RESUMEN
BACKGROUND: CD2-associated protein (CD2AP), a slit diaphragm-associated scaffolding protein involved in survival and regulation of the cytoskeleton in podocytes, is considered a "stabilizer" of the slit diaphragm complex that connects the slit diaphragm protein nephrin to the cytoskeleton of the cell. Tyrosine phosphorylation of slit diaphragm molecules can influence their surface expression, but it is unknown whether tyrosine phosphorylation events of CD2AP are also physiologically relevant to slit diaphragm stability. METHODS: We used isoelectric focusing, western blot analysis, and immunofluorescence to investigate phosphorylation of CD2AP, and phospho-CD2AP antibodies and site-directed mutagenesis to define the specific phosphorylated tyrosine residues. We used cross-species rescue experiments in Cd2apKD zebrafish and in Drosophila cindrRNAi mutants to define the physiologic relevance of CD2AP phosphorylation of the tyrosine residues. RESULTS: We found that VEGF-A stimulation can induce a tyrosine phosphorylation response in CD2AP in podocytes, and that these phosphorylation events have an important effect on slit diaphragm protein localization and functionality in vivo. We demonstrated that tyrosine in position Y10 of the SH3-1 domain of CD2AP is indispensable for CD2AP function in vivo. We found that the binding affinity of nephrin to CD2AP is significantly enhanced in the absence of Y10; however, unexpectedly, this increased affinity leads not to stabilization but to functional impairment of the glomerular filtration barrier. CONCLUSIONS: Our findings provide insight into CD2AP and its phosphorylation in the context of slit diaphragm functionality, and indicate a fine-tuned affinity balance of CD2AP and nephrin that is influenced by receptor tyrosine kinase stimulation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de la Membrana/química , Tirosina/metabolismo , Animales , Drosophila melanogaster , Células HEK293 , Humanos , Proteínas de la Membrana/metabolismo , Fosforilación , Podocitos/metabolismo , Estabilidad Proteica , Factor A de Crecimiento Endotelial Vascular/farmacología , Pez CebraRESUMEN
The presence of B-cell clusters in allogenic T cell-mediated rejection (TCMR) of kidney allografts is linked to more severe disease entities. In this study we characterized B-cell infiltrates in patients with TCMR and examined the role of serum CXCL-13 in these patients and experimentally. CXCL-13 serum levels were analyzed in 73 kidney allograft recipients at the time of allograft biopsy. In addition, four patients were evaluated for CXCL13 levels during the first week after transplantation. ELISA was done to measure CXCL-13 serum levels. For further mechanistic understanding, a translational allogenic kidney transplant (ktx) mouse model for TCMR was studied in BalbC recipients of fully mismatched transplants with C57BL/6 donor kidneys. CXCL-13 serum levels were measured longitudinally, CD20 and CD3 composition and CXCL13 mRNA in tissue were examined by flow cytometry and kidneys were examined by histology and immunohistochemistry. We found significantly higher serum levels of the B-cell chemoattractant CXCL13 in patients with TCMR compared to controls and patients with borderline TCMR. Moreover, in patients with acute rejection within the first week after ktx, a >5-fold CXCL13 increase was measured and correlated with B-cell infiltrates in the biopsies. In line with the clinical findings, TCMR in mice correlated with increased systemic serum-CXCL13 levels. Moreover, renal allografts had significantly higher CXCL13 mRNA expression than isogenic controls and showed interstitial CD20+ B-cell clusters and CD3+ cell infiltrates accumulating in the vicinity of renal vessels. CXCL13 blood levels correlate with B-cell involvement in TCMR and might help to identify patients at risk of a more severe clinical course of rejection.
