RESUMEN
Recent advances in stem cell biology have enabled the generation of kidney organoids in vitro, and further maturation of these organoids is observed after experimental transplantation. However, the current organoids remain immature and their precise maturation stages are difficult to determine because of limited information on developmental stage-dependent gene expressions in the kidney in vivo. To establish relevant molecular coordinates, we performed single-cell RNA sequencing (scRNA-seq) on developing kidneys at different stages in the mouse. By selecting genes that exhibited upregulation at birth compared with embryonic day 15.5 as well as cell lineage-specific expression, we generated gene lists correlated with developmental stages in individual cell lineages. Application of these lists to transplanted embryonic kidneys revealed that most cell types, other than the collecting ducts, exhibited similar maturation to kidneys at the neonatal stage in vivo, revealing non-synchronous maturation across the cell lineages. Thus, our scRNA-seq data can serve as useful molecular coordinates to assess the maturation of developing kidneys and eventually of kidney organoids.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Riñón/crecimiento & desarrollo , Riñón/metabolismo , Animales , Animales Recién Nacidos , Linaje de la Célula , Regulación hacia Abajo , Riñón/citología , Riñón/embriología , Glomérulos Renales/citología , Glomérulos Renales/embriología , Glomérulos Renales/crecimiento & desarrollo , Glomérulos Renales/metabolismo , Trasplante de Riñón , Túbulos Renales/citología , Túbulos Renales/embriología , Túbulos Renales/crecimiento & desarrollo , Túbulos Renales/metabolismo , Ratones , Podocitos/citología , Podocitos/metabolismo , RNA-Seq , Análisis de la Célula Individual , Células Madre/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia ArribaRESUMEN
Steroid-resistant nephrotic syndrome (SRNS) is characterized by high-range proteinuria and most often focal and segmental glomerulosclerosis (FSGS). Identification of mutations in genes causing SRNS has improved our understanding of disease mechanisms and highlighted defects in the podocyte, a highly specialized glomerular epithelial cell, as major factors in disease pathogenesis. By exome sequencing, we identified missense mutations in TBC1D8B in two families with an X-linked early-onset SRNS with FSGS. TBC1D8B is an uncharacterized Rab-GTPase-activating protein likely involved in endocytic and recycling pathways. Immunofluorescence studies revealed TBC1D8B presence in human glomeruli, and affected individual podocytes displayed architectural changes associated with migration defects commonly found in FSGS. In zebrafish we demonstrated that both knockdown and knockout of the unique TBC1D8B ortholog-induced proteinuria and that this phenotype was rescued by human TBC1D8B mRNA injection, but not by either of the two mutated mRNAs. We also showed an interaction between TBC1D8B and Rab11b, a key protein in vesicular recycling in cells. Interestingly, both internalization and recycling processes were dramatically decreased in affected individuals' podocytes and fibroblasts, confirming the crucial role of TBC1D8B in the cellular recycling processes, probably as a Rab11b GTPase-activating protein. Altogether, these results confirmed that pathogenic variations in TBC1D8B are involved in X-linked podocytopathy and points to alterations in recycling processes as a mechanism of SRNS.
Asunto(s)
Proteínas de Unión al Calcio/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Mutación con Pérdida de Función , Síndrome Nefrótico/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Pez Cebra/genética , Animales , Transporte Biológico/genética , Proteínas de Unión al Calcio/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Glomérulos Renales/metabolismo , Masculino , Podocitos/citología , Podocitos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Secuenciación del Exoma , Pez Cebra , Proteínas de Pez Cebra/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Progressive podocyte loss is a feature of healthy ageing. While previous studies have reported age-related changes in podocyte number, density and size and associations with proteinuria and glomerulosclerosis, few studies have examined how the response of remaining podocytes to podocyte depletion changes with age. Mild podocyte depletion was induced in PodCreiDTR mice aged 1, 6, 12 and 18 months via intraperitoneal administration of diphtheria toxin. Control mice received intraperitoneal vehicle. Podometrics, proteinuria and glomerular pathology were assessed, together with podocyte expression of p-rp-S6, a phosphorylation target that represents activity of the mammalian target of rapamycin (mTOR). Podocyte number per glomerulus did not change in control mice in the 18-month time period examined. However, control mice at 18 months had the largest podocytes and the lowest podocyte density. Podocyte depletion at 1, 6 and 12 months resulted in mild albuminuria but no glomerulosclerosis, whereas similar levels of podocyte depletion at 18 months resulted in both albuminuria and glomerulosclerosis. Following podocyte depletion at 6 and 12 months, the number of p-rp-S6 positive podocytes increased significantly, and this was associated with an adaptive increase in podocyte volume. However, at 18 months of age, remaining podocytes were unable to further elevate mTOR expression or undergo hypertrophic adaptation in response to mild podocyte depletion, resulting in marked glomerular pathology. These findings demonstrate the importance of mTORC1-mediated podocyte hypertrophy in both physiological (ageing) and adaptive settings, highlighting a functional limit to podocyte hypertrophy reached under physiological conditions.
