RESUMEN
Introduction Activation of the focal adhesion kinase (FAK) in podocytes is involved in the pathogenesis of minimal change disease (MCD), but the pathway leading to its activation in this disease is unknown. Here, we tested whether podocyte β1 integrin is the upstream modulator of FAK activation and podocyte injury in experimental models of MCD-like injury. Methods We used lipopolysaccharide (LPS) and MCD sera to induce MCD-like changes in vivo and in cultured human podocytes, respectively. We performed functional studies using specific β1 integrin inhibitors in vivo and in vitro, and integrated histological analysis, western blotting, and immunofluorescence to assess for morphological and molecular changes in podocytes. By ELISA, we measured serum LPS levels in 35 children with MCD or presumed MCD (idiopathic nephrotic syndrome [INS]) and in 18 healthy controls. Results LPS-injected mice showed morphological (foot process effacement, and normal appearing glomeruli on light microscopy) and molecular features (synaptopodin loss, nephrin mislocalization, FAK phosphorylation) characteristic of human MCD. Administration of a β1 integrin inhibitor to mice abrogated FAK phosphorylation, and ameliorated proteinuria and podocyte injury following LPS. Children with MCD/INS in relapse had higher serum LPS levels than controls. In cultured human podocytes, β1 integrin blockade prevented cytoskeletal rearrangements following exposure to MCD sera in relapse. Conclusions Podocyte β1 integrin activation is an upstream mediator of FAK phosphorylation and podocyte injury in models of MCD-like injury (AU)
Antecedentes La activación de la quinasa de adhesión focal (FAK) en podocitos juega un papel en la patogénesis de la enfermedad de cambios mínimos (ECM), pero su mecanismo de activación en dicha enfermedad es desconocido. En este estudio investigamos si la integrina β1 de los podocitos modula la activación de FAK y del daño podocitario en modelos experimentales de la ECM. Métodos Utilizamos lipopolisacárido (LPS) y suero de pacientes con ECM para inducir daño podocitario in vivo e in vitro, respectivamente. Realizamos estudios funcionales usando inhibidores específicos de la integrina β1 in vivo e in vitro, así como estudios histológicos, western blots y técnicas de inmunofluorescencia para evaluar cambios morfológicos y moleculares en podocitos. Usando ELISA medimos los niveles séricos de LPS en 35 niños con ECM o sospecha de ECM (síndrome nefrótico idiopático [SNI]) y en 18 individuos sanos. Resultados Los ratones inyectados con LPS desarrollaron cambios morfológicos (fusión de pedicelos, con apariencia normal de los glomérulos) y moleculares (pérdida de la expresión de sinaptopodina, cambio en la localización de la nefrina fosforilada y fosforilzación de FAK), que son característicos de la ECM en humanos. La administración de un inhibidor de la integrina β1 en ratones disminuyó la fosforilación de FAK, proteinuria y daño podocitario que ocurre tras la inyección de LPS. En niños con ECM/SNI, los niveles séricos de LPS fueron más elevados que en controles. En cultivos de podocitos humanos, la adicción de un inhibidor de la integrina β1 al suero de niños con ECM en recaída evitó cambios en el citoesqueleto. Conclusiones La integrina β1 de los podocitos actúa como mediador de la activación de la FAK y del daño podocitario en modelos experimentales de la ECM (AU)
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Animales , Ratones , Podocitos/metabolismo , Integrina beta1/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Enfermedades Renales/metabolismo , Modelos Animales de Enfermedad , PolisacáridosRESUMEN
INTRODUCTION: Activation of the focal adhesion kinase (FAK) in podocytes is involved in the pathogenesis of minimal change disease (MCD), but the pathway leading to its activation in this disease is unknown. Here, we tested whether podocyte ß1 integrin is the upstream modulator of FAK activation and podocyte injury in experimental models of MCD-like injury. METHODS: We used lipopolysaccharide (LPS) and MCD sera to induce MCD-like changes in vivo and in cultured human podocytes, respectively. We performed functional studies using specific ß1 integrin inhibitors in vivo and in vitro, and integrated histological analysis, western blotting, and immunofluorescence to assess for morphological and molecular changes in podocytes. By ELISA, we measured serum LPS levels in 35 children with MCD or presumed MCD (idiopathic nephrotic syndrome [INS]) and in 18 healthy controls. RESULTS: LPS-injected mice showed morphological (foot process effacement, and normal appearing glomeruli on light microscopy) and molecular features (synaptopodin loss, nephrin mislocalization, FAK phosphorylation) characteristic of human MCD. Administration of a ß1 integrin inhibitor to mice abrogated FAK phosphorylation, and ameliorated proteinuria and podocyte injury following LPS. Children with MCD/INS in relapse had higher serum LPS levels than controls. In cultured human podocytes, ß1 integrin blockade prevented cytoskeletal rearrangements following exposure to MCD sera in relapse. CONCLUSIONS: Podocyte ß1 integrin activation is an upstream mediator of FAK phosphorylation and podocyte injury in models of MCD-like injury.