Asunto(s)
Quimiocina CXCL13/sangre , Rechazo de Injerto/sangre , Trasplante de Riñón/efectos adversos , Adulto , Animales , Linfocitos B/inmunología , Biomarcadores/sangre , Rechazo de Injerto/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Persona de Mediana Edad , Linfocitos T/inmunologíaRESUMEN
Different glomerular diseases that affect podocyte homeostasis can clinically present as nephrotic syndrome with massive proteinuria, hypoalbuminemia, hyperlipidemia and edema. Up to now, no drugs that specifically target the actin cytoskeleton of podocytes are on the market and model systems for library screenings to develop anti-proteinuric drugs are of high interest. We developed a standardized proteinuria model in zebrafish using puromycin aminonucleoside (PAN) via treatment in the fish water to allow for further drug testing to develop anti-proteinuric drugs for the treatment of glomerular diseases. We noticed that fish that carry the nacre-mutation show a significantly higher susceptibility for the disruption of the glomerular filtration barrier following PAN treatment, which results in a more pronounced proteinuria phenotype. Nacre zebrafish inherit a mutation yielding a truncated version of microphthalmia-associated transcription factor/melanogenesis associated transcription factor (mitf). We hypothesized that the nacre mutation may lead to reduced formin expression and defects in cytoskeletal rearrangement. Based on the observations in zebrafish, we carried out a PAN treatment on cultured human podocytes after knockdown with MITF siRNA causing a rearrangement of the actin cytoskeleton.
RESUMEN
Kidney disease has been linked to dysregulated signaling via PKC in kidney cells such as podocytes. PKCα is a conventional isoform of PKC and a well-known binding partner of ß-catenin, which promotes its degradation. ß-Catenin is the main effector of the canonical Wnt pathway and is critical in cell adhesion. However, whether other PKC isoforms interact with ß-catenin has not been studied systematically. Here we demonstrate that PKCϵ-deficient mice, which develop proteinuria and glomerulosclerosis, display lower ß-catenin expression compared with PKC wild-type mice, consistent with an altered phenotype of podocytes in culture. Remarkably, ß-catenin showed a reversed subcellular localization pattern: Although ß-catenin exhibited a perinuclear pattern in undifferentiated wild-type cells, it predominantly localized to the nucleus in PKCϵ knockout cells. Phorbol 12-myristate 13-acetate stimulation of both cell types revealed that PKCϵ positively regulates ß-catenin expression and stabilization in a glycogen synthase kinase 3ß-independent manner. Further, ß-catenin overexpression in PKCϵ-deficient podocytes could restore the wild-type phenotype, similar to rescue with a PKCϵ construct. This effect was mediated by up-regulation of P-cadherin and the ß-catenin downstream target fascin1. Zebrafish studies indicated three PKCϵ-specific phosphorylation sites in ß-catenin that are required for full ß-catenin function. Co-immunoprecipitation and pulldown assays confirmed PKCϵ and ß-catenin as binding partners and revealed that ablation of the three PKCϵ phosphorylation sites weakens their interaction. In summary, we identified a novel pathway for regulation of ß-catenin levels and define PKCϵ as an important ß-catenin interaction partner and signaling opponent of other PKC isoforms in podocytes.