Asunto(s)
Envejecimiento , Podocitos , Albuminuria/metabolismo , Albuminuria/patología , Animales , Femenino , Hipertrofia/metabolismo , Hipertrofia/patología , Masculino , Ratones , Podocitos/citología , Proteinuria , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
FKBP12 was identified as a binding protein of tacrolimus (Tac). Tac binds to FKBP12 and exhibits immunosuppressive effects in T cells. Although it is reported that Tac treatment directly ameliorates the dysfunction of the podocyte in nephrotic syndrome, the precise pharmacological mechanism of Tac is not well understood yet. It is also known that FKBP12 functions independently of Tac. However, the localization and the physiological function of FKBP12 are not well elucidated. In this study, we observed that FKBP12 is highly expressed in glomeruli, and the FKBP12 in glomeruli is restricted in podocytes. FKBP12 in cultured podocytes was expressed along the actin cytoskeleton and associated with filamentous actin (F-actin). FKBP12 interacted with the actin-associated proteins 14-3-3 and synaptopodin. RNA silencing for FKBP12 reduced 14-3-3 expression, F-actin staining, and process formation in cultured podocytes. FKBP12 expression was decreased in the nephrotic model caused by adriamycin (ADR) and the cultured podocyte treated with ADR. The process formation was deteriorated in the podocytes treated with ADR. Tac treatment ameliorated these decreases. Tac treatment to the normal cells increased the expression of FKBP12 at F-actin in processes and enhanced process formation. Tac enhanced the interaction of FKBP12 with synaptopodin. These observations suggested that FKBP12 at actin cytoskeleton participates in the maintenance of processes, and Tac treatment ameliorates podocyte injury by restoring FKBP12 at actin cytoskeleton.
Asunto(s)
Síndrome Nefrótico/metabolismo , Podocitos/metabolismo , Proteinuria/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Tacrolimus/farmacología , Citoesqueleto de Actina/metabolismo , Animales , Femenino , Células HEK293 , Humanos , Podocitos/citología , Ratas , Ratas WistarRESUMEN
The current understanding of mammalian kidney development is largely based on mouse models. Recent landmark studies revealed pervasive differences in renal embryogenesis between mouse and human. The scarcity of detailed gene expression data in humans therefore hampers a thorough understanding of human kidney development and the possible developmental origin of kidney diseases. In this paper, we present a single-cell transcriptomics study of the human fetal kidney. We identified 22 cell types and a host of marker genes. Comparison of samples from different developmental ages revealed continuous gene expression changes in podocytes. To demonstrate the usefulness of our data set, we explored the heterogeneity of the nephrogenic niche, localized podocyte precursors, and confirmed disease-associated marker genes. With close to 18,000 renal cells from five different developmental ages, this study provides a rich resource for the elucidation of human kidney development, easily accessible through an interactive web application.
