RESUMO
Transient receptor potential canonical channels (TRPCs) are non-selective cationic channels that play a role in signal transduction, especially in G -protein-mediated signaling cascades. TRPC5 is expressed predominantly in the brain but also in the kidney. However, its role in kidney physiology and pathophysiology is controversial. Some studies have suggested that TRPC5 drives podocyte injury and proteinuria, particularly after small GTPase Rac1 activation to induce the trafficking of TRPC5 to the plasma membrane. Other studies using TRPC5 gain-of-function transgenic mice have questioned the pathogenic role of TRPC5 in podocytes. Here, we show that TRPC5 over-expression or inhibition does not ameliorate proteinuria induced by the expression of constitutively active Rac1 in podocytes. Additionally, single-cell patch-clamp studies did not detect functional TRPC5 channels in primary cultures of podocytes. Thus, we conclude that TRPC5 plays a role redundant to that of TRPC6 in podocytes and is unlikely to be a useful therapeutic target for podocytopathies.
Assuntos
Glomerulosclerose Segmentar e Focal , Proteínas Monoméricas de Ligação ao GTP , Podócitos , Camundongos , Animais , Podócitos/patologia , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Canal de Cátion TRPC6/genética , Canal de Cátion TRPC6/metabolismo , Proteinúria/patologia , Camundongos Transgênicos , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Acute kidney injury is common, with a major effect on morbidity and health care utilization. Soluble urokinase plasminogen activator receptor (suPAR) is a signaling glycoprotein thought to be involved in the pathogenesis of kidney disease. We investigated whether a high level of suPAR predisposed patients to acute kidney injury in multiple clinical contexts, and we used experimental models to identify mechanisms by which suPAR acts and to assess it as a therapeutic target. METHODS: We measured plasma levels of suPAR preprocedurally in patients who underwent coronary angiography and patients who underwent cardiac surgery and at the time of admission to the intensive care unit in critically ill patients. We assessed the risk of acute kidney injury at 7 days as the primary outcome and acute kidney injury or death at 90 days as a secondary outcome, according to quartile of suPAR level. In experimental studies, we used a monoclonal antibody to urokinase plasminogen activator receptor (uPAR) as a therapeutic strategy to attenuate acute kidney injury in transgenic mice receiving contrast material. We also assessed cellular bioenergetics and generation of reactive oxygen species in human kidney proximal tubular (HK-2) cells that were exposed to recombinant suPAR. RESULTS: The suPAR level was assessed in 3827 patients who were undergoing coronary angiography, 250 who were undergoing cardiac surgery, and 692 who were critically ill. Acute kidney injury developed in 318 patients (8%) who had undergone coronary angiography. The highest suPAR quartile (vs. the lowest) had an adjusted odds ratio of 2.66 (95% confidence interval [CI], 1.77 to 3.99) for acute kidney injury and 2.29 (95% CI, 1.71 to 3.06) for acute kidney injury or death at 90 days. Findings were similar in the surgical and critically ill cohorts. The suPAR-overexpressing mice that were given contrast material had greater functional and histologic evidence of acute kidney injury than wild-type mice. The suPAR-treated HK-2 cells showed heightened energetic demand and mitochondrial superoxide generation. Pretreatment with a uPAR monoclonal antibody attenuated kidney injury in suPAR-overexpressing mice and normalized bioenergetic changes in HK-2 cells. CONCLUSIONS: High suPAR levels were associated with acute kidney injury in various clinical and experimental contexts. (Funded by the National Institutes of Health and others.).
Assuntos
Injúria Renal Aguda/sangue , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Angiografia Coronária/efeitos adversos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/prevenção & controle , Idoso , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Biomarcadores/sangue , Estado Terminal , Modelos Animais de Doenças , Feminino , Humanos , Unidades de Terapia Intensiva , Túbulos Renais/citologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Razão de Chances , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Complicações Pós-Operatórias/sangue , Complicações Pós-Operatórias/etiologia , Medição de Risco/métodos , Ativador de Plasminogênio Tipo Uroquinase/farmacologiaRESUMO
Chemokines and other chemoattractants direct leukocyte migration and are essential for the development and delivery of immune and inflammatory responses. To probe the molecular mechanisms that underlie chemoattractant-guided migration, we did an RNA-mediated interference screen that identified several members of the synaptotagmin family of calcium-sensing vesicle-fusion proteins as mediators of cell migration: SYT7 and SYTL5 were positive regulators of chemotaxis, whereas SYT2 was a negative regulator of chemotaxis. SYT7-deficient leukocytes showed less migration in vitro and in a gout model in vivo. Chemoattractant-induced calcium-dependent lysosomal fusion was impaired in SYT7-deficient neutrophils. In a chemokine gradient, SYT7-deficient lymphocytes accumulated lysosomes in their uropods and had impaired uropod release. Our data identify a molecular pathway required for chemotaxis that links chemoattractant-induced calcium flux to exocytosis and uropod release.