Asunto(s)
Podocitos/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Procesamiento Proteico-Postraduccional , beta Catenina/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Sustitución de Aminoácidos , Animales , Bioensayo , Carcinógenos/toxicidad , Línea Celular Transformada , Regulación de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células HEK293 , Humanos , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Mutación , Fosforilación/efectos de los fármacos , Podocitos/citología , Podocitos/efectos de los fármacos , Proteína Quinasa C-epsilon/antagonistas & inhibidores , Proteína Quinasa C-epsilon/genética , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/toxicidad , Técnicas de Cultivo de Tejidos , beta Catenina/antagonistas & inhibidores , beta Catenina/química , beta Catenina/genéticaRESUMEN
Diabetic nephropathy (DN) is the major cause of end-stage renal disease worldwide. Podocytes are important for glomerular filtration barrier function and maintenance of size selectivity in protein filtration in the kidney. Podocyte damage is the basis of many glomerular diseases characterized by loss of interdigitating foot processes and decreased expression of components of the slit diaphragm. Nephrin, a podocyte-specific protein, is the main component of the slit diaphragm. Loss of nephrin is observed in human and rodent models of diabetic kidney disease. The long isoform of CIN85 (RukL) is a binding partner of nephrin that mediates nephrin endocytosis via ubiquitination in podocytes. Here we demonstrate that the loss of nephrin expression and the onset of proteinuria in diabetic mice correlate with an increased accumulation of ubiquitinated proteins and expression of CIN85/RukL in podocytes. CIN85/RukL deficiency preserved nephrin surface expression on the slit diaphragm and reduced proteinuria in diabetic mice, whereas overexpression of CIN85 in zebrafish induced severe edema and disruption of the filtration barrier. Thus, CIN85/RukL is involved in endocytosis of nephrin in podocytes under diabetic conditions, causing podocyte depletion and promoting proteinuria. CIN85/RukL expression therefore shows potential to be a novel target for antiproteinuric therapy in diabetes.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Endocitosis/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteinuria/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Creatinina/metabolismo , Diabetes Mellitus Experimental/genética , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/metabolismo , Endocitosis/genética , Genotipo , Humanos , Ratones , Microscopía Electrónica de Transmisión , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Podocitos/metabolismo , Podocitos/ultraestructura , Proteinuria/genéticaRESUMEN
Dysregulation of the actin cytoskeleton in podocytes represents a common pathway in the pathogenesis of proteinuria across a spectrum of chronic kidney diseases (CKD). The GTPase dynamin has been implicated in the maintenance of cellular architecture in podocytes through its direct interaction with actin. Furthermore, the propensity of dynamin to oligomerize into higher-order structures in an actin-dependent manner and to cross-link actin microfilaments into higher-order structures has been correlated with increased actin polymerization and global organization of the actin cytoskeleton in the cell. We found that use of the small molecule Bis-T-23, which promotes actin-dependent dynamin oligomerization and thus increased actin polymerization in injured podocytes, was sufficient to improve renal health in diverse models of both transient kidney disease and CKD. In particular, administration of Bis-T-23 in these renal disease models restored the normal ultrastructure of podocyte foot processes, lowered proteinuria, lowered collagen IV deposits in the mesangial matrix, diminished mesangial matrix expansion and extended lifespan. These results further establish that alterations in the actin cytoskeleton of kidney podocytes is a common hallmark of CKD, while also underscoring the substantial regenerative potential of injured glomeruli and identifying the oligomerization cycle of dynamin as an attractive potential therapeutic target to treat CKD.
Asunto(s)
Ácidos Cumáricos/administración & dosificación , Cianoacrilatos/administración & dosificación , Dinaminas/metabolismo , Podocitos/efectos de los fármacos , Proteinuria/tratamiento farmacológico , Insuficiencia Renal Crónica/tratamiento farmacológico , Acrilamida/administración & dosificación , Citoesqueleto de Actina/efectos de los fármacos , Animales , Dinaminas/química , Dinaminas/efectos de los fármacos , Humanos , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/patología , Glomérulos Renales/ultraestructura , Ratones , Modelos Animales , Podocitos/patología , Podocitos/ultraestructura , Proteinuria/metabolismo , Proteinuria/patología , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Pez CebraRESUMEN
Zinc-α2-glycoprotein (AZGP1) is a secreted protein synthesized by epithelial cells and adipocytes that has roles in lipid metabolism, cell cycling, and cancer progression. Our previous findings in AKI indicated a new role for AZGP1 in the regulation of fibrosis, which is a unifying feature of CKD. Using two models of chronic kidney injury, we now show that mice with genetic AZGP1 deletion develop significantly more kidney fibrosis. This destructive phenotype was rescued by injection of recombinant AZGP1. Exposure of AZGP1-deficient mice to cardiac stress by thoracic aortic constriction revealed that antifibrotic effects were not restricted to the kidney but were cardioprotective. In vitro, recombinant AZGP1 inhibited kidney epithelial dedifferentiation and antagonized fibroblast activation by negatively regulating TGF-ß signaling. Patient sera with high levels of AZGP1 similarly attenuated TGF-ß signaling in fibroblasts. Taken together, these findings indicate a novel role for AZGP1 as a negative regulator of fibrosis progression, suggesting that recombinant AZGP1 may have translational effect for treating fibrotic disease.