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Regulación del Desarrollo de la Expresión Génica , Riñón/metabolismo , Organogénesis/genética , Podocitos/metabolismo , Transcriptoma , Diferenciación Celular , Linaje de la Célula/genética , Conjuntos de Datos como Asunto , Femenino , Desarrollo Fetal , Feto , Perfilación de la Expresión Génica , Ontología de Genes , Edad Gestacional , Humanos , Riñón/citología , Riñón/crecimiento & desarrollo , Masculino , Anotación de Secuencia Molecular , Podocitos/citología , Análisis de la Célula IndividualRESUMEN
Parietal epithelial cells (PECs) are epithelial cells in the kidney, surrounding Bowman's space. When activated, PECs increase in cell volume, proliferate, migrate to the glomerular tuft and excrete extracellular matrix. Activated PECs are crucially involved in the formation of sclerotic lesions, seen in focal segmental glomerulosclerosis (FSGS). In FSGS, a number of glomeruli show segmental sclerotic lesions. Further disease progression will lead to increasing number of involved glomeruli and gradual destruction of the affected glomeruli. Although the involvement of PECs in FSGS has been acknowledged, little is known about the molecular processes driving PEC activation. To get more insights in this process, accurate in vivo and in vitro models are needed. Here, we describe the development and characterization of a novel conditionally immortalized human PEC (ciPEC) line. We demonstrated that ciPECs are differentiated when grown under growth-restrictive conditions and express important PEC-specific markers, while lacking podocyte and endothelial markers. In addition, ciPECs showed PEC-like morphology and responded to IL-1ß treatment. We therefore conclude that we have successfully generated a novel PEC line, which can be used for future studies on the role of PECs in FSGS.
Asunto(s)
Células Epiteliales/citología , Matriz Extracelular/metabolismo , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomérulos Renales/citología , Humanos , Receptores de Hialuranos/metabolismo , Riñón/citología , Podocitos/citologíaRESUMEN
Apical-basal polarity is a key feature of most epithelial cells and it is regulated by highly conserved protein complexes. In mammalian podocytes, which emerge from columnar epithelial cells, this polarity is preserved and the tight junctions are converted to the slit diaphragms, establishing the filtration barrier. In Drosophila, nephrocytes show several structural and functional similarities with mammalian podocytes and proximal tubular cells. However, in contrast to podocytes, little is known about the role of apical-basal polarity regulators in these cells. In this study, we used expansion microscopy and found the apical polarity determinants of the PAR/aPKC and Crb-complexes to be predominantly targeted to the cell cortex in proximity to the nephrocyte diaphragm, whereas basolateral regulators also accumulate intracellularly. Knockdown of PAR-complex proteins results in severe endocytosis and nephrocyte diaphragm defects, which is due to impaired aPKC recruitment to the plasma membrane. Similar, downregulation of most basolateral polarity regulators disrupts Nephrin localization but had surprisingly divergent effects on endocytosis. Our findings suggest that morphology and slit diaphragm assembly/maintenance of nephrocytes is regulated by classical apical-basal polarity regulators, which have distinct functions in endocytosis.
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Polaridad Celular , Proteínas de Drosophila/metabolismo , Endocitosis , Uniones Intercelulares/fisiología , Proteínas de la Membrana/metabolismo , Podocitos/fisiología , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Podocitos/citología , Podocitos/metabolismo , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismoRESUMEN
The effects of healthy aging on the kidney, and how these effects intersect with superimposed diseases, are highly relevant in the context of the population's increasing longevity. Age-associated changes to podocytes, which are terminally differentiated glomerular epithelial cells, adversely affect kidney health. This review discusses the molecular and cellular mechanisms underlying podocyte aging, how these mechanisms might be augmented by disease in the aged kidney, and approaches to mitigate progressive damage to podocytes. Furthermore, we address how biologic pathways such as those associated with cellular growth confound aging in humans and rodents.
