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1.
J Am Soc Nephrol ; 30(7): 1220-1237, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31235616

RESUMO

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.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/química , Tirosina/metabolismo , Animais , Drosophila melanogaster , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Fosforilação , Podócitos/metabolismo , Estabilidade Proteica , Fator A de Crescimento do Endotélio Vascular/farmacologia , Peixe-Zebra
2.
J Am Soc Nephrol ; 25(9): 1991-2002, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24676636

RESUMO

FSGS is characterized by segmental scarring of the glomerulus and is a leading cause of kidney failure. Identification of genes causing FSGS has improved our understanding of disease mechanisms and points to defects in the glomerular epithelial cell, the podocyte, as a major factor in disease pathogenesis. Using a combination of genome-wide linkage studies and whole-exome sequencing in a kindred with familial FSGS, we identified a missense mutation R431C in anillin (ANLN), an F-actin binding cell cycle gene, as a cause of FSGS. We screened 250 additional families with FSGS and found another variant, G618C, that segregates with disease in a second family with FSGS. We demonstrate upregulation of anillin in podocytes in kidney biopsy specimens from individuals with FSGS and kidney samples from a murine model of HIV-1-associated nephropathy. Overexpression of R431C mutant ANLN in immortalized human podocytes results in enhanced podocyte motility. The mutant anillin displays reduced binding to the slit diaphragm-associated scaffold protein CD2AP. Knockdown of the ANLN gene in zebrafish morphants caused a loss of glomerular filtration barrier integrity, podocyte foot process effacement, and an edematous phenotype. Collectively, these findings suggest that anillin is important in maintaining the integrity of the podocyte actin cytoskeleton.


Assuntos
Glomerulosclerose Segmentar e Focal/genética , Proteínas dos Microfilamentos/genética , Mutação , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Idoso , Sequência de Aminoácidos , Animais , Movimento Celular/genética , Sequência Conservada , Proteínas Contráteis/genética , Proteínas do Citoesqueleto/metabolismo , Análise Mutacional de DNA , Modelos Animais de Doenças , Exoma , Feminino , Técnicas de Silenciamento de Genes , Barreira de Filtração Glomerular/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas Mutantes/genética , Linhagem , Podócitos/metabolismo , Homologia de Sequência de Aminoácidos , Regulação para Cima , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
3.
Kidney Int ; 86(6): 1161-73, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24940800

RESUMO

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.


Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Podócitos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Animais , Arginina/metabolismo , Células HEK293/metabolismo , Humanos , Glomérulos Renais/química , Lisina/metabolismo , Masculino , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Camundongos , Podócitos/química , Proteinúria/induzido quimicamente , Salicilatos/farmacologia , Transdução de Sinais , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/análise , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação/efeitos dos fármacos , Transfecção , Enzimas de Conjugação de Ubiquitina/análise
4.
Am J Pathol ; 183(6): 1945-1959, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24096077

RESUMO

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.


Assuntos
Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/fisiologia , Isoenzimas/metabolismo , Proteínas Nucleares/metabolismo , Podócitos/metabolismo , Proteína Quinase C/metabolismo , Citoesqueleto de Actina/genética , Animais , Membrana Celular/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Isoenzimas/genética , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Podócitos/citologia , Proteína Quinase C/genética , Proteína Quinase C-épsilon/genética , Proteína Quinase C-épsilon/metabolismo
5.
J Biol Chem ; 285(33): 25285-95, 2010 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-20457601

RESUMO

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.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Podócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Western Blotting , Linhagem Celular , Células Cultivadas , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Endocitose/fisiologia , Ensaio de Imunoadsorção Enzimática , Fatores de Crescimento de Fibroblastos/farmacologia , Imunofluorescência , Humanos , Imuno-Histoquímica , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Podócitos/efeitos dos fármacos , Ligação Proteica/genética , Ligação Proteica/fisiologia , Proteinúria/metabolismo , RNA Interferente Pequeno , Ubiquitinação/efeitos dos fármacos
6.
Am J Physiol Renal Physiol ; 297(6): F1656-67, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19828679