Assuntos
Movimento Celular/fisiologia , Sinaptotagminas/metabolismo , Animais , Quimiocina CXCL12/metabolismo , Quimiotaxia , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase , Receptores CXCR4/metabolismo , Sinaptotagmina II/metabolismo , Sinaptotagminas/genética , Linfócitos T/imunologiaRESUMO
The selectivity of the glomerular filter is established by physical, chemical, and signaling interplay among its three core constituents: glomerular endothelial cells, the glomerular basement membrane, and podocytes. Functional impairment or injury of any of these three components can lead to proteinuria. Podocytes are injured in many forms of human and experimental glomerular disease, including minimal change disease, focal segmental glomerulosclerosis, and diabetes mellitus. One of the earliest signs of podocyte injury is loss of their distinct structure, which is driven by dysregulated dynamics of the actin cytoskeleton. The status of the actin cytoskeleton in podocytes depends on a set of actin binding proteins, nucleators and inhibitors of actin polymerization, and regulatory GTPases. Mutations that alter protein function in each category have been implicated in glomerular diseases in humans and animal models. In addition, a growing body of studies suggest that pharmacological modifications of the actin cytoskeleton have the potential to become novel therapeutics for podocyte-dependent chronic kidney diseases. This review presents an overview of the essential proteins that establish actin cytoskeleton in podocytes and studies demonstrating the feasibility of drugging actin cytoskeleton in kidney diseases.
Assuntos
Citoesqueleto de Actina , Podócitos , Citoesqueleto de Actina/fisiologia , Animais , Humanos , Podócitos/metabolismoRESUMO
Proteinuria encompasses diverse causes including both genetic diseases and acquired forms such as diabetic and hypertensive nephropathy. The basis of proteinuria is a disturbance in size selectivity of the glomerular filtration barrier, which largely depends on the podocyte: a terminally differentiated epithelial cell type covering the outer surface of the glomerulus. Compromised podocyte structure is one of the earliest signs of glomerular injury. The phenotype of diverse animal models and podocyte cell culture firmly established the essential role of the actin cytoskeleton in maintaining functional podocyte structure. Podocyte foot processes, actin-based membrane extensions, contain 2 molecularly distinct "hubs" that control actin dynamics: a slit diaphragm and focal adhesions. Although loss of foot processes encompasses disassembly of slit diaphragm multiprotein complexes, as long as cells are attached to the glomerular basement membrane, focal adhesions will be the sites in which stress due to filtration flow is counteracted by forces generated by the actin network in foot processes. Numerous studies within last 20 years have identified actin binding and regulatory proteins as well as integrins as essential components of signaling and actin dynamics at focal adhesions in podocytes, suggesting that some of them may become novel, druggable targets for proteinuric kidney diseases. Here we review evidence supporting the idea that current treatments for chronic kidney diseases beneficially and directly target the podocyte actin cytoskeleton associated with focal adhesions and suggest that therapeutic reagents that target the focal adhesion-regulated actin cytoskeleton in foot processes have potential to modernize treatments for chronic kidney diseases.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Adesões Focais/metabolismo , Membrana Basal Glomerular/metabolismo , Podócitos/metabolismo , Proteinúria/metabolismo , Insuficiência Renal Crônica/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/patologia , Animais , Adesões Focais/efeitos dos fármacos , Adesões Focais/patologia , Membrana Basal Glomerular/efeitos dos fármacos , Membrana Basal Glomerular/patologia , Membrana Basal Glomerular/fisiopatologia , Taxa de Filtração Glomerular , Humanos , Terapia de Alvo Molecular , Podócitos/efeitos dos fármacos , Podócitos/patologia , Proteinúria/tratamento farmacológico , Proteinúria/genética , Proteinúria/patologia , Fármacos Renais/uso terapêutico , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Transdução de SinaisRESUMO