Asunto(s)
Fallo Renal Crónico/genética , Riñón/metabolismo , Miocardio/metabolismo , Proteínas de Plasma Seminal/metabolismo , Adipoquinas , Animales , Aorta/patología , Proteínas Portadoras/metabolismo , Diferenciación Celular , Epitelio/patología , Fibroblastos/metabolismo , Fibrosis/patología , Eliminación de Gen , Glicoproteínas/metabolismo , Células HEK293 , Humanos , Riñón/patología , Enfermedades Renales/metabolismo , Fallo Renal Crónico/metabolismo , Masculino , Ratones , Miocardio/patología , Fosforilación , Biosíntesis de Proteínas , Ratas , Proteínas Recombinantes/química , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Obstrucción Ureteral/patología , Zn-alfa-2-GlicoproteínaRESUMEN
The early glomerular changes in diabetes include a podocyte phenotype with loss of slit diaphragm proteins, changes in the actin cytoskeleton and foot process architecture. This review focuses on the role of the protein kinase C (PKC) family in podocytes and points out the differential roles of classical, novel, and atypical PKCs in podocytes. Some PKC isoforms are indispensable for proper glomerular development and slit diaphragm maintenance, whereas others might be harmful when activated in the diabetic milieu. Therefore, some might be interesting treatment targets in the early phase of diabetes.
RESUMEN
Podocyte effacement and the reformation of foot processes and slit diaphragms can be induced within minutes experimentally. Therefore, it seems likely that the slit diaphragm proteins underlie orchestrated recycling mechanisms under the control of posttranslational modifiers. One of these modifiers, SUMO (small ubiquitin-like modifier), is an ubiquitin-like protein with a 20% corresponding identity to ubiquitin. Modification by SUMOs to proteins on lysine residues can block the ubiquitination of the same site leading to the stabilization of the target protein. Here we found in vitro and in vivo that nephrin is a substrate modified by SUMO proteins thereby increasing its steady-state level and expression at the plasma membrane. A conversion of lysines to arginines at positions 1114 and 1224 of the intracellular tail of murine nephrin led to decreased stability of nephrin, decreased expression at the plasma membrane, and decreased PI3K/AKT signaling. Furthermore, treatment of podocytes with the SUMOylation inhibitor ginkgolic acid led to reduced membrane expression of nephrin. Similarly, the conversion of lysine to arginine at position 1100 of human nephrin caused decreased stability and expression at the plasma membrane. As SUMOylation is a reversible process, our results suggest that SUMOylation participates in the tight orchestration of nephrin turnover at the slit diaphragm.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Podocitos , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación , Animales , Arginina/metabolismo , Células HEK293/metabolismo , Humanos , Glomérulos Renales/química , Lisina/metabolismo , Masculino , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Ratones , Podocitos/química , Proteinuria/inducido químicamente , Salicilatos/farmacología , Transducción de Señal , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/análisis , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Sumoilación/efectos de los fármacos , Transfección , Enzimas Ubiquitina-Conjugadoras/análisisRESUMEN
BACKGROUND: The majority of sera from patients with primary membranous nephropathy have autoantibodies against the M-type phospholipase A2 receptor (PLA2R) which is expressed on human podocytes. The rabbit variant of PLA2R attaches to collagen type IV via the fibronectin type II domain, which is also present in the human variant of PLA2R. DESIGN: To assess whether the human PLA2R variant is also involved in attachment to collagen type IV, we conducted a cell adhesion assay on a collagen-coated surface using PLA2R-transfected and mock-transfected human embryonic kidney (HEK) cells. To test the hypothesis that sera from patients containing anti-PLA2R antibodies interfere with the adhesion of podocytes to collagen, we performed cell adhesion assays on a collagen type IV-coated surface using positive and negative serum samples from patients and cultured human podocytes in vitro expressing PLA2R. RESULTS: The HEK cell adhesion assay confirmed an enhanced attachment of PLA2R-transfected cells to collagen type IV. We confirmed diminished podocyte adhesion in the presence of serum with anti-PLA2R antibodies. The concentration of anti-PLA2R antibodies correlated with proteinuria and to the degree of diminished adhesion of podocytes. CONCLUSIONS: We demonstrated that serum of patients containing autoantibodies directed to PLA2R interferes with the ability of podocytes to attach to collagen type IV in vitro, providing evidence of a serum soluble pathogenic factor interfering with podocyte adhesion in membranous nephropathy.