RESUMO

Podocytes are an important component of the glomerular filtration barrier and are the major source of vascular endothelial growth factor (VEGF) in the glomerulus. The role of VEGF for the phenotype of the glomerular endothelium has been intensely studied; however, the direct effects of autocrine VEGF on the podocyte are largely unknown. In this study we characterized the expression of VEGF isoforms and VEGF receptors in cultured human podocytes and examined direct effects on cell signaling and apoptosis after stimulation with exogenous VEGF or ablation of autocrine VEGF. We identified VEGF-A and VEGF-C as the dominant isoforms in human podocytes and showed that autocrine levels of both are important for the intracellular activation of antiapoptotic phosphoinositol 3-kinase/AKT and suppression of the proapoptotic p38MAPK via VEGFR-2. We demonstrated that ablation of VEGF-A or VEGF-C as well as treatment with bevacizumab or a VEGFR-2/-3 tyrosine kinase inhibitor led to reduced podocyte survival. In contrast, ablation of VEGF-B had no effect on podocyte survival. Treatment with exogenous VEGF-C reversed the effect of VEGF-A neutralization, and exogenous VEGF-A abrogated the effect of VEGF-C ablation in human podocytes. Our results underline the importance of autocrine VEGF for podocyte survival and indicate the delicate balance of VEGF-A and VEGF-C to influence progression of glomerular diseases.


Assuntos
Comunicação Autócrina/fisiologia , Podócitos/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Bevacizumab , Diferenciação Celular , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Podócitos/citologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/farmacologia , Fator B de Crescimento do Endotélio Vascular/farmacologia , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator C de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores
7.
Cell Physiol Biochem ; 24(5-6): 627-34, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19910703

RESUMO

BACKGROUND: Progressive loss of podocytes has been documented as an early lesion in the development of glomerular disease. In a variety of glomerular diseases, including diabetic nephropathy the activation of transforming growth factor-beta (TGF-beta) has been demonstrated to promote podocyte death and the development of glomerulosclerosis. In this manuscript we analyzed the role of PKC-alpha (PKCalpha) on TGF-beta1 induced apoptosis in podocytes. METHODS: To accomplish this we generated stable murine PKCalpha deficient podocyte cell lines and examined survival- and pro-apoptotic signaling signatures as well as caspase activation after stimulation with TGF-beta. RESULTS: After stimulation with TGF-beta we can demonstrate an enhanced and prolonged activation of PI3K/AKT and ERK1/2 in PKCalpha-knockout (PKCalpha-/-) podocytes compared to PKCalpha-wildtype (PKCalpha+/ +) podocytes, whereas proapoptotic signaling via p38MAPK is significantly reduced. Interestingly, activation of the Smad-pathway is also prolonged in the PKCalpha-/-podocytes. When we analyzed the underlying mechanisms we found a TGF-beta inducible interaction of PKCalpha with the TGF-beta-type-I-receptor (TGFbetaRI). Moreover, endocytosis assays showed that the TGFbetaRI is less internalized in PKCalpha-/- podocytes. CONCLUSION: Since we can demonstrate a key role for PKCalpha in the signaling response after stimulation with TGF-beta we conclude that PKCalpha might be an interesting target molecule as a "podocyte protective" therapy.


Assuntos
Podócitos/enzimologia , Proteína Quinase C-alfa/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Apoptose , Linhagem Celular , Endocitose , Técnicas de Inativação de Genes , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C-alfa/deficiência , Proteína Quinase C-alfa/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
8.
Nephrol Dial Transplant ; 23(10): 3138-45, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18458033

RESUMO

BACKGROUND: The detection of viable podocytes in the urine of patients with proteinuric diseases has been described as a non-invasive method to monitor disease activity. Most of the published studies use podocalyxin (PDX) as a podocyte specific marker. METHODS: We examined the excretion of viable PDX-positive cells in a random set of spot urine from patients with biopsy-proven focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MGN) or membranoproliferative glomerulonephritis (MPGN) and characterized the excreted cells for podocyte and parietal epithelia markers as well as for proliferation activity. RESULTS: We found that untreated patients with active disease excrete high numbers of PDX-positive cells in their urine. In contrast to that we were not able to detect significant amounts of PDX-positive cells in the urine of patients with active minimal change disease (MCD) and patients with FSGS or MGN in full remission. When we further characterized the cells we rarely detected expression of podocyte specific markers in the PDX-positive cells, but at least 50% of the PDX-positive cells were double positive for cytokeratin (CK8-18). Immunohistochemistry of the corresponding renal biopsies showed that 100% of podocytes and parietal cells stained positive for PDX. Semiquantitative analysis revealed that 45% of parietal cells were positive for CK8-18 and 100% of proximal tubular cells. No cells of the glomerular epithelial layer stained positive for CK8-18. CONCLUSIONS: PDX-positive cells are lost in the urine in disease states that require podocyte regeneration and are a useful non-invasive marker for glomerular disease activity. These cells are possibly derived from the parietal epithelial layer.