BACKGROUND: Relatively high plasma levels of soluble urokinase-type plasminogen activator receptor (suPAR) have been associated with focal segmental glomerulosclerosis and poor clinical outcomes in patients with various conditions. It is unknown whether elevated suPAR levels in patients with normal kidney function are associated with future decline in the estimated glomerular filtration rate (eGFR) and with incident chronic kidney disease. METHODS: We measured plasma suPAR levels in 3683 persons enrolled in the Emory Cardiovascular Biobank (mean age, 63 years; 65% men; median suPAR level, 3040 pg per milliliter) and determined renal function at enrollment and at subsequent visits in 2292 persons. The relationship between suPAR levels and the eGFR at baseline, the change in the eGFR over time, and the development of chronic kidney disease (eGFR <60 ml per minute per 1.73 m(2) of body-surface area) were analyzed with the use of linear mixed models and Cox regression after adjustment for demographic and clinical variables. RESULTS: A higher suPAR level at baseline was associated with a greater decline in the eGFR during follow-up; the annual change in the eGFR was -0.9 ml per minute per 1.73 m(2) among participants in the lowest quartile of suPAR levels as compared with -4.2 ml per minute per 1.73 m(2) among participants in the highest quartile (P<0.001). The 921 participants with a normal eGFR (≥ 90 ml per minute per 1.73 m(2)) at baseline had the largest suPAR-related decline in the eGFR. In 1335 participants with a baseline eGFR of at least 60 ml per minute per 1.73 m(2), the risk of progression to chronic kidney disease in the highest quartile of suPAR levels was 3.13 times as high (95% confidence interval, 2.11 to 4.65) as that in the lowest quartile. CONCLUSIONS: An elevated level of suPAR was independently associated with incident chronic kidney disease and an accelerated decline in the eGFR in the groups studied. (Funded by the Abraham J. and Phyllis Katz Foundation and others.).
Assuntos
Taxa de Filtração Glomerular , Rim/fisiologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Insuficiência Renal Crônica/diagnóstico , Idoso , Biomarcadores/sangue , Progressão da Doença , Feminino , Humanos , Incidência , Estimativa de Kaplan-Meier , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Estudos Prospectivos , ProteinúriaRESUMO
Rho family GTPases, the prototypical members of which are Cdc42, Rac1, and RhoA, are molecular switches best known for regulating the actin cytoskeleton. In addition to the canonical small GTPases, the large GTPase dynamin has been implicated in regulating the actin cytoskeleton via direct dynamin-actin interactions. The physiologic role of dynamin in regulating the actin cytoskeleton has been linked to the maintenance of the kidney filtration barrier. Additionally, the small molecule Bis-T-23, which promotes actin-dependent dynamin oligomerization and thus, increases actin polymerization, improved renal health in diverse models of CKD, implicating dynamin as a potential therapeutic target for the treatment of CKD. Here, we show that treating cultured mouse podocytes with Bis-T-23 promoted stress fiber formation and focal adhesion maturation in a dynamin-dependent manner. Furthermore, Bis-T-23 induced the formation of focal adhesions and stress fibers in cells in which the RhoA signaling pathway was downregulated by multiple experimental approaches. Our study suggests that dynamin regulates focal adhesion maturation by a mechanism parallel to and synergistic with the RhoA signaling pathway. Identification of dynamin as one of the essential and autonomous regulators of focal adhesion maturation suggests a molecular mechanism that underlies the beneficial effect of Bis-T-23 on podocyte physiology.
Assuntos
Dinaminas/fisiologia , Adesões Focais/fisiologia , Podócitos/fisiologia , Citoesqueleto de Actina/fisiologia , Animais , Camundongos , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
Dynamin is a 96-kDa protein that has multiple oligomerization states that influence its GTPase activity. A number of different dynamin effectors, including lipids, actin filaments, and SH3-domain-containing proteins, have been implicated in the regulation of dynamin oligomerization, though their roles in influencing dynamin oligomerization have been studied predominantly in vitro using recombinant proteins. Here, we identify higher order dynamin oligomers such as rings and helices in vitro and in live cells using fluorescence lifetime imaging microscopy (FLIM). FLIM detected GTP- and actin-dependent dynamin oligomerization at distinct cellular sites, including the cell membrane and transition zones where cortical actin transitions into stress fibers. Our study identifies a major role for direct dynamin-actin interactions and dynamin's GTPase activity in the regulation of dynamin oligomerization in cells.