Asunto(s)
Autoanticuerpos/farmacología , Adhesión Celular/fisiología , Colágeno Tipo IV/fisiología , Podocitos/fisiología , Receptores de Fosfolipasa A2/inmunología , Suero/fisiología , Adulto , Anciano , Estudios de Casos y Controles , Colágeno Tipo IV/metabolismo , Femenino , Glomerulonefritis Membranosa/fisiopatología , Células HEK293 , Humanos , Masculino , Persona de Mediana Edad , Receptores de Fosfolipasa A2/metabolismo , Adulto JovenRESUMEN
The atypical protein kinase C (aPKC) isotypes PKCλ/ι and PKCζ are both expressed in podocytes; however, little is known about differences in their function. Previous studies in mice have demonstrated that podocyte-specific loss of PKCλ/ι leads to a severe glomerular phenotype, whereas mice deficient in PKCζ develop no renal phenotype. We analyzed various effects caused by PKCλ/ι and PKCζ deficiency in cultured murine podocytes. In contrast to PKCζ-deficient podocytes, PKCλ/ι-deficient podocytes exhibited a severe actin cytoskeletal phenotype, reduced cell size, decreased number of focal adhesions, and increased activation of small GTPases. Comparative microarray analysis revealed that the guanine nucleotide exchange factor Def-6 was specifically up-regulated in PKCλ/ι-deficient podocytes. In vivo Def-6 expression is significantly increased in podocytes of PKCλ/ι-deficient mice. Cultured PKCλ/ι-deficient podocytes exhibited an enhanced membrane association of Def-6, indicating enhanced activation. Overexpression of aPKCλ/ι in PKCλ/ι-deficient podocytes could reduce the membrane-associated expression of Def-6 and rescue the actin phenotype. In the present study, PKCλ/ι was identified as an important factor for actin cytoskeletal regulation in podocytes and Def-6 as a specific downstream target of PKCλ/ι that regulates the activity of small GTPases and subsequently the actin cytoskeleton of podocytes.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/fisiología , Isoenzimas/metabolismo , Proteínas Nucleares/metabolismo , Podocitos/metabolismo , Proteína Quinasa C/metabolismo , Citoesqueleto de Actina/genética , Animales , Membrana Celular/genética , Células Cultivadas , Proteínas de Unión al ADN/genética , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Factores de Intercambio de Guanina Nucleótido , Isoenzimas/genética , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Podocitos/citología , Proteína Quinasa C/genética , Proteína Quinasa C-epsilon/genética , Proteína Quinasa C-epsilon/metabolismoRESUMEN
The phospholipase A2 receptor (PLA2R) was recently discovered as a target autoantigen in patients with idiopathic membranous nephropathy (IMN). Published evidence suggests that the autoantibodies directed towards a conformation dependent epitope are currently effectively detected by a cell based assay (CBA) utilizing indirect immunofluorescence (IIF) on tissue culture cells transfected with the PLA2R cDNA. Limitations of such IIF-CBA assays include observer dependent subjective evaluation of semi-quantitative test results and the protocols are not amenable to high throughput diagnostic testing. We developed a quantitative, observer independent, high throughput capture immunoassay for detecting PLA2R autoantibodies on an addressable laser bead immunoassay (ALBIA) platform. Since reactive domains of PLA2R (i.e. epitopes) could be used to improve diagnostic tests by using small peptides in various high throughput diagnostic platforms, we identified PLA2R epitopes that bound autoantibodies of IMN patients. These studies confirmed that inter-molecular epitope spreading occurs in IMN but use of the cognate synthetic peptides in immunoassays was unable to conclusively distinguish between IMN patients and normal controls. However, combinations of these peptides were able to effectively absorb anti-PLA2R reactivity in IIF-CBA and an immunoassay that employed a lysate derived from HEK cells tranfected with and overexpressing PLA2R. While we provide evidence of intermolecular epitope spreading, our data indicates that in addition to conformational epitopes, human anti-PLA2R reactivity in a commercially available CBA and an addressable laser bead immunoassay is significantly absorbed by peptides representing epitopes of PLA2R.