Assuntos
Glomerulonefrite Membranoproliferativa/urina , Glomerulonefrite Membranosa/urina , Glomerulosclerose Segmentar e Focal/urina , Podócitos/patologia , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Glomerulonefrite Membranoproliferativa/metabolismo , Glomerulonefrite Membranoproliferativa/patologia , Glomerulonefrite Membranosa/metabolismo , Glomerulonefrite Membranosa/patologia , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Queratinas/metabolismo , Rim/metabolismo , Rim/patologia , Masculino , Pessoa de Meia-Idade , Podócitos/metabolismo , Sialoglicoproteínas/metabolismo , Coloração e Rotulagem , Urina/citologia , Adulto Jovem
9.
Kidney Blood Press Res ; 31(6): 411-5, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19096223

RESUMO

OBJECTIVE: Erythropoietin (EPO) has cytoprotective effects apart from its hematopoietic effects. We studied the effects of different EPO molecules on podocyte signaling in vitro and on podocyte survival in an experimental model of diabetic kidney injury (db/db mouse). METHODS: We elucidated intracellular signaling by epoetin-beta, darbepoetin-alpha, and the continuous erythropoietin receptor activator (CERA) in immortalized murine podocyte cultures. Moreover, we treated db/db mice with placebo or with CERA in a chronic (14-week) randomized controlled study. We also studied non-diabetic db/m mice as controls. RESULTS: We could clearly demonstrate phosphorylation of the JAK/PI3K pathway and Akt signaling in podocytes by epoetin-beta, darbepoetin-alpha and CERA. In the long-term animal study we found significantly reduced podocyte numbers in placebo-treated db/db mice compared to db/m control mice (7.4 +/- 0.2 vs. 10.2 +/- 0.9 per glomerular field; p < 0.05). Chronic CERA treatment ameliorated podocyte loss in kidneys of diabetic animals (8.5 +/- 0.5 per glomerular field; p < 0.05 vs. placebo-treated db/db mice). CONCLUSION: EPO activates pro-survival intracellular pathways in podocytes in vitro, and ameliorates diabetes-induced podocyte loss in vivo. Chronic EPO administration may be a feasible way to protect podocyte from diabetic injury.


Assuntos
Complicações do Diabetes/prevenção & controle , Diabetes Mellitus Experimental/patologia , Eritropoetina/uso terapêutico , Podócitos/patologia , Animais , Células Cultivadas , Darbepoetina alfa , Diabetes Mellitus Experimental/tratamento farmacológico , Eritropoetina/análogos & derivados , Eritropoetina/farmacologia , Janus Quinases/metabolismo , Camundongos , Podócitos/efeitos dos fármacos , Polietilenoglicóis/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes , Transdução de Sinais
10.
Diabetes ; 65(12): 3667-3679, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27531950

RESUMO

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.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Endocitose/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteinúria/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Creatinina/metabolismo , Diabetes Mellitus Experimental/genética , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Endocitose/genética , Genótipo , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Podócitos/metabolismo , Podócitos/ultraestrutura , Proteinúria/genética
11.
Nat Med ; 21(6): 601-9, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25962121

RESUMO

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.


Assuntos
Ácidos Cumáricos/administração & dosagem , Cianoacrilatos/administração & dosagem , Dinaminas/metabolismo , Podócitos/efeitos dos fármacos , Proteinúria/tratamento farmacológico , Insuficiência Renal Crônica/tratamento farmacológico , Acrilamida/administração & dosagem , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Dinaminas/química , Dinaminas/efeitos dos fármacos , Humanos , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Camundongos , Modelos Animais , Podócitos/patologia , Podócitos/ultraestrutura , Proteinúria/metabolismo , Proteinúria/patologia , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Peixe-Zebra
12.
Artigo em Inglês | MEDLINE | ID: mdl-25414693

RESUMO

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.

13.
Semin Nephrol ; 32(4): 368-76, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22958491

RESUMO

Renal fibrosis is the major determinant in progression of acute and chronic kidney diseases. Transforming growth factor-ß (TGF-ß) has been shown to be an important mediator of progressive fibrosis. Several studies have implicated that TGF-ß1 is involved in the tight balance of survival and apoptotic responses in podocytes that are Smad-dependent or independent. Bone morphogenic protein-7 (BMP-7), another member of the TGF-ß superfamily, has to date been involved primarily in kidney development and was described as an active blocker of TGF-ß-induced profibrotic effects. Here, we summarize the direct effects of these two cytokines on podocytes. We describe their involvement in podocyte survival and apoptosis pathways with the potential to modify the critical steps in podocyte apoptosis induction. Our group has analyzed the cross-talk of BMP-7 and TGF-ß1 signaling in podocytes and we describe BMP-7 as a cytoprotective factor that could antagonize proapoptotic TGF-ß signals. In addition, we identified various extracellular and intracellular modifiers that can influence this sensitive cross-talk. On the basis of our work and the work of others we conclude that the balance of TGF-ß1 and BMP-7 signaling and involvement of extracellular and intracellular modifiers in these cascades are important parts of podocyte physiology and pathophysiology.


Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Podócitos/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Apoptose , Proteína Morfogenética Óssea 7/farmacologia , Sobrevivência Celular , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Sistema de Sinalização das MAP Quinases , Podócitos/efeitos dos fármacos , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/farmacologia
14.
Mol Cell Biol ; 32(6): 1068-79, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22203040

RESUMO

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.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Podócitos/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , Proteínas do Citoesqueleto/genética , Deleção de Genes , Regulação da Expressão Gênica , Humanos , Camundongos , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Filogenia
15.
PLoS One ; 5(4): e10185, 2010 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-20419132

RESUMO

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.


Assuntos
Diabetes Mellitus/enzimologia , Endocitose , Proteínas de Membrana/metabolismo , Podócitos/metabolismo , Proteína Quinase C-alfa/metabolismo , Animais , Diabetes Mellitus/metabolismo , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/patologia , Regulação Enzimológica da Expressão Gênica , Glucose/farmacologia , Humanos , Camundongos , Podócitos/fisiologia , Proteína Quinase C-alfa/genética
16.
PLoS One ; 5(9): e12626, 2010 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-20838616

RESUMO

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.


Assuntos
Cofilina 1/genética , Cofilina 1/metabolismo , Inativação Gênica , Proteinúria/metabolismo , Peixe-Zebra , Citoesqueleto de Actina/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Humanos , Camundongos , Podócitos/metabolismo , Proteinúria/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
17.
Cell Cycle ; 7(24): 3858-68, 2008 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19066472

RESUMO

Tyrosine phosphorylation of the cell cycle regulator p27(Kip1) plays a crucial role in its binding to cyclin dependent kinases and its subcellular localization. While Src and Bcr-Abl were shown to be responsible for tyrosine phosphorylation, no data are available on the dephosphorylation of p27(Kip1) and the phosphatase involved. Considering the associated dephosphorylation as a pivotal event in the regulation of cell cycle proteins, we focused on the tyrosine phosphatase SHP-2, which is regulated in promyelocytic leukemia cells on G-CSF stimulation. SHP-2 was thus found in association with p27(Kip1) and the G-CSF receptor, and we observed a nuclear translocation of SHP-2 on G-CSF stimulation. Using a catalytically inactive form of SHP-2 and siRNA directed against SHP-2, we could demonstrate the involvement of SHP-2 in tyrosine dephosphorylation of p27(Kip1). Moreover, SHP-2 was strongly activated on G-CSF stimulation and specifically dephosphorylated p27(Kip1) in vitro. Most importantly, we could illustrate that SHP-2 modulates p27(Kip1) stability and contributes to p27(Kip1)-mediated cell cycle progression. Taken together, our results demonstrate that SHP-2 is a key regulator of p27(Kip1) tyrosine phosphorylation.


Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Linhagem Celular , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Humanos , Fosforilação , RNA Interferente Pequeno , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Proteínas Tirosina Fosfatases Contendo o Domínio SH2/metabolismo
18.
Am J Physiol Renal Physiol ; 293(4): F1355-62, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17686962

RESUMO

Progressive tubulointerstitial fibrosis is the common end point leading to end-stage renal disease in experimental and clinical settings. Since the peptide hormone leptin is involved not only in the regulation of obesity but also in the regulation of inflammation and fibrosis, we tested the hypothesis whether leptin deficiency has an impact on tubulointerstitial fibrosis in mice. Leptin-deficient (ob/ob) and leptin receptor-deficient mice (db/db) were exposed to 14 days of unilateral ureteral obstruction (UUO). The degree of fibrosis and inflammation was compared with that in sham-operated mice by performing immunohistochemistry, quantitative PCR, and Western blotting. We found that tubulointerstitial fibrosis was significantly reduced in the obstructed kidneys of ob/ob compared with db/db mice or control mice. Detailed analysis of infiltrating inflammatory cells by immunohistochemistry revealed a significant reduction of CD4(+) cells at 14 days after UUO in both ob/ob and db/db mice. In contrast, we could not detect significant differences in CD8(+) cells and macrophage content. Transforming growth factor (TGF)-beta mRNA levels, TGF-beta-induced Smad-2/3 activation, and the upregulation of downstream target genes were significantly reduced in ob/ob mice. In addition, we demonstrated that leptin could enhance TGF-beta signaling in normal rat kidney fibroblasts in vitro. We conclude that leptin can serve as a cofactor of TGF-beta activation and thus plays an important role in renal tubulointerstitial fibrosis. Therefore, selective blockade of the leptin axis might provide a therapeutic possibility to prevent or delay fibrotic kidney disease.