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Nefrosis Lipoidea , Síndrome Nefrótico , Podocitos , Niño , Ratones , Humanos , Animales , Nefrosis Lipoidea/inducido químicamente , Integrina beta1/metabolismo , Lipopolisacáridos/metabolismo , Modelos Teóricos , RecurrenciaRESUMEN
Renal arginine vasopressin receptor 2 (AVPR2) plays a crucial role in osmoregulation. Engagement of ligand with AVPR2 results in aquaporin 2 movement to the apical membrane and water reabsorption from the urinary filtrate. Despite this essential role, little is known about transcriptional regulation of Avpr2. Here, we identify novel roles for PAX2, a transcription factor crucial for kidney development, and its adaptor protein, Pax transcription interacting protein (PTIP), for epigenetic regulation of Avpr2 and thus body water balance. Chromatin immunoprecipitation (ChIP) from murine inner medulla cells (IMCD-3) identified the minimal DNA-binding region of PAX2 on the Avpr2 promoter. Regulation of Avpr2 by PAX2 was confirmed using a heterologous DNA expression system. PAX2 recruits the adaptor protein PTIP and its associated histone methyltransferase (HMT) complex to Avpr2 promoter, imposing epigenetic marks on this region and throughout the coding sequence that modulate Avpr2 gene transcription. Reduction of PAX2 or PTIP protein levels by siRNA prevented histone lysine methylation and expression of Avpr2. ChIP using mouse or human kidneys determined that PAX2 is highly enriched in the AVPR2 promoter alongside PTIP and HMT proteins, leading to high levels of histone H3 lysine trimethylation within the promoter and throughout the gene. In conclusion, PAX2 provides locus specificity for PTIP, allowing the HMT complex to impart epigenetic changes at the Avpr2 locus and regulate Avpr2 transcription. These finding have major implications for understanding regulation of body water balance.NEW & NOTEWORTHY The transcription factor PAX2 plays an indispensable role in kidney development. In the adult kidney, we identified the first described protein this protein regulates. PAX2 and its interacting partner Pax transcription interacting protein recruit a histone methyltransferase complex to the promoter and epigentically regulate the expression of arginine vasopressin receptor 2, a protein that plays a crucial role in osmoregulation in the distal tubule.
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Proteínas Portadoras/metabolismo , Epigénesis Genética/fisiología , Factor de Transcripción PAX2/metabolismo , Receptores de Vasopresinas/metabolismo , Animales , Núcleo Celular/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas Nucleares/metabolismoRESUMEN
BACKGROUND: Urinary CD80 has emerged as potential biomarker in idiopathic nephrotic syndrome (INS). However, its cellular source remains controversial. The aim of the study was to assess whether CD80 is truly expressed by glomerular cells in INS patients during relapse and in the LPS mouse model of podocyte injury. METHODS: The presence of CD80 in glomeruli was evaluated by combining immunostaining, immunogold labeling, and in situ hybridization techniques. RESULTS: CD80 was present along the surface of glomerular endothelial cells (GEC) and rarely in podocytes in six of nine minimal change disease (MCD) patients in relapse, two of eleven patients with focal segmental glomerulosclerosis in relapse, and absent in controls. In mice, CD80 was upregulated at mRNA and protein level in GEC and podocytes, in a similar pattern to that seen in MCD patients. CONCLUSIONS: Glomerular endothelial cells and podocytes can express CD80 in patients with MCD during relapse. A better understanding of the role of CD80 in glomerular cells may provide further insights into the mechanisms of proteinuria in INS.
