miR-146a-deficient mice (miR-146a-/-) showed accelerated development of glomerulopathy and albuminuria upon streptozotocin (STZ)-induced hyperglycemia. The miR-146a targets, Notch-1 and ErbB4, were also significantly up-regulated in the glomeruli of diabetic patients and mice, suggesting induction of the downstream TGFß signaling. Treatment with a pan-ErbB kinase inhibitor erlotinib with nanomolar activity against ErbB4 significantly suppressed diabetic glomerular injury and albuminuria in both WT and miR-146a-/- animals. Treatment of podocytes in vitro with TGF-ß1 resulted in increased expression of Notch-1, ErbB4, pErbB4, and pEGFR, the heterodimerization partner of ErbB4, suggesting increased ErbB4/EGFR signaling. TGF-ß1 also increased levels of inflammatory cytokine monocyte chemoattractant protein-1 (MCP-1) and MCP-1 induced protein-1 (MCPIP1), a suppressor of miR-146a, suggesting an autocrine loop. Inhibition of ErbB4/EGFR with erlotinib co-treatment of podocytes suppressed this signaling. Our findings suggest a novel role for miR-146a in protecting against diabetic glomerulopathy and podocyte injury. They also point to ErbB4/EGFR as a novel, druggable target for therapeutic intervention, especially because several pan-ErbB inhibitors are clinically available.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Nefropatías Diabéticas/metabolismo , MicroARNs/metabolismo , Podocitos/metabolismo , Receptor ErbB-4/biosíntesis , Receptor Notch1/biosíntesis , Regulación hacia Arriba , Animales , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/patología , Clorhidrato de Erlotinib/farmacología , Ratones , Ratones Noqueados , MicroARNs/genética , Podocitos/patología , Receptor ErbB-4/genética , Receptor Notch1/genética , Ribonucleasas/genética , Ribonucleasas/metabolismo , Factores de Riesgo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
In diseases of many parenchymatous organs, heterogeneous deterioration of individual functional units determines the clinical prognosis. However, the molecular characterization at the level of such individual subunits remains a technological challenge that needs to be addressed in order to better understand pathological mechanisms. Proteinuric glomerular kidney diseases are frequent and assorted diseases affecting a fraction of glomeruli and their draining tubules to variable extents, and for which no specific treatment exists. Here, we developed and applied a mass spectrometry-based methodology to investigate heterogeneity of proteomes from individually isolated nephron segments from mice with proteinuric kidney disease. In single glomeruli from two different mouse models of sclerotic glomerular disease, we identified a coherent protein expression module consisting of extracellular matrix protein deposition (reflecting glomerular sclerosis), glomerular albumin (reflecting proteinuria) and LAMP1, a lysosomal protein. This module was associated with a loss of podocyte marker proteins while genetic ablation of LAMP1-correlated lysosomal proteases could ameliorate glomerular damage in vivo. Furthermore, proteomic analyses of individual glomeruli from patients with genetic sclerotic and non-sclerotic proteinuric diseases revealed increased abundance of lysosomal proteins, in combination with a decreased abundance of mutated gene products. Thus, altered protein homeostasis (proteostasis) is a conserved key mechanism in proteinuric kidney diseases. Moreover, our technology can capture intra-individual variability in diseases of the kidney and other tissues at a sub-biopsy scale.