Assuntos
Actinas/metabolismo , Dinaminas/metabolismo , Guanosina Trifosfato/metabolismo , Multimerização Proteica , Actinas/química , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Dinaminas/química , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Podocyte injury and loss mark an early step in the pathogenesis of various glomerular diseases, making these cells excellent targets for therapeutics. However, cell-based high-throughput screening assays for the rational development of podocyte-directed therapeutics are currently lacking. Here, we describe a novel high-content screening-based phenotypic assay that analyzes thousands of podocytes per assay condition in 96-well plates to quantitatively measure dose-dependent changes in multiple cellular features. Our assay consistently produced a Z' value >0.44, making it suitable for compound screening. On screening with >2100 pharmacologically active agents, we identified 24 small molecules that protected podocytes against injury in vitro (1% hit rate). Among the identified hits, we confirmed an ß1-integrin agonist, pyrintegrin, as a podocyte-protective agent. Treatment with pyrintegrin prevented damage-induced decreases in F-actin stress fibers, focal adhesions, and active ß1-integrin levels in cultured cells. In vivo, administration of pyrintegrin protected mice from LPS-induced podocyte foot process effacement and proteinuria. Analysis of the murine glomeruli showed that LPS administration reduced the levels of active ß1 integrin in the podocytes, which was prevented by cotreatment with pyrintegrin. In rats, pyrintegrin reduced peak proteinuria caused by puromycin aminonucleoside-induced nephropathy. Our findings identify pyrintegrin as a potential therapeutic candidate and show the use of podocyte-based screening assays for identifying novel therapeutics for proteinuric kidney diseases.
Assuntos
Hidroxiquinolinas/química , Integrina beta1/metabolismo , Glomérulos Renais/metabolismo , Podócitos/citologia , Sulfonamidas/química , Actinas/metabolismo , Albuminúria/metabolismo , Animais , Movimento Celular , Células Epiteliais/efeitos dos fármacos , Adesões Focais/metabolismo , Ensaios de Triagem em Larga Escala , Nefropatias/metabolismo , Lipopolissacarídeos/química , Camundongos , Microscopia Confocal , Fenótipo , Proteinúria/patologia , Puromicina Aminonucleosídeo/química , RatosRESUMO
The GTPase dynamin has captivated researchers for over two decades, even managing to establish its own research field. Dynamin's allure is partly due to its unusual biochemical properties as well as its essential role in multiple cellular processes, which include the regulation of clathrin-mediated endocytosis and of actin cytoskeleton. On the basis of the classic model, dynamin oligomerization into higher order oligomers such as rings and helices directly executes the final fission reaction in endocytosis, which results in the generation of clathrin-coated vesicles. Dynamin's role in the regulation of actin cytoskeleton is mostly explained by its interactions with a number of actin-binding and -regulating proteins; however, the molecular mechanism of dynamin's action continues to elude us. Recent insights into the mechanism and role of dynamin oligomerization in the regulation of actin polymerization point to a novel role for dynamin oligomerization in the cell.
Assuntos
Dinaminas/metabolismo , Endocitose , Citoesqueleto de Actina/metabolismo , Animais , Invaginações Revestidas da Membrana Celular/metabolismo , Humanos , Microtúbulos/metabolismo , Multimerização ProteicaRESUMO
Podocytes are highly differentiated cells and critical elements for the filtration barrier of the kidney. Loss of their foot process (FP) architecture (FP effacement) results in urinary protein loss. Here we show a novel role for the neutral amino acid glutamine in structural and functional regulation of the kidney filtration barrier. Metabolic flux analysis of cultured podocytes using genetic, toxic, and immunologic injury models identified increased glutamine utilization pathways. We show that glutamine uptake is increased in diseased podocytes to couple nutrient support to increased demand during the disease state of FP effacement. This feature can be utilized to transport increased amounts of glutamine into damaged podocytes. The availability of glutamine determines the regulation of podocyte intracellular pH (pHi). Podocyte alkalinization reduces cytosolic cathepsin L protease activity and protects the podocyte cytoskeleton. Podocyte glutamine supplementation reduces proteinuria in LPS-treated mice, whereas acidification increases glomerular injury. In summary, our data provide a metabolic opportunity to combat urinary protein loss through modulation of podocyte amino acid utilization and pHi.