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Antígeno B7-1/metabolismo , Células Endoteliales/metabolismo , Glomeruloesclerosis Focal y Segmentaria/diagnóstico , Nefrosis Lipoidea/diagnóstico , Podocitos/metabolismo , Adulto , Animales , Antígeno B7-1/orina , Biomarcadores/metabolismo , Biomarcadores/orina , Biopsia , Células Endoteliales/ultraestructura , Femenino , Glomeruloesclerosis Focal y Segmentaria/patología , Glomeruloesclerosis Focal y Segmentaria/orina , Humanos , Glomérulos Renales/citología , Glomérulos Renales/patología , Glomérulos Renales/ultraestructura , Masculino , Ratones , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Nefrosis Lipoidea/patología , Nefrosis Lipoidea/orina , Podocitos/ultraestructura , Recurrencia , Adulto JovenRESUMEN
Nephrin (Nphs1) is an adhesion protein that is expressed at the podocyte intercellular junction in the glomerulus. Nphs1 mutations in humans or deletion in animal genetic models results in a developmental failure of foot process formation. A number of studies have shown decrease in expression of nephrin in various proteinuric kidney diseases as well as in animal models of glomerular disease. Decrease in nephrin expression has been suggested to precede podocyte loss and linked to the progression of kidney disease. Whether the decrease in expression of nephrin is related to loss of podocytes or lead to podocyte detachment is unclear. To answer this central question we generated an inducible model of nephrin deletion (Nphs1Tam-Cre) in order to lower nephrin expression in healthy adult mice. Following tamoxifen-induction there was a 75% decrease in nephrin expression by 14 days. The Nphs1Tam-Cre mice had normal foot process ultrastructure and intact filtration barriers up to 4-6 weeks post-induction. Despite the loss of nephrin expression, the podocyte number and density remained unchanged during the initial period. Unexpectedly, nephrin expression, albeit at low levels persisted at the slit diaphragm up to 16-20 weeks post-tamoxifen induction. The mice became progressively proteinuric with glomerular hypertrophy and scarring reminiscent of focal and segmental glomerulosclerosis at 20 weeks. Four week-old Nphs1 knockout mice subjected to protamine sulfate model of podocyte injury demonstrated failure to recover from foot process effacement following heparin sulfate. Similarly, Nphs1 knockout mice failed to recover following nephrotoxic serum (NTS) with persistence of proteinuria and foot process effacement. Our results suggest that as in development, nephrin is necessary for maintenance of a healthy glomerular filter. In contrast to the developmental phenotype, lowering nephrin expression in a mature glomerulus resulted in a slowly progressive disease that histologically resembles FSGS a disease linked closely with podocyte depletion. Podocytes with low levels of nephrin expression are both susceptible and unable to recover following perturbation. Our results suggest that decreased nephrin expression independent of podocyte loss occurring as an early event in proteinuric kidney diseases might play a role in disease progression.
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Glomérulos Renales/citología , Glomérulos Renales/lesiones , Proteínas de la Membrana/metabolismo , Podocitos/metabolismo , Animales , Membrana Celular/metabolismo , Eliminación de Gen , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Podocitos/citología , Estabilidad Proteica , Proteinuria/genéticaRESUMEN
Nephrin is expressed at the basolateral aspect of podocytes and is an important signaling protein at the glomerular slit diaphragm. In vitro studies have demonstrated that Nephrin phosphorylation-dependent signaling is able to assemble a protein complex that is able to polymerize actin. However, proximal signaling events that result in nephrin tyrosine phosphorylation are not well understood. Nephrin deletion in mice and human nephrin mutations result in developmental failure of the podocyte intercellular junction resutling in proteinuria. This has been presumed to be due to a failure to respond to an external polarized cue in the absence of nephrin or a failure to transduce an outside-in signal in patients with nephrin mutations. The nephrin extracellular domain binds to itself or neph1 across the foot process intercellular junction. Nephrin is tyrosine phosphorylation-silent in healthy glomeruli when presumably the nephrin extracellular domain is in an engaged state. These observations raise the possibility of an alternate proximal signaling mechanism that might be responsible for nephrin tyrosine phosphorylation. Here we present data showing that integrin engagement at the basal aspect of cultured podocytes results in nephrin tyrosine phosphorylation. This is abrogated by incubating podocytes with an antibody that prevents integrin ß1 ligation and activation in response to binding to extracellular matrix. Furthermore, nephrin tyrosine phosphorylation was observed in podocytes expressing a membrane-targeted nephrin construct that lacks the extracellular domain. We propose, integrin-activation based signaling might be responsible for nephrin phosphorylation rather than engagment of the nephrin extracellular domain by a ligand.