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Glomerulonefritis/metabolismo , Nefronas/metabolismo , Proteinuria/metabolismo , Proteoma , Proteómica/métodos , Espectrometría de Masas en Tándem , Animales , Variación Biológica Individual , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/metabolismo , Glomerulonefritis/genética , Glomerulonefritis/patología , Glomerulonefritis/fisiopatología , Humanos , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Masculino , Ratones , Ratones Noqueados , Nefronas/patología , Nefronas/fisiopatología , Síndrome Nefrótico/genética , Síndrome Nefrótico/metabolismo , Síndrome Nefrótico/patología , Síndrome Nefrótico/fisiopatología , Podocitos/metabolismo , Podocitos/patología , Proteinuria/genética , Proteinuria/patología , Proteinuria/fisiopatología , Proteostasis , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Reproducibilidad de los Resultados , Albúmina Sérica/metabolismo , Proteínas WT1RESUMEN
Renal proximal tubular cells constantly recycle nutrients to ensure minimal loss of vital substrates into the urine. Although most of the transport mechanisms have been discovered at the molecular level, little is known about the factors regulating these processes. Here, we show that mTORC1 and mTORC2 specifically and synergistically regulate PTC endocytosis and transport processes. Using a conditional mouse genetic approach to disable nonredundant subunits of mTORC1, mTORC2, or both, we showed that mice lacking mTORC1 or mTORC1/mTORC2 but not mTORC2 alone develop a Fanconi-like syndrome of glucosuria, phosphaturia, aminoaciduria, low molecular weight proteinuria, and albuminuria. Interestingly, proteomics and phosphoproteomics of freshly isolated kidney cortex identified either reduced expression or loss of phosphorylation at critical residues of different classes of specific transport proteins. Functionally, this resulted in reduced nutrient transport and a profound perturbation of the endocytic machinery, despite preserved absolute expression of the main scavenger receptors, MEGALIN and CUBILIN. Our findings highlight a novel mTOR-dependent regulatory network for nutrient transport in renal proximal tubular cells.
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Endocitosis/fisiología , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Complejos Multiproteicos/fisiología , Serina-Treonina Quinasas TOR/fisiología , Animales , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Transporte de ProteínasRESUMEN
Regulated intracellular proteostasis, controlled in part by proteolysis, is essential in maintaining the integrity of podocytes and the glomerular filtration barrier of the kidney. We applied a novel proteomics technology that enables proteome-wide identification, mapping, and quantification of protein N-termini to comprehensively characterize cleaved podocyte proteins in the glomerulus in vivo We found evidence that defined proteolytic cleavage results in various proteoforms of important podocyte proteins, including those of podocin, nephrin, neph1, α-actinin-4, and vimentin. Quantitative mapping of N-termini demonstrated perturbation of protease action during podocyte injury in vitro, including diminished proteolysis of α-actinin-4. Differentially regulated protease substrates comprised cytoskeletal proteins as well as intermediate filaments. Determination of preferential protease motifs during podocyte damage indicated activation of caspase proteases and inhibition of arginine-specific proteases. Several proteolytic processes were clearly site-specific, were conserved across species, and could be confirmed by differential migration behavior of protein fragments in gel electrophoresis. Some of the proteolytic changes discovered in vitro also occurred in two in vivo models of podocyte damage (WT1 heterozygous knockout mice and puromycin aminonucleoside-treated rats). Thus, we provide direct and systems-level evidence that the slit diaphragm and podocyte cytoskeleton are regulated targets of proteolytic modification, which is altered upon podocyte damage.