Asunto(s)
Envejecimiento/fisiología , Podocitos/citología , Adulto , Anciano , Animales , Autofagia , Restricción Calórica , Ciclo Celular , Forma de la Célula , Células Cultivadas , Senescencia Celular , Daño del ADN , Femenino , Expresión Génica , Humanos , Inflamasomas , Glomérulos Renales/citología , Glomérulos Renales/crecimiento & desarrollo , Masculino , Ratones , Persona de Mediana Edad , Mitocondrias/metabolismo , Modelos Animales , Oligopéptidos/farmacología , Estrés Oxidativo , Podocitos/metabolismo , Ratas , Muerte Celular Regulada , Sirtuinas/metabolismo , Especificidad de la Especie , Adulto JovenRESUMEN
BACKGROUND: Microscopic analysis of urine sediment is probably the most commonly used diagnostic procedure in nephrology. The urinary cells, however, have not yet undergone careful unbiased characterization. METHODS: Single-cell transcriptomic analysis was performed on 17 urine samples obtained from five subjects at two different occasions, using both spot and 24-hour urine collection. A pooled urine sample from multiple healthy individuals served as a reference control. In total 23,082 cells were analyzed. Urinary cells were compared with human kidney and human bladder datasets to understand similarities and differences among the observed cell types. RESULTS: Almost all kidney cell types can be identified in urine, such as podocyte, proximal tubule, loop of Henle, and collecting duct, in addition to macrophages, lymphocytes, and bladder cells. The urinary cell-type composition was subject specific and reasonably stable using different collection methods and over time. Urinary cells clustered with kidney and bladder cells, such as urinary podocytes with kidney podocytes, and principal cells of the kidney and urine, indicating their similarities in gene expression. CONCLUSIONS: A reference dataset for cells in human urine was generated. Single-cell transcriptomics enables detection and quantification of almost all types of cells in the kidney and urinary tract.
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Riñón/citología , Anciano , Código de Barras del ADN Taxonómico , Femenino , Biblioteca de Genes , Humanos , Riñón/metabolismo , Enfermedades Renales/genética , Enfermedades Renales/patología , Enfermedades Renales/orina , Masculino , Persona de Mediana Edad , Podocitos/citología , Podocitos/metabolismo , RNA-Seq , Análisis de la Célula Individual/métodos , Análisis de la Célula Individual/estadística & datos numéricos , Transcriptoma , Vejiga Urinaria/citología , Vejiga Urinaria/metabolismo , Orina/citologíaRESUMEN
The filtering component of the kidney, the nephron, arises from a single progenitor population. These nephron progenitor cells (NPCs) both self-renew and differentiate throughout the course of kidney development ensuring sufficient nephron endowment. An appropriate balance of these processes must be struck as deficiencies in nephron numbers are associated with hypertension and kidney disease. This review will discuss the mechanisms and molecules supporting NPC maintenance and differentiation. A focus on recent work will highlight new molecular insights into NPC regulation and their dynamic behavior in both space and time.
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Riñón/citología , Nefronas/citología , Podocitos/citología , Células Madre/citología , Animales , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Riñón/embriología , Riñón/metabolismo , Nefronas/metabolismo , Organogénesis/genética , Podocitos/metabolismo , Células Madre/metabolismoRESUMEN
Chronic kidney disease can be understood as a pathological reduction in the number of functional glomeruli. It is a frequent medical problem and one of the major independent risk factors for cardiovascular morbidity and mortality. In humans, glomeruli/nephrons are generated during the prenatal period (glomerular endowment), which may be impaired by multiple conditions. After birth, glomeruli are progressively lost - mostly due to glomerular scarring (focal segmental glomerulosclerosis; FSGS). Multiple independent studies have shown that significant loss of glomerular visceral epithelial cells (podocytes) is sufficient to induce FSGS. It is generally believed that podocytes cannot renew themselves and it has been generally assumed that their number is determined at birth (podocyte endowment). However, there are several lines of experimental evidence showing that podocytes can be replenished in the postnatal period. First, a limited reserve of podocytes has been reported on Bowman's capsule, which may be associated with body growth and increases in glomerular size between childhood and adulthood. Second, two intrinsic progenitor cell niches have been proposed to replenish podocytes throughout adult life and in association with glomerular injury and podocyte loss: parietal epithelial cells and/or cells of the renin lineage. While there is increasing evidence supporting postnatal podocyte gain, controversy remains about the involved signalling pathways and the efficiency of these sources to prevent nephron loss.