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Integrinas/metabolismo , Proteínas de la Membrana/metabolismo , Tirosina/metabolismo , Animales , Células HEK293 , Humanos , Integrina beta1/análisis , Integrina beta1/metabolismo , Proteínas de la Membrana/análisis , Ratones , Fosforilación , Transducción de SeñalRESUMEN
The mechanisms by which the glomerular filtration barrier prevents the loss of large macromolecules and simultaneously, maintains the filter remain poorly understood. Recent studies proposed that podocytes have an active role in both the endocytosis of filtered macromolecules and the maintenance of the filtration barrier. Deletion of a key endosomal trafficking regulator, the class 3 phosphatidylinositol (PtdIns) 3-kinase vacuolar protein sorting 34 (Vps34), in podocytes results in aberrant endosomal membrane morphology and podocyte dysfunction. We recently showed that the vacuolation phenotype in cultured Vps34-deficient podocytes is caused by the absence of a substrate for the Vps34 downstream effector PtdIns 3-phosphate 5-kinase (PIKfyve), which phosphorylates Vps34-generated PtdIns(3)P to produce PtdIns (3,5)P2. PIKfyve perturbation and PtdIns(3,5)P2 reduction result in massive membrane vacuolation along the endosomal system, but the cell-specific functions of PIKfyve in vivo remain unclear. We show here that the genetic deletion of PIKfyve in endocytically active proximal tubular cells resulted in the development of large cytoplasmic vacuoles caused by arrested endocytic traffic progression at a late-endosome stage. In contrast, deletion of PIKfyve in glomerular podocytes did not significantly alter the endosomal morphology, even in age 18-month-old mice. However, on culturing, the PIKfyve-deleted podocytes developed massive cytoplasmic vacuoles. In summary, these data suggest that glomerular podocytes and proximal tubules have different requirements for PIKfyve function, likely related to distinct in vivo needs for endocytic flux.
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Fosfatidilinositol 3-Quinasas Clase III/fisiología , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/enzimología , Fosfatidilinositol 3-Quinasas/fisiología , Podocitos/enzimología , Animales , Endosomas , Glomérulos Renales , Ratones , Fosfatidilinositol 3-Quinasas/genéticaRESUMEN
In most forms of glomerular diseases, loss of size selectivity by the kidney filtration barrier is associated with changes in the morphology of podocytes. The kidney filtration barrier is comprised of the endothelial lining, the glomerular basement membrane, and the podocyte intercellular junction, or slit diaphragm. The cell adhesion proteins nephrin and neph1 localize to the slit diaphragm and transduce signals in a Src family kinase Fyn-mediated tyrosine phosphorylation-dependent manner. Studies in cell culture suggest nephrin phosphorylation-dependent signaling events are primarily involved in regulation of actin dynamics and lamellipodium formation. Nephrin phosphorylation is a proximal event that occurs both during development and following podocyte injury. We hypothesized that abrogation of nephrin phosphorylation following injury would prevent nephrin-dependent actin remodeling and foot process morphological changes. Utilizing a biased screening approach, we found nonreceptor Src homology 2 (sh2) domain-containing phosphatase Shp2 to be associated with phosphorylated nephrin. We observed an increase in nephrin tyrosine phosphorylation in the presence of Shp2 in cell culture studies. In the human glomerulopathies minimal-change nephrosis and membranous nephropathy, there is an increase in Shp2 phosphorylation, a marker of increased Shp2 activity. Mouse podocytes lacking Shp2 do not develop foot process spreading when subjected to podocyte injury in vivo using protamine sulfate or nephrotoxic serum (NTS). In the NTS model, we observed a lack of foot process spreading in mouse podocytes with Shp2 deleted and smaller amounts of proteinuria. Taken together, these results suggest that Shp2-dependent signaling events are necessary for changes in foot process structure and function following injury.