Assuntos
Leptina/metabolismo , Nefrite Intersticial/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Obstrução Ureteral/metabolismo , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Rim/metabolismo , Rim/patologia , Rim/fisiologia , Leptina/genética , Masculino , Camundongos , Camundongos Mutantes , Nefrite Intersticial/etiologia , Nefrite Intersticial/prevenção & controle , Receptores para Leptina/genética , Receptores para Leptina/metabolismo , Transdução de Sinais/fisiologia , Obstrução Ureteral/complicações
19.
J Biol Chem ; 282(10): 7457-64, 2007 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-17213204

RESUMO

Defects in podocyte signaling are the basis of many inherited glomerular diseases leading to glomerulosclerosis. CD2-associated protein (CD2AP) is highly expressed in podocytes and is considered to play an important role in the maintenance of the glomerular slit diaphragm. Mice deficient for CD2AP (CD2AP(-/-)) appear normal at birth but develop a rapid onset nephrotic syndrome at 3 weeks of age. We demonstrate that impaired intracellular signaling with subsequent podocyte damage is the reason for this delayed podocyte injury in CD2AP(-/-) mice. We document that CD2AP deficiency in podocytes leads to diminished signal initiation and termination of signaling pathways mediated by receptor tyrosine kinases (RTKs). In addition, we demonstrate that CIN85, a paralog of CD2AP, is involved in termination of RTK signaling in podocytes. CIN85 protein expression is increased in CD2AP(-/-) podocytes in vitro. Stimulation of CD2AP(-/-) podocytes with various growth factors, including insulin-like growth factor 1, vascular endothelial growth factor, and fibroblast growth factor, resulted in a significantly decreased phosphatidylinositol 3-kinase/AKT and ERK signaling response. Moreover, increased CIN85 protein is detectable in podocytes in diseased CD2AP(-/-) mice, leading to decreased base-line activation of ERK and decreased phosphorylation after growth factor stimulation in vivo. Because repression of CIN85 protein leads to a restored RTK signaling response, our results support an important role of CD2AP/CIN85 protein balance in the normal signaling response of podocytes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas do Citoesqueleto/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Podócitos/metabolismo , Receptores Proteína Tirosina Quinases/fisiologia , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína Adaptadora GRB2/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo
20.
J Am Soc Nephrol ; 17(6): 1644-56, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16672319

RESUMO

Podocyte apoptosis initiates progressive glomerulosclerosis in TGF-beta1 transgenic and CD2AP-knockout (CD2AP-/-) mice. It was previously shown that in both mouse models, activation of the TGF-beta pathway is the key event during development of podocyte apoptosis. Furthermore, CD2AP is an important modifier of TGF-beta-induced survival signaling via activation of the phosphoinositol 3-kinase/AKT signaling pathway. This article presents IGF-binding protein-3 (IGFBP-3) as a new modulator of apoptosis and survival signaling in glomerular podocytes. High expression of IGFBP-3 protein in the urine of diseased CD2AP-/- mice was discovered, and IGFBP-3 expression in glomerular podocytes and parietal cells was detected. IGFBP-3 can induce changes in podocyte actin cytoskeleton, leads to apoptosis in cultured murine podocytes, and can enhance TGF-beta1-induced apoptosis in vitro. For studying this process on a molecular level, proapoptotic p38 mitogen-activated protein kinase pathways and antiapoptotic phosphoinositol 3-kinase/AKT pathways were examined in cultured murine podocytes. It was found that IGFBP-3 increments the level of TGF-beta1-induced phosphorylated p38 mitogen-activated protein kinase and decreases the phosphorylation of antiapoptotic AKT. This effect is specific for the co-stimulation of IGFBP-3 with TGF-beta1 because a combination of IGFBP-3 with bone morphogenic protein-7 (BMP-7), another member of the TGF-beta superfamily, results in apoptosis opposing signaling effects with a strong increase of phosphorylated AKT and subsequent functional effects. These results demonstrate that the IGF/IGFBP axis plays an important role in the development of podocyte apoptosis by modulation of TGF-beta and BMP-7-induced pro- and antiapoptotic signals.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Glomerulosclerose Segmentar e Focal/metabolismo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/fisiologia , Podócitos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Feminino , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Transdução de Sinais
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