Asunto(s)
Autoantígenos/química , Autoantígenos/inmunología , Mapeo Epitopo/métodos , Glomerulonefritis Membranosa/diagnóstico , Inmunoensayo/métodos , Receptores de Fosfolipasa A2/química , Receptores de Fosfolipasa A2/inmunología , Secuencia de Aminoácidos , Autoanticuerpos/análisis , Autoanticuerpos/inmunología , Células HEK293 , Humanos , Rayos Láser , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Estructura Terciaria de ProteínaRESUMEN
Diabetic nephropathy is the most common cause of chronic renal failure in industrialized countries. Depletion of podocytes plays an important role in the progression of diabetic glomerulopathy. Various factors in the diabetic milieu lead to serious podocyte stress driving the cells toward cell cycle arrest (p27(Kip1)), hypertrophy, detachment, and apoptosis. Mitochondria are responsible for oxidative phosphorylation and energy supply in podocytes. Recent studies indicated that mitochondrial dysfunction is a key factor in diabetic nephropathy. In the present study, we investigated metabolic profiles of podocytes under diabetic conditions. We examined oxygen consumption rates (OCRs) and oxidative phosphorylation complex activities in murine podocytes. Cells were exposed to high glucose for 48 hours, cultured for 10 passages under high-glucose conditions (30 mmol/L), or incubated with transforming growth factor-ß (5 ng/mL) for 24 hours. After prolonged exposure to high glucose, podocytes showed a significantly increased OCR at baseline and also a higher OCR after addition of oligomycin, indicating significant changes in mitochondrial energy metabolism. Higher OCRs after inhibition of respiration by rotenone also indicated changes in nonmitochondrial respiration. Podocytes stimulated with a proapoptotic concentration of transforming growth factor-ß displayed similar bioenergetic profiles, even with decreased citrate synthase activity. In all tested conditions, we found a higher cellular nicotinamide adenine dinucleotide content and changes in activities of respiratory chain complexes. In summary, we provide for the first time evidence that key factors of the diabetic milieu induce changes in glucose metabolism and mitochondrial function in podocytes.