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Riñón/patología , Proteínas de la Membrana/metabolismo , Nefritis/patología , Podocitos/patología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Tirosina/metabolismo , Secuencia de Aminoácidos , Animales , Modelos Animales de Enfermedad , Eliminación de Gen , Humanos , Riñón/metabolismo , Proteínas de la Membrana/química , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Nefritis/inducido químicamente , Nefritis/metabolismo , Fosforilación , Podocitos/metabolismo , Protaminas , Mapas de Interacción de Proteínas , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Tirosina/análisisRESUMEN
The evolutionarily conserved PIKfyve, which synthesizes PtdIns5P from PtdIns, and PtdIns(3,5)P2 from PtdIns3P, requires PtdIns3P as both an enzyme substrate and a membrane recruitment signal. Whereas the PtdIns3P source is undetermined, class III PI3K (Vps34), the only evolutionarily conserved of the eight mammalian PI3Ks, is presumed as a main candidate. A hallmark of PIKfyve deficiency is formation of multiple translucent cytoplasmic vacuoles seen by light microscopy in cells cultured in complete media. Such an aberrant phenotype is often observed in cells from conditional Vps34 knockout (KO) mice. To clarify the mechanism of Vps34 KO-triggered vacuolation and the PtdIns3P source for PIKfyve functionality, here we have characterized a podocyte cell type derived from Vps34fl/fl mice, which, upon Cre-mediated gene KO, robustly formed cytoplasmic vacuoles resembling those in PikfyveKO MEFs. Vps34wt, expressed in Vps34KO podocytes restored the normal morphology, but only if the endogenous PIKfyve activity was intact. Conversely, expressed PIKfyvewt rescued completely the vacuolation only in PikfyveKO MEFs but not in Vps34KO podocytes. Analyses of phosphoinositide profiles by HPLC and localization patterns by a PtdIns3P biosensor revealed that Vps34 is the main supplier of localized PtdIns3P not only for PIKfyve activity but also for membrane recruitment. Concordantly, Vps34KO podocytes had severely reduced steady-state levels of both PtdIns(3,5)P2 and PtdIns5P, along with PtdIns3P. We further revealed a plausible physiologically-relevant Vps34-independent PtdIns3P supply for PIKfyve, operating through activated class I PI3Ks. Our data provide the first evidence that the vacuolation phenotype in Vps34KO podocytes is due to PIKfyve dysfunction and that Vps34 is a main PtdIns3P source for constitutive PIKfyve functionality.
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Membrana Celular/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Homeostasis , Membranas Intracelulares/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Podocitos/metabolismo , Transducción de Señal , Animales , Membrana Celular/ultraestructura , Medios de Cultivo , Eliminación de Gen , Ratones Noqueados , Fenotipo , Podocitos/ultraestructura , Especificidad por Sustrato , Vacuolas/metabolismo , Vacuolas/ultraestructuraRESUMEN
The reduction in podocyte density to levels below a threshold value drives glomerulosclerosis and progression to ESRD. However, technical demands prohibit high-throughput application of conventional morphometry for estimating podocyte density. We evaluated a method for estimating podocyte density using single paraffin-embedded formalin-fixed sections. Podocyte nuclei were imaged using indirect immunofluorescence detection of antibodies against Wilms' tumor-1 or transducin-like enhancer of split 4. To account for the large size of podocyte nuclei in relation to section thickness, we derived a correction factor given by the equation CF=1/(D/T+1), where T is the tissue section thickness and D is the mean caliper diameter of podocyte nuclei. Normal values for D were directly measured in thick tissue sections and in 3- to 5-µm sections using calibrated imaging software. D values were larger for human podocyte nuclei than for rat or mouse nuclei (P<0.01). In addition, D did not vary significantly between human kidney biopsies at the time of transplantation, 3-6 months after transplantation, or with podocyte depletion associated with transplant glomerulopathy. In rat models, D values also did not vary with podocyte depletion, but increased approximately 10% with old age and in postnephrectomy kidney hypertrophy. A spreadsheet with embedded formulas was created to facilitate individualized podocyte density estimation upon input of measured values. The correction factor method was validated by comparison with other methods, and provided data comparable with prior data for normal human kidney transplant donors. This method for estimating podocyte density is applicable to high-throughput laboratory and clinical use.