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Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Enfermedades Renales/metabolismo , Podocitos/metabolismo , Proteolisis , Animales , Células Cultivadas , Humanos , Masculino , Ratones Noqueados , Proteoma , Proteómica/métodos , RatasRESUMEN
MAGI-1 is a multidomain cytosolic scaffolding protein that in the kidney is specifically located at the podocyte slit diaphragm, a specialized junction that is universally injured in proteinuric diseases. There it interacts with several essential molecules, including nephrin and neph1, which are required for slit diaphragm formation and as an intracellular signaling hub. Here, we show that diminished MAGI-1 expression in cultured podocytes reduced nephrin and neph1 membrane localization and weakened tight junction integrity. Global magi1 knock-out mice, however, demonstrated normal glomerular histology and function into adulthood. We hypothesized that a second mild but complementary genetic insult might induce glomerular disease susceptibility in these mice. To identify such a gene, we utilized the developing fly eye to test for functional complementation between MAGI and its binding partners. In this way, we identified diminished expression of fly Hibris (nephrin) or Roughest (neph1) as dramatically exacerbating the effects of MAGI depletion. Indeed, when these combinations were studied in mice, the addition of nephrin, but not neph1, heterozygosity to homozygous deletion of MAGI-1 resulted in spontaneous glomerulosclerosis. In cultured podocytes, MAGI-1 depletion reduced intercellular contact-induced Rap1 activation, a pathway critical for proper podocyte function. Similarly, magi1 knock-out mice showed diminished glomerular Rap1 activation, an effect dramatically enhanced by concomitant nephrin haploinsufficiency. Finally, combined overexpression of MAGI-1 and nephrin increased Rap1 activation, but not when substituting a mutant MAGI-1 that cannot bind nephrin. We conclude that the interaction between nephrin and MAGI-1 regulates Rap1 activation in podocytes to maintain long term slit diaphragm structure.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de la Membrana/metabolismo , Podocitos/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Moléculas de Adhesión Celular , Activación Enzimática , Glomeruloesclerosis Focal y Segmentaria/genética , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomeruloesclerosis Focal y Segmentaria/patología , Guanilato-Quinasas , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Proteínas de Unión al GTP rap1/genéticaRESUMEN
BACKGROUND & AIMS: Severe cholestasis may cause cholemic nephropathy that can be modeled in common bile duct ligated (CBDL) mice. We aimed to explore the therapeutic efficacy and mechanisms of norursodeoxycholic acid (norUDCA) in cholemic nephropathy. METHODS: In 8-week CBDL mice fed with norUDCA (prior or post CBDL) or chow we evaluated serum urea levels, urine cytology and urinary neutrophil gelatinase associated lipocalin (uNGAL), kidney and liver tissue quantification of fibrosis by hydroxyproline content and gene chip expression looking at key genes of inflammation and fibrosis. Moreover, we comprehensively analysed bile acid profiles in liver, kidney, serum and urine samples. RESULTS: NorUDCA-fed CBDL mice had significantly lower serum urea and uNGAL levels and less severe cholemic nephropathy as demonstrated by normal urine cytology, significantly reduced tubulointerstitial nephritis, and renal fibrosis as compared to controls. NorUDCA underwent extensive metabolism to produce even more hydrophilic compounds that were significantly enriched in kidneys. CONCLUSION: NorUDCA ameliorates cholemic nephropathy due to the formation of highly hydrophilic metabolites enriched in kidney. Consequently, norUDCA may represent a medical treatment for cholemic nephropathy. LAY SUMMARY: The term cholemic nephropathy describes renal dysfunction together with characteristic morphological alterations of the kidney in obstructive cholestasis that can be mimicked by ligation of the common bile duct in mice. Feeding the hydrophilic bile acid norUDCA to bile duct ligated mice leads to a significant amelioration of the renal phenotype due to the formation of highly hydrophilic metabolites enriched in the kidney and may therefore represent a medical treatment for cholemic nephropathy.
Asunto(s)
Colestasis/complicaciones , Enfermedades Renales/tratamiento farmacológico , Ácido Ursodesoxicólico/análogos & derivados , Animales , Ácidos y Sales Biliares/orina , Modelos Animales de Enfermedad , Fibrosis , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Ligadura , Lipocalina 2/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Nefritis Intersticial/tratamiento farmacológico , Ácido Ursodesoxicólico/metabolismo , Ácido Ursodesoxicólico/uso terapéuticoRESUMEN
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.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Fertilidad/efectos de los fármacos , Complejos Multiproteicos/efectos de los fármacos , Vesículas Seminales/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/efectos de los fármacos , Animales , Masculino , Mamíferos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones Transgénicos , Fosforilación , Vesículas Seminales/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The last 5 years have witnessed tremendous advances in both light- and electron-microscopic techniques in the biomedical sciences. Application of these new cutting-edge methods to glomerular biology has advanced considerably and, in part, completed our endeavor to draw a detailed map of the glomerular tuft. The scope of this review is to illustrate these new insights within both the morphometry of podocyte cells and the architecture of the glomerular filtration barrier and to assess whether these findings have indeed had an impact on our biological understanding of glomerular function.