Asunto(s)
Glucemia/metabolismo , Nefropatías Diabéticas/metabolismo , Hiperglucemia/metabolismo , Glomérulos Renales , Mitocondrias/metabolismo , Consumo de Oxígeno , Podocitos/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Células Cultivadas , Diabetes Mellitus Experimental , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/fisiopatología , Fluorometría/métodos , Membrana Basal Glomerular/efectos de los fármacos , Membrana Basal Glomerular/metabolismo , Barrera de Filtración Glomerular/efectos de los fármacos , Transportador de Glucosa de Tipo 1/metabolismo , Hiperglucemia/etiología , Inmunohistoquímica , Indicadores y Reactivos , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Glomérulos Renales/fisiopatología , Masculino , Ratones , Ratones Endogámicos , Mitocondrias/enzimología , Oxazinas , Oxidación-Reducción , Fosforilación , Podocitos/efectos de los fármacos , Podocitos/enzimología , Podocitos/patología , Especies Reactivas de Oxígeno/metabolismo , Succinato Citocromo c Oxidorreductasa/metabolismo , Succinato Deshidrogenasa/metabolismo , XantenosRESUMEN
ADF/cofilins are the major regulators of actin dynamics in mammalian cells. The activation of ADF/cofilins is controlled by a variety of regulatory mechanisms. Dysregulation of ADF/cofilin may result in loss of a precisely organized actin cytoskeletal architecture and can reduce podocyte migration and motility. Recent studies suggest that cofilin-1 can be regulated through several extracellular signals and slit diaphragm proteins. Cofilin knockdown and knockout animal models show dysfunction of glomerular barrier and filtration with foot process effacement and loss of secondary foot processes. This indicates that cofilin-1 is necessary for modulating actin dynamics in podocytes. Podocyte alterations in actin architecture may initiate or aid the progression of a large variety of glomerular diseases, and cofilin activity is required for reorganization of an intact filtration barrier. Since almost all proteinuric diseases result from a similar phenotype with effacement of the foot processes, we propose that cofilin-1 is at the centre stage of the development of proteinuria and thus may be an attractive drug target for antiproteinuric treatment strategies.
RESUMEN
Podocytes are highly differentiated and polarized epithelial cells located on the visceral side of the glomerulus. They form an indispensable component of the glomerular filter, the slit diaphragm, formed by several transmembrane proteins and adaptor molecules. Disruption of the slit diaphragm can lead to massive proteinuria and nephrotic syndrome in mice and humans. CD2AP is an adaptor protein that is important for the maintenance of the slit diaphragm. Together with its paralogue, CIN85, CD2AP belongs to a family of adaptor proteins that are primarily described as being involved in endocytosis and downregulation of receptor tyrosine kinase activity. We have shown that full-length CIN85 is upregulated in podocytes in the absence of CD2AP, whereas in wild-type cells, full-length CIN85 is not detectable. In this study, we show that full-length CIN85 is postranslationally modified by SUMOylation in wild-type podocytes. We can demonstrate that CIN85 is SUMOylated by SUMO-1, -2, and -3 and that SUMOylation is enhanced in the presence of CD2AP. Conversion of lysine 598 to arginine completely abolishes SUMOylation and leads to increased binding of CIN85 to nephrin. Our results indicate a novel role for CD2AP in regulating posttranslational modification of CIN85.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Podocitos/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular , Proteínas del Citoesqueleto/genética , Eliminación de Gen , Regulación de la Expresión Génica , Humanos , Ratones , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/genética , FilogeniaRESUMEN
BACKGROUND: Podocytes are highly specialized epithelial cells on the visceral side of the glomerulus. Their interdigitating primary and secondary foot processes contain an actin based contractile apparatus that can adjust to changes in the glomerular perfusion pressure. Thus, the dynamic regulation of actin bundles in the foot processes is critical for maintenance of a well functioning glomerular filtration barrier. Since the actin binding protein, cofilin-1, plays a significant role in the regulation of actin dynamics, we examined its role in podocytes to determine the impact of cofilin-1 dysfunction on glomerular filtration. METHODS AND FINDINGS: We evaluated zebrafish pronephros function by dextran clearance and structure by TEM in cofilin-1 morphant and mutant zebrafish and we found that cofilin-1 deficiency led to foot process effacement and proteinuria. In vitro studies in murine and human podocytes revealed that PMA stimulation induced activation of cofilin-1, whereas treatment with TGF-ß resulted in cofilin-1 inactivation. Silencing of cofilin-1 led to an accumulation of F-actin fibers and significantly decreased podocyte migration ability. When we analyzed normal and diseased murine and human glomerular tissues to determine cofilin-1 localization and activity in podocytes, we found that in normal kidney tissues unphosphorylated, active cofilin-1 was distributed throughout the cell. However, in glomerular diseases that affect podocytes, cofilin-1 was inactivated by phosphorylation and observed in the nucleus. CONCLUSIONS: Based on these in vitro and in vivo studies we concluded cofilin-1 is an essential regulator for actin filament recycling that is required for the dynamic nature of podocyte foot processes. Therefore, we describe a novel pathomechanism of proteinuria development.