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Técnica del Anticuerpo Fluorescente Indirecta , Podocitos/citología , Animales , Biopsia , Recuento de Células , Femenino , Humanos , Masculino , Ratones , Proteínas Nucleares/análisis , Proteínas Nucleares/inmunología , Ratas , Estándares de Referencia , Proteínas Represoras/análisis , Proteínas Represoras/inmunología , Manejo de Especímenes , Proteínas WT1/análisis , Proteínas WT1/inmunologíaRESUMEN
BACKGROUND: Proteinuria and/or albuminuria are widely used for noninvasive assessment of kidney diseases. However, proteinuria is a nonspecific marker of diverse forms of kidney injury, physiologic processes and filtration of small proteins of monoclonal and other pathologic processes. The opportunity to develop new glomerular disease biomarkers follows the realization that the degree of podocyte depletion determines the degree of glomerulosclerosis, and if persistent, determines the progression to end-stage kidney disease (ESKD). Podocyte cell lineage-specific mRNAs can be recovered in urine pellets of model systems and in humans. In model systems, progressive glomerular disease is associated with decreased nephrin mRNA steady-state levels compared with podocin mRNA. Thus, the urine podocin:nephrin mRNA ratio (PNR) could serve as a useful progression biomarker. The use of podocyte-specific transcript ratios also circumvents many problems inherent to urine assays. METHODS: To test this hypothesis, the human diphtheria toxin receptor (hDTR) rat model of progression was used to evaluate potentially useful urine mRNA biomarkers. We compared histologic progression parameters (glomerulosclerosis score, interstitial fibrosis score and percent of podocyte depletion) with clinical biomarkers [serum creatinine, systolic blood pressure (BP), 24-h urine volume, 24-h urine protein excretion and the urine protein:creatinine ratio(PCR)] and with the novel urine mRNA biomarkers. RESULTS: The PNR correlated with histologic outcome as well or better than routine clinical biomarkers and other urine mRNA biomarkers in the model system with high specificity and sensitivity, and a low coefficient of assay variation. CONCLUSIONS: We concluded that the PNR, used in combination with proteinuria, will be worth testing for its clinical diagnostic and decision-making utility.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Enfermedades Renales/metabolismo , Glomérulos Renales/metabolismo , Proteínas de la Membrana/metabolismo , Podocitos/metabolismo , Proteinuria/metabolismo , ARN Mensajero/orina , Animales , Biomarcadores/metabolismo , Biomarcadores/orina , Humanos , Péptidos y Proteínas de Señalización Intracelular/orina , Enfermedades Renales/patología , Glomérulos Renales/patología , Proteínas de la Membrana/orina , Podocitos/patología , Proteinuria/patología , Ratas , Ratas Endogámicas F344 , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Podocyte depletion leads to glomerulosclerosis, but whether an impaired capacity of podocytes to respond to hypertrophic stress also causes glomerulosclerosis is unknown. We generated transgenic Fischer 344 rats that express a dominant negative AA-4E-BP1 transgene driven by the podocin promoter; a member of the mammalian target of rapamycin complex 1 (mTORC1) pathway, 4E-BP1 modulates cap-dependent translation, which is a key determinant of a cell's hypertrophic response to nutrients and growth factors. AA-4E-BP1 rat podocytes expressed the transgene and had normal kidney histology and protein excretion at 100 g of body weight but developed ESRD by 12 months. Proteinuria and glomerulosclerosis were linearly related to both increasing body weight and transgene dose. Uni-nephrectomy reduced the body weight at which proteinuria first developed by 40%-50%. The initial histologic manifestation of disease was the appearance of bare areas of glomerular basement membrane from the pulling apart of podocyte foot processes, followed by adhesions to the Bowman capsule. Morphometric analysis confirmed the mismatch between glomerular tuft volume and total podocyte volume (number × size) per tuft in relation to weight gain and nephrectomy. Proteinuria and glomerulosclerosis did not develop if dietary calorie restriction prevented weight gain and glomerular enlargement. In summary, failure of podocytes to match glomerular tuft growth in response to growth signaling through the mTORC1 pathway can trigger proteinuria, glomerulosclerosis, and progression to ESRD. Reducing body weight and glomerular growth may be useful adjunctive therapies to slow or prevent progression to ESRD.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Glomeruloesclerosis Focal y Segmentaria/etiología , Glomérulos Renales/crecimiento & desarrollo , Fosfoproteínas/metabolismo , Podocitos/fisiología , Aumento de Peso , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Restricción Calórica , Proteínas de Ciclo Celular , Glomeruloesclerosis Focal y Segmentaria/prevención & control , Heterocigoto , Homocigoto , Humanos , Hipertrofia , Masculino , Nefrectomía , Fosfoproteínas/genética , Podocitos/patología , Proteinuria/etiología , Proteinuria/prevención & control , Ratas , Ratas Endogámicas F344 , Ratas TransgénicasRESUMEN