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Células Epiteliales/citología , Glomeruloesclerosis Focal y Segmentaria/fisiopatología , Glomérulos Renales/citología , Nefronas/citología , Podocitos/citología , Animales , Modelos Animales de Enfermedad , Glomeruloesclerosis Focal y Segmentaria/patología , Humanos , Glomérulos Renales/crecimiento & desarrollo , Nefronas/crecimiento & desarrollo , Nicho de Células Madre , Células Madre/citologíaRESUMEN
Focal segmental glomerulosclerosis (FSGS) is a chronic glomerular disease with poor clinical outcomes. Podocyte loss via apoptosis is one important mechanism underlying the pathogenesis of FSGS. Recently, Yes-associated-protein (YAP), a key downstream protein in the Hippo pathway, was identified as an activator for multiple gene transcriptional factors in the nucleus to control cell proliferation and apoptosis. To investigate the potential role of YAP in the progression of FSGS, we examined kidney samples from patients with minimal change disease or FSGS and found that increases in podocyte apoptosis is positively correlated with the cytoplasmic distribution of YAP in human FSGS. Utilizing an established mT/mG transgenic mouse model and primary cultured podocytes, we found that YAP was distributed uniformly in nucleus and cytoplasm in the podocytes of control animals. Adriamycin treatment induced gradual nuclear exclusion of YAP with enhanced phospho-YAP/YAP ratio, accompanied by the induction of podocyte apoptosis both in vivo and in vitro. Moreover, we used verteporfin to treat an Adriamycin-induced FSGS mouse model, and found YAP inhibition by verteporfin induced nuclear exclusion of YAP, thus increasing podocyte apoptosis and accelerating disease progression. Therefore, our findings suggest that YAP nuclear distribution and activation in podocytes is an important endogenous anti-apoptotic mechanism during the progression of FSGS.
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Proteínas Adaptadoras Transductoras de Señales , Apoptosis/genética , Glomeruloesclerosis Focal y Segmentaria , Podocitos , Factores de Transcripción , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Doxorrubicina/efectos adversos , Glomeruloesclerosis Focal y Segmentaria/genética , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomeruloesclerosis Focal y Segmentaria/patología , Humanos , Riñón/citología , Riñón/metabolismo , Riñón/patología , Masculino , Ratones , Ratones Transgénicos , Podocitos/citología , Podocitos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAPRESUMEN
BACKGROUND/AIMS: Podocytes are lost in most glomerular diseases, leading to glomerulosclerosis and progressive kidney disease. It is generally assumed, that podocytes are exposed to the filtration flow and thus to significant shear forces driving their detachment from the glomerular basement membrane (GBM). In this context, foot process effacement has been proposed as potential adaptive response to increase adhesion of podocytes to the GBM. METHODS: We have tested these hypotheses using optical clearing and high-resolution 3-dimensional morphometric analysis in the isolated perfused murine kidney. We investigated the dynamics of podocyte detachment at different perfusion pressures (50, 300 and more than 450 mmHg) in healthy young or old mice (20 vs. 71 weeks of age), or mice injected with anti-GBM serum to induce global foot process effacement. RESULTS: Results show that healthy podocytes in young mice are tightly attached onto the GBM and even supramaximal pressures did not cause significant detachment. Compared to young mice, in aged mice and mice with anti-GBM nephritis and foot process effacement, gradual progressive loss of podocytes had occurred already before perfusion. High perfusion pressures resulted in a relatively minor additional loss of podocytes in aged mice. In mice with anti-GBM nephritis significant additional podocyte loss occurred at this early time point when increasing perfusion pressures to 300 mmHg or higher. CONCLUSION: This work provides the first experimental evidence that podocytes are extraordinarily resistant to acutely increased perfusion pressures in an ex vivo isolated kidney perfusion model. Only in glomerular disease, significant numbers of injured podocytes detached following acute increases in perfusion pressure.