Asunto(s)
Cofilina 1/genética , Cofilina 1/metabolismo , Silenciador del Gen , Proteinuria/metabolismo , Pez Cebra , Citoesqueleto de Actina/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Ratones , Podocitos/metabolismo , Proteinuria/genética , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Podocyte damage is the basis of many glomerular diseases with ultrastructural changes and decreased expression of components of the slit diaphragm such as nephrin and podocin. Under physiological conditions it is likely that the slit diaphragm underlies permanent renewal processes to indemnify its stability in response to changes in filtration pressure. This would require constant reorganization of the podocyte foot process and the renewal of slit diaphragm components. Thus far, the mechanisms underlying the turnover of slit diaphragm proteins are largely unknown. In this manuscript we examined a mechanism of nephrin endocytosis via CIN85/Ruk(L)-mediated ubiquitination. We can demonstrate that the loss of nephrin expression and onset of the proteinuria in CD2AP(-/-) mice correlates with an increased accumulation of ubiquitinated proteins and expression of CIN85/Ruk(L) in podocytes. In cultured murine podocytes CD2AP deficiency leads to an early ubiquitination of nephrin and podocin after stimulation with fibroblast growth factor-4. Binding assays with different CIN85/Ruk isoforms and mutants showed that nephrin and podocin are binding to the coiled-coil domain of CIN85/Ruk(L). We found that in the presence of CIN85/Ruk(L), which is involved in down-regulation of receptor-tyrosine kinases, nephrin is internalized after stimulation with fibroblast growth factor-4. Interestingly, coexpression of CIN85/Ruk(L) with CD2AP led to a decreased binding of CIN85/Ruk(L) to nephrin and podocin, which indicates a functional competition between CD2AP and CIN85/Ruk(L). Our results support a novel role for CIN85/Ruk(L) in slit diaphragm turnover and proteinuria.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Podocitos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Western Blotting , Línea Celular , Células Cultivadas , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Endocitosis/fisiología , Ensayo de Inmunoadsorción Enzimática , Factores de Crecimiento de Fibroblastos/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Ratones , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Podocitos/efectos de los fármacos , Unión Proteica/genética , Unión Proteica/fisiología , Proteinuria/metabolismo , ARN Interferente Pequeño , Ubiquitinación/efectos de los fármacosRESUMEN
BACKGROUND: Microalbuminuria is an early lesion during the development of diabetic nephropathy. The loss of high molecular weight proteins in the urine is usually associated with decreased expression of slit diaphragm proteins. Nephrin, is the major component of the glomerular slit diaphragm and loss of nephrin has been well described in rodent models of experimental diabetes as well as in human diabetic nephropathy. METHODOLOGY/PRINCIPAL FINDINGS: In this manuscript we analyzed the role of PKC-alpha (PKCalpha) on endocytosis of nephrin in podocytes. We found that treatment of diabetic mice with a PKCalpha-inhibitor (GO6976) leads to preserved nephrin expression and reduced proteinuria. In vitro, we found that high glucose stimulation would induce PKCalpha protein expression in murine and human podocytes. We can demonstrate that PKCalpha mediates nephrin endocytosis in podocytes and that overexpression of PKCalpha leads to an augmented endocytosis response. After PKC-activation, we demonstrate an inducible association of PKCalpha, PICK1 and nephrin in podocytes. Moreover, we can demonstrate a strong induction of PKCalpha in podocytes of patients with diabetic nephropathy. CONCLUSIONS/SIGNIFICANCE: We therefore conclude that activation of PKCalpha is a pathomechanistic key event during the development of diabetic nephropathy. PKCalpha is involved in reduction of nephrin surface expression and therefore PKCalpha inhibition might be a novel target molecule for anti-proteinuric therapy.