Podocyte depletion is a major mechanism driving glomerulosclerosis. Progression is the process by which progressive glomerulosclerosis leads to end stage kidney disease (ESKD). In order to determine mechanisms contributing to persistent podocyte loss, we used a human diphtheria toxin transgenic rat model. After initial diphtheria toxin-induced podocyte injury (over 30% loss in 4 weeks), glomeruli became destabilized, resulting in continued autonomous podocyte loss causing global podocyte depletion (ESKD) by 13 weeks. This was monitored by urine mRNA analysis and by quantitating podocytes in glomeruli. Similar patterns of podocyte depletion were found in the puromycin aminonucleoside and 5/6 nephrectomy rat models of progressive end-stage disease. Angiotensin II blockade (combined enalapril and losartan) restabilized the glomeruli, and prevented continuous podocyte loss and progression to ESKD. Discontinuing angiotensin II blockade resulted in recurrent glomerular destabilization, podocyte loss, and progression to ESKD. Reduction in blood pressure alone did not reduce proteinuria or prevent podocyte loss from destabilized glomeruli. The protective effect of angiotensin II blockade was entirely accounted for by reduced podocyte loss. Thus, an initiating event resulting in a critical degree of podocyte depletion can destabilize glomeruli and initiate a superimposed angiotensin II-dependent podocyte loss process that accelerates progression resulting in eventual global podocyte depletion and ESKD. These events can be monitored noninvasively in real-time through urine mRNA assays.
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Angiotensina II/metabolismo , Fallo Renal Crónico/metabolismo , Fallo Renal Crónico/patología , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Podocitos/metabolismo , Podocitos/patología , Angiotensina II/antagonistas & inhibidores , Animales , Antihipertensivos/farmacología , Modelos Animales de Enfermedad , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Fallo Renal Crónico/tratamiento farmacológico , Fallo Renal Crónico/genética , Masculino , Proteínas de la Membrana/genética , Podocitos/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Ratas TransgénicasRESUMEN
Actin dynamics has emerged at the forefront of podocyte biology. Slit diaphragm junctional adhesion protein Nephrin is necessary for development of the podocyte morphology and transduces phosphorylation-dependent signals that regulate cytoskeletal dynamics. The present study extends our understanding of Nephrin function by showing in cultured podocytes that Nephrin activation induced actin dynamics is necessary for lamellipodia formation. Upon activation Nephrin recruits and regulates a protein complex that includes Ship2 (SH2 domain containing 5' inositol phosphatase), Filamin and Lamellipodin, proteins important in regulation of actin and focal adhesion dynamics, as well as lamellipodia formation. Using the previously described CD16-Nephrin clustering system, Nephrin ligation or activation resulted in phosphorylation of the actin crosslinking protein Filamin in a p21 activated kinase dependent manner. Nephrin activation in cell culture results in formation of lamellipodia, a process that requires specialized actin dynamics at the leading edge of the cell along with focal adhesion turnover. In the CD16-Nephrin clustering model, Nephrin ligation resulted in abnormal morphology of actin tails in human podocytes when Ship2, Filamin or Lamellipodin were individually knocked down. We also observed decreased lamellipodia formation and cell migration in these knock down cells. These data provide evidence that Nephrin not only initiates actin polymerization but also assembles a protein complex that is necessary to regulate the architecture of the generated actin filament network and focal adhesion dynamics.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Contráctiles/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Complejos Multiproteicos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Seudópodos/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Membrana Celular/metabolismo , Filaminas , Técnicas de Silenciamiento del Gen , Humanos , Inositol Polifosfato 5-Fosfatasas , Ratones , Modelos Biológicos , Proteínas Oncogénicas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Fosforilación , Fosfotirosina/metabolismo , Podocitos/metabolismo , Podocitos/patología , Protaminas/metabolismo , Unión Proteica , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Quinasas p21 Activadas/metabolismo , Dominios Homologos srcRESUMEN