Asunto(s)
Membrana Basal Glomerular/patología , Enfermedades Renales/patología , Podocitos/patología , Envejecimiento , Animales , Adhesión Celular , Supervivencia Celular , Femenino , Membrana Basal Glomerular/citología , Masculino , Ratones , Perfusión , Podocitos/citología , PresiónRESUMEN
BACKGROUND/AIMS: Podocyte differentiation is essential for proper blood filtration in the kidney. It is well known that transcription factors play an essential role to maintain the differentiation of podocytes. The present study is focused on the basic helix-loop-helix (bHLH) transcription factor Tcf21 (Pod1) which is essential for the development of podocytes in vivo. Since parietal epithelial cells (PECs) are still under debate to be progenitor cells which can differentiate into podocytes, we wanted to find out whether the expression of Tcf21 induces a transition of PECs into podocytes. METHODS: We transfected PECs with Tcf21-GFP and analyzed the expression of PEC- and podocyte-specific markers. Furthermore, we performed ChIP-Seq analysis to identify new putative interaction partners and target genes of Tcf21. RESULTS: By gene arrays analysis, we found that podocytes express high levels of Tcf21 in vivo in contrast to cultured podocytes and parietal epithelial cells (PECs) in vitro. After the expression of Tcf21 in PECs, we observed a downregulation of specific PEC markers like caveolin1, ß-catenin and Pax2. Additionally, we found that the upregulation of Tcf21 induced multi-lobulation of cell nuclei, budding and a formation of micronuclei (MBM). Furthermore, a high number of PECs showed a tetraploid set of chromosomes. By qRT-PCR and Western blot analysis, we revealed that the transcription factor YY1 is downregulated by Tcf21. Interestingly, co-expression of YY1 and Tcf21 rescues MBM and reduced tetraploidy. By ChIP-Seq analysis, we identified a genome-wide Tcf21-binding site (CAGCTG), which matched the CANNTG sequence, a common E-box binding motif used by bHLH transcription factors. Using this technique, we identified additional Tcf21 targets genes that are involved in the regulation of the cell cycle (e.g. Mdm2, Cdc45, Cyclin D1, Cyclin D2), on the stability of microtubules (e.g. Mapt) as well as chromosome segregation. CONCLUSION: Taken together, we demonstrate that Tcf21 inhibits the expression of PEC-specific markers and of the transcription factor YY1, induces MBM as well as regulates the cell cycle suggesting that Tcf21 might be important for PEC differentiation into podocyte-like cells.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células Epiteliales/citología , Podocitos/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular , Transdiferenciación Celular , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Podocitos/metabolismo , TransfecciónRESUMEN
Roundabout guidance receptor 2 (ROBO2) plays an important role during early kidney development. ROBO2 is expressed in podocytes, inhibits nephrin-induced actin polymerization, down-regulates nonmuscle myosin IIA activity, and destabilizes kidney podocyte adhesion. However, the role of ROBO2 during kidney injury, particularly in mature podocytes, is not known. Herein, we report that loss of ROBO2 in podocytes [Robo2 conditional knockout (cKO) mouse] is protective from glomerular injuries. Ultrastructural analysis reveals that Robo2 cKO mice display less foot process effacement and better-preserved slit-diaphragm density compared with wild-type littermates injured by either protamine sulfate or nephrotoxic serum (NTS). The Robo2 cKO mice also develop less proteinuria after NTS injury. Further studies reveal that ROBO2 expression in podocytes is up-regulated after glomerular injury because its expression levels are higher in the glomeruli of NTS injured mice and passive Heymann membranous nephropathy rats. Moreover, the amount of ROBO2 in the glomeruli is also elevated in patients with membranous nephropathy. Finally, overexpression of ROBO2 in cultured mouse podocytes compromises cell adhesion. Taken together, these findings suggest that kidney injury increases glomerular ROBO2 expression that might compromise podocyte adhesion and, thus, loss of Robo2 in podocytes could protect from glomerular injury by enhancing podocyte adhesion that helps maintain foot process structure. Our findings also suggest that ROBO2 is a therapeutic target for podocyte injury and podocytopathy.