Assuntos
Podócitos/metabolismo , Proteinúria/metabolismo , Animais , Transporte Biológico Ativo/genética , Transporte Biológico Ativo/imunologia , Células Cultivadas , Citoesqueleto/genética , Citoesqueleto/imunologia , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Knockout , Podócitos/imunologia , Podócitos/patologia , Proteinúria/genética , Proteinúria/imunologia , Proteinúria/patologiaRESUMO
Proteinuric chronic kidney disease (CKD), once a rare affliction believed to be mainly caused by genetic mutations, has become a global pandemic that severely diminishes the quality of life for millions. Despite the changing face of CKD, treatment options and resources remain woefully antiquated and have failed to arrest or reverse the effects of kidney-related diseases. Histological and genetic data strongly implicate one promising target: the podocyte. Podocytes are terminally differentiated cells of the kidney glomerulus that are essential for the integrity of the kidney filter. Their function is primarily based on their intricate structure, which includes foot processes. Loss of these actin-driven membrane extensions is tightly connected to the presence of protein in the urine, podocyte loss, development of CKD, and ultimately renal failure.
Assuntos
Glomérulos Renais/patologia , Podócitos/fisiologia , Predisposição Genética para Doença , Humanos , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologiaRESUMO
Gain-of-function mutations in the calcium channel TRPC6 lead to autosomal dominant focal segmental glomerulosclerosis and podocyte expression of TRPC6 is increased in some acquired human glomerular diseases, particularly in membranous nephropathy. These observations led to the hypothesis that TRPC6 overactivation is deleterious to podocytes through pathological calcium signaling, both in genetic and acquired diseases. Here, we show that the effects of TRPC6 on podocyte function are context-dependent. Overexpression of TRPC6 alone did not directly affect podocyte morphology and cytoskeletal structure. Unexpectedly, however, overexpression of TRPC6 protected podocytes from complement-mediated injury, whereas genetic or pharmacological TRPC6 inactivation increased podocyte susceptibility to complement. Mechanistically, this effect was mediated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation. Podocyte-specific TRPC6 transgenic mice showed stronger CaMKII activation, reduced podocyte foot process effacement and reduced levels of proteinuria during nephrotoxic serum nephritis, whereas TRPC6 null mice exhibited reduced CaMKII activation and higher levels of proteinuria compared with wild type littermates. Human membranous nephropathy biopsy samples showed podocyte staining for active CaMKII, which correlated with the degree of TRPC6 expression. Together, these data suggest a dual and context dependent role of TRPC6 in podocytes where acute activation protects from complement-mediated damage, but chronic overactivation leads to focal segmental glomerulosclerosis.
Assuntos
Proteínas do Sistema Complemento/metabolismo , Glomerulosclerose Segmentar e Focal/metabolismo , Podócitos/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Sinalização do Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Ativação Enzimática , Glomerulonefrite Membranosa/metabolismo , Glomerulonefrite Membranosa/patologia , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Podócitos/patologia , Proteinúria/metabolismo , Canais de Cátion TRPC/genética , Canal de Cátion TRPC6RESUMO
The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis. Dynamin also regulates the actin cytoskeleton through an unknown, GTPase-dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles. Point mutations in the actin-binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin-capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments. Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin-CPs.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Dinaminas/metabolismo , Gelsolina/metabolismo , Fibras de Estresse/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Dinaminas/antagonistas & inibidores , Dinaminas/genética , Endocitose/fisiologia , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Podócitos/metabolismo , Ligação Proteica , RNA Interferente Pequeno/farmacologia , Coelhos , Homologia de Sequência de AminoácidosRESUMO
In genetic disease, an accurate expression landscape of disease genes and faithful animal models will enable precise genetic diagnoses and therapeutic discoveries, respectively. We previously discovered that variants in NOS1AP , encoding nitric oxide synthase 1 (NOS1) adaptor protein, cause monogenic nephrotic syndrome (NS). Here, we determined that an intergenic splice product of N OS1AP / Nos1ap and neighboring C1orf226/Gm7694 , which precludes NOS1 binding, is the predominant isoform in mammalian kidney transcriptional and proteomic data. Gm7694 -/- mice, whose allele exclusively disrupts the intergenic product, developed NS phenotypes. In two human NS subjects, we identified causative NOS1AP splice variants, including one predicted to abrogate intergenic splicing but initially misclassified as benign based on the canonical transcript. Finally, by modifying genetic background, we generated a faithful mouse model of NOS1AP -associated NS, which responded to anti-proteinuric treatment. This study highlights the importance of intergenic splicing and a potential treatment avenue in a mendelian disorder.