Actin dynamics determines podocyte morphology during development and in response to podocyte injury and might be necessary for maintaining normal podocyte morphology. Because podocyte intercellular junction receptor Nephrin plays a role in regulating actin dynamics, and given the described role of cofilin in actin filament polymerization and severing, we hypothesized that cofilin-1 activity is regulated by Nephrin and is necessary in normal podocyte actin dynamics. Nephrin activation induced cofilin dephosphorylation via intermediaries that include phosphatidylinositol 3-kinase, SSH1, 14-3-3, and LIMK in a cell culture model. This Nephrin-induced cofilin activation required a direct interaction between Nephrin and the p85 subunit of phosphatidylinositol 3-kinase. In a similar fashion, cofilin-1 dephosphorylation was observed in a rat model of podocyte injury at a time when foot process spreading is initially observed. To investigate the necessity of cofilin-1 in the glomerulus, podocyte-specific Cfl1 null mice were generated. Cfl1 null podocytes developed normally. However, these mice developed persistent proteinuria by 3 months of age, although they did not exhibit foot process spreading until 8 months, when the rate of urinary protein excretion became more exaggerated. In a mouse model of podocyte injury, protamine sulfate perfusion of the Cfl1 mutant mouse induced a broadened and flattened foot process morphology that was distinct from that observed following perfusion of control kidneys, and mutant podocytes did not recover normal structure following additional perfusion with heparin sulfate. We conclude that cofilin-1 is necessary for maintenance of normal podocyte architecture and for actin structural changes that occur during induction and recovery from podocyte injury.
Asunto(s)
Cofilina 1/metabolismo , Podocitos/metabolismo , Actinas/metabolismo , Albuminuria/metabolismo , Animales , Línea Celular , Femenino , Eliminación de Gen , Técnicas de Silenciamiento del Gen , Humanos , Quinasas Lim/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Especificidad de Órganos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Podocitos/patología , Podocitos/ultraestructura , Protaminas , Unión Proteica , Seudópodos/metabolismo , Seudópodos/ultraestructura , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Because loss of podocytes associates with glomerulosclerosis, monitoring podocyte loss by measuring podocyte products in urine may be clinically useful. To determine whether a single episode of podocyte injury would cause persistent podocyte loss, we induced limited podocyte depletion using a diphtheria toxin receptor (hDTR) transgenic rat. We monitored podocyte loss by detecting nephrin and podocin mRNA in urine particulates with quantitative reverse transcriptase-PCR. Aquaporin 2 mRNA served as a kidney reference gene to account for variable kidney contribution to RNA amount and quality. We found that a single injection of diphtheria toxin resulted in an initial peak of proteinuria and podocyte mRNAs (podocin and nephrin) followed 8 d later by a second peak of proteinuria and podocyte mRNAs that were podocin positive but nephrin negative. Proteinuria that persisted for months correlated with podocin-positive, nephrin-negative mRNAs in urine. Animals with persistent podocyte mRNA in urine progressed to ESRD with global podocyte depletion and interstitial scarring. Podocytes in ectatic tubules expressed podocalyxin and podocin proteins but not nephrin, compatible with detached podocytes' having an altered phenotype. Parallel human studies showed that biopsy-proven glomerular injury associated with increased urinary podocin:aquaporin 2 and nephrin:aquaporin 2 molar ratios. We conclude that a single episode of podocyte injury can trigger glomerular destabilization, resulting in persistent podocyte loss and an altered phenotype of podocytes recovered from urine. Podocyte mRNAs in urine may be a useful clinical tool for the diagnosis and monitoring of glomerular diseases.