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Enfermedades Renales/prevención & control , Glomérulos Renales/citología , Podocitos/citología , Sustancias Protectoras/metabolismo , Receptores Inmunológicos/deficiencia , Adulto , Animales , Femenino , Humanos , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Glomérulos Renales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Podocitos/metabolismo , Proteinuria/metabolismo , Proteinuria/patología , Proteinuria/prevención & control , RatasRESUMEN
Ephrin-B1 plays a critical role at slit diaphragm. Partitioning-defective (Par)-6 is down-regulated in podocyte of ephrin-B1 knockout mouse, suggesting that Par-6 is associated with ephrin-B1. Par polarity complex, consisting of Par-6, Par-3, and atypical protein kinase C, is essential for tight junction formation. In this study, the expression of Par-6 was analyzed in the normal and nephrotic syndrome model rats, and the molecular association of Par-6, Par-3, ephrin-B1, and nephrin was assessed with the human embryonic kidney 293 cell expression system. Par-6 was concentrated at slit diaphragm. Par 6 interacted with ephrin-B1 but not with nephrin, and Par-3 interacted with nephrin but not with ephrin-B1. The complexes of Par-6-ephrin-B1 and Par-3-nephrin were linked via extracellular sites of ephrin-B1 and nephrin. The Par-6-ephrin-B1 complex was delinked from the Par-3-nephrin complex, and Par-6 and ephrin-B1 were clearly down-regulated already at early phase of nephrotic model. The alteration of Par-6/ephrin-B1 advanced that of Par-3/nephrin. Stimulation to nephrin phosphorylated not only nephrin but also ephrin-B1, and consequently inhibited the interaction between ephrin-B1 and Par-6. Par-6 appeared at presumptive podocyte of early developmental stage and moved to basal area at capillary loop stage to participate in slit diaphragm formation at the final stage. Par-6-ephrin-B1 interaction is crucial for formation and maintenance of slit diaphragm of podocyte.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas Portadoras/metabolismo , Efrina-B1/metabolismo , Glomérulos Renales/citología , Proteínas de la Membrana/metabolismo , Síndrome Nefrótico/patología , Podocitos/citología , Animales , Animales Recién Nacidos , Proteínas Portadoras/genética , Diafragma , Efrina-B1/genética , Células HEK293 , Humanos , Glomérulos Renales/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Síndrome Nefrótico/metabolismo , Fosforilación , Podocitos/metabolismo , Ratas , Ratas WistarRESUMEN
The actin cytoskeleton of podocytes plays a central role in the functioning of the filtration barrier in the kidney. Calcium entry into podocytes via TRPC6 (Transient Receptor Potential Canonical 6) channels leads to actin cytoskeleton rearrangement, thereby affecting the filtration barrier. We hypothesized that there is feedback from the cytoskeleton that modulates the activity of TRPC6 channels. Experiments using scanning ion-conductance microscopy demonstrated a change in migration properties in podocyte cell cultures treated with cytochalasin D, a pharmacological agent that disrupts the actin cytoskeleton. Cell-attached patch-clamp experiments revealed that cytochalasin D increases the activity of TRPC6 channels in CHO (Chinese Hamster Ovary) cells overexpressing the channel and in podocytes from freshly isolated glomeruli. Furthermore, it was previously reported that mutation in ACTN4, which encodes α-actinin-4, causes focal segmental glomerulosclerosis and solidifies the actin network in podocytes. Therefore, we tested whether α-actinin-4 regulates the activity of TRPC6 channels. We found that co-expression of mutant α-actinin-4 K255E with TRPC6 in CHO cells decreases TRPC6 channel activity. Therefore, our data demonstrate a direct interaction between the structure of the actin cytoskeleton and TRPC6 activity.
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Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Calcio/metabolismo , Glomérulos Renales/metabolismo , Podocitos/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Glomérulos Renales/citología , Masculino , Podocitos/citología , Ratas , Ratas WistarRESUMEN
Nephrin is an immunoglobulin-type cell-adhesion molecule with a key role in the glomerular interpodocyte slit diaphragm. Mutations in the nephrin gene are associated with defects in the slit diaphragm, leading to early-onset nephrotic syndrome, typically resistant to treatment. Although the endocytic trafficking of nephrin is essential for the assembly of the slit diaphragm, nephrin's specific endocytic motifs remain unknown. To search for endocytic motifs, here we performed a multisequence alignment of nephrin and identified a canonical YXXØ-type motif, Y1139RSL, in the nephrin cytoplasmic tail, expressed only in primates. Using site-directed mutagenesis, various biochemical methods, single-plane illumination microscopy, a human podocyte line, and a human nephrin-expressing zebrafish model, we found that Y1139RSL is a novel endocytic motif and a structural element for clathrin-mediated nephrin endocytosis that functions as a phosphorylation-sensitive signal. We observed that Y1139RSL motif-mediated endocytosis helps to localize nephrin to specialized plasma membrane domains in podocytes and is essential for normal foot process organization into a functional slit diaphragm between neighboring foot processes in zebrafish. The importance of nephrin Y1139RSL for healthy podocyte development was supported by population-level analyses of genetic variations at this motif, revealing that such variations are very rare, suggesting that mutations in this motif have autosomal-recessive negative effects on kidney health. These findings expand our understanding of the mechanism underlying nephrin endocytosis and may lead to improved diagnostic tools or therapeutic strategies for managing early-onset, treatment-resistant nephrotic syndrome.