RESUMO
Chromosomal instability and the subsequent genetic mutations are considered to be critical factors in the development of the majority of solid tumors. Here, we describe how the nucleoside diphosphate kinase Nm23-H1, a protein with a known link to cancer progression, regulates a critical step during cytokinesis. Nm23-H1 acts to provide a local source of GTP for the GTPase dynamin. Loss of Nm23-H1 in diploid cells leads to cytokinetic furrow regression, followed by cytokinesis failure and generation of tetraploid cells. Loss of dynamin phenocopies loss of Nm23-H1, and ectopic overexpression of WT dynamin complements the loss of Nm23-H1. In the absence of p53 signaling, the tetraploid cells resulting from loss of Nm23-H1 continue cycling and develop classic hallmarks of tumor cells. We thus provide evidence that the loss of Nm23-H1, an event suspected to promote metastasis, may additionally function at an earlier stage of tumor development to drive the acquisition of chromosomal instability.
Assuntos
Instabilidade Cromossômica/genética , Citocinese/genética , Dinaminas/genética , Nucleosídeo NM23 Difosfato Quinases/genética , Western Blotting , Ciclo Celular/genética , Linhagem Celular , Células Cultivadas , Senescência Celular/genética , Dinaminas/metabolismo , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Mitose/genética , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Poliploidia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Soluble urokinase plasminogen activator receptor (suPAR) is a risk factor for kidney diseases. In addition to suPAR, proteolysis of membrane-bound uPAR results in circulating D1 and D2D3 proteins. We showed that when exposed to a high-fat diet, transgenic mice expressing D2D3 protein developed progressive kidney disease marked by microalbuminuria, elevated serum creatinine, and glomerular hypertrophy. D2D3 transgenic mice also exhibited insulin-dependent diabetes mellitus evidenced by decreased levels of insulin and C-peptide, impaired glucose-stimulated insulin secretion, decreased pancreatic ß cell mass, and high fasting blood glucose. Injection of anti-uPAR antibody restored ß cell mass and function in D2D3 transgenic mice. At the cellular level, the D2D3 protein impaired ß cell proliferation and inhibited the bioenergetics of ß cells, leading to dysregulated cytoskeletal dynamics and subsequent impairment in the maturation and trafficking of insulin granules. D2D3 protein was predominantly detected in the sera of patients with nephropathy and insulin-dependent diabetes mellitus. These sera inhibited glucose-stimulated insulin release from human islets in a D2D3-dependent manner. Our study showed that D2D3 injures the kidney and pancreas and suggests that targeting this protein could provide a therapy for kidney diseases and insulin-dependent diabetes mellitus.
Assuntos
Diabetes Mellitus Tipo 1 , Hiperglicemia , Imunotoxinas , Nefropatias , Animais , Camundongos , Humanos , Receptores de Ativador de Plasminogênio Tipo Uroquinase , InsulinaRESUMO
Chronic kidney diseases and acute kidney injury are mechanistically distinct kidney diseases. While chronic kidney diseases are associated with podocyte injury, acute kidney injury affects renal tubular epithelial cells. Despite these differences, a cardinal feature of both acute and chronic kidney diseases is dysregulated actin cytoskeleton. We have shown that pharmacological activation of GTPase dynamin ameliorates podocyte injury in murine models of chronic kidney diseases by promoting actin polymerization. Here we establish dynamin's role in modulating stiffness and polarity of renal tubular epithelial cells by crosslinking actin filaments into branched networks. Activation of dynamin's crosslinking capability by a small molecule agonist stabilizes the actomyosin cortex of the apical membrane against injury, which in turn preserves renal function in various murine models of acute kidney injury. Notably, a dynamin agonist simultaneously attenuates podocyte and tubular injury in the genetic murine model of Alport syndrome. Our study provides evidence for the feasibility and highlights the benefits of novel holistic nephron-protective therapies.
Assuntos
Injúria Renal Aguda , Podócitos , Insuficiência Renal Crônica , Citoesqueleto de Actina , Injúria Renal Aguda/prevenção & controle , Animais , Dinaminas , Feminino , Humanos , Rim/fisiologia , Masculino , Camundongos , Insuficiência Renal Crônica/tratamento farmacológicoRESUMO
The GTPase dynamin is essential for endocytosis, but its mechanism of action remains uncertain. Structures of its GTPase domain, as well as that of assembled dynamin, have led to major advances in understanding the structural basis of its mode of action. Novel data point more clearly than ever towards a role for this protein in the actin cytoskeleton, mitogen-activated protein kinase signaling and apoptosis, suggesting that dynamin might be a signaling GTPase.