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Glomérulos Renales/metabolismo , Proteínas de la Membrana/metabolismo , Secuencias de Aminoácidos , Animales , Línea Celular , Membrana Celular/metabolismo , Movimiento Celular , Clatrina/metabolismo , Embrión no Mamífero/metabolismo , Endocitosis , Humanos , Glomérulos Renales/ultraestructura , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Morfolinos/metabolismo , Mutagénesis Sitio-Dirigida , Fosforilación , Podocitos/citología , Podocitos/metabolismo , Pez Cebra/crecimiento & desarrolloRESUMEN
Prostaglandin E2 receptor EP1 (PGE2/EP1) promotes diabetic renal injury, and EP1 receptor deletion improves hyperfiltration, albuminuria, and fibrosis. The role of EP1 receptors in hypertensive kidney disease (HKD) remains controversial. We examined the contribution of EP1 receptors to HKD. EP1 null (EP1-/-) mice were bred with hypertensive TTRhRen mice (Htn) to evaluate kidney function and injury at 24 weeks. EP1 deletion had no effect on elevation of systolic blood pressure in Htn mice (HtnEP1-/-) but resulted in pronounced albuminuria and reduced FITC-inulin clearance, compared with Htn or wild-type (WT) mice. Ultrastructural injury to podocytes and glomerular endothelium was prominent in HtnEP1-/- mice; including widened subendothelial space, subendothelial lucent zones and focal lifting of endothelium from basement membrane, with focal subendothelial cell debris. Cortex COX2 mRNA was increased by EP1 deletion. Glomerular EP3 mRNA was reduced by EP1 deletion, and EP4 by Htn and EP1 deletion. In WT mice, PGE2 increased chloride reabsorption via EP1 in isolated perfused thick ascending limb (TAL), but PGE2 or EP1 deletion did not affect vasopressin-mediated chloride reabsorption. In WT and Htn mouse inner medullary collecting duct (IMCD), PGE2 inhibited vasopressin-water transport, but not in EP1-/- or HtnEP1-/- mice. Overall, EP1 mediated TAL and IMCD transport in response to PGE2 is unaltered in Htn, and EP1 is protective in HKD.
Asunto(s)
Hipertensión Renal , Podocitos , Subtipo EP1 de Receptores de Prostaglandina E , Animales , Modelos Animales de Enfermedad , Células Endoteliales/citología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Eliminación de Gen , Tasa de Filtración Glomerular/genética , Hipertensión Renal/metabolismo , Hipertensión Renal/patología , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Masculino , Ratones , Ratones Transgénicos , Podocitos/citología , Podocitos/metabolismo , Podocitos/patología , Subtipo EP1 de Receptores de Prostaglandina E/genética , Subtipo EP1 de Receptores de Prostaglandina E/metabolismoRESUMEN
The autophagy, which can be regulated by lysosomal membrane proteins, plays a critical role in maintaining normal podocyte function. TM7SF1 is a novel lysosomal membrane protein, but its effect on autophagy is still unknown. This study aimed to identify the role of TM7SF1 in mouse podocyte (MPC5) autophagy. Interestingly, we detected an increase in LC3BII and SQSTM1/P62 in MPC5 through inhibiting TM7SF1, and which can be completely corrected after blocking the autolysosome degradation with chloroquine (CQ). Moreover, inhibition of TM7SF1 expression did not increase the mRNA level of SQSTM1/P62. Theses results suggested that inhibition of TM7SF1 led to impaired degradation of autophagy products, which manifest as an abnormal accumulation of LC3BII and SQSTM1/P62. Further studies showed that the downregulation of TM7SF1 resulted in a significant decrease in the number of acid lysosomes, which directly led to decreases in the number and function of autolysosomes. In conclusion, TM7SF1 is therefore essential for autolysosomes degradation pathway at the end of autophagy flow, and for the maintenance of podocyte function.