RESUMO
Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain-containing protein tyrosine phosphatase-2-dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high-affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an in vitro model of cultured podocytes and an ex vivo model of Drosophila nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity.
Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Proteínas de Membrana/metabolismo , Animais , Linhagem Celular , Taxa de Filtração Glomerular/fisiologia , Fator de Crescimento de Hepatócito/fisiologia , Humanos , Junções Intercelulares/metabolismo , Rim/patologia , Glomérulos Renais/metabolismo , Proteínas de Membrana/genética , Camundongos , Peptídeos/metabolismo , Fosforilação , Podócitos/metabolismo , Ligação Proteica/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Myosin 1c (Myo1c) is an unconventional myosin that modulates signaling pathways involved in tissue injury and repair. In this study, we observed that Myo1c expression is significantly upregulated in human chronic liver disease such as nonalcoholic steatohepatitis (NASH) and in animal models of liver fibrosis. High throughput data from the GEO-database identified similar Myo1c upregulation in mice and human liver fibrosis. Notably, transforming growth factor-ß1 (TGF-ß1) stimulation to hepatic stellate cells (HSCs), the liver pericyte and key cell type responsible for the deposition of extracellular matrix, upregulates Myo1c expression, whereas genetic depletion or pharmacological inhibition of Myo1c blunted TGF-ß-induced fibrogenic responses, resulting in repression of α-smooth muscle actin (α-SMA) and collagen type I α 1 chain (Col1α1) mRNA. Myo1c deletion also decreased fibrogenic processes such as cell proliferation, wound healing response, and contractility when compared with vehicle-treated HSCs. Importantly, phosphorylation of mothers against decapentaplegic homolog 2 (SMAD2) and mothers against decapentaplegic homolog 3 (SMAD3) were significantly blunted upon Myo1c inhibition in GRX cells as well as Myo1c knockout (Myo1c-KO) mouse embryonic fibroblasts (MEFs) upon TGF-ß stimulation. Using the genetic Myo1c-KO mice, we confirmed that Myo1c is critical for fibrogenesis, as Myo1c-KO mice were resistant to carbon tetrachloride (CCl4)-induced liver fibrosis. Histological and immunostaining analysis of liver sections showed that deposition of collagen fibers and α-SMA expression were significantly reduced in Myo1c-KO mice upon liver injury. Collectively, these results demonstrate that Myo1c mediates hepatic fibrogenesis by modulating TGF-ß signaling and suggest that inhibiting this process may have clinical application in treating liver fibrosis.NEW & NOTEWORTHY The incidences of liver fibrosis are growing at a rapid pace and have become one of the leading causes of end-stage liver disease. Although TGF-ß1 is known to play a prominent role in transforming cells to produce excessive extracellular matrix that lead to hepatic fibrosis, the therapies targeting TGF-ß1 have achieved very limited clinical impact. This study highlights motor protein myosin-1c-mediated mechanisms that serve as novel regulators of TGF-ß1 signaling and fibrosis.
Assuntos
Fibroblastos/metabolismo , Cirrose Hepática/metabolismo , Fígado/metabolismo , Miosina Tipo I/metabolismo , Animais , Cadeia alfa 1 do Colágeno Tipo I , Fibroblastos/patologia , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Fígado/patologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Camundongos , Miosina Tipo I/genética , Fosforilação , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
The exocyst is a highly conserved protein complex found in most eukaryotic cells and is associated with many functions, including protein translocation in the endoplasmic reticulum, vesicular basolateral targeting, and ciliogenesis in the kidney. To investigate the exocyst functions, here we exchanged proline for alanine in the highly conserved VXPX ciliary targeting motif of EXOC5 (exocyst complex component 5), a central exocyst gene/protein, and generated stable EXOC5 ciliary targeting sequence-mutated (EXOC5CTS-m) Madin-Darby canine kidney (MDCK) cells. The EXOC5CTS-m protein was stable and could bind other members of the exocyst complex. Culturing stable control, EXOC5-overexpressing (OE), Exoc5-knockdown (KD), and EXOC5CTS-m MDCK cells on Transwell filters, we found that primary ciliogenesis is increased in EXOC5 OE cells and inhibited in Exoc5-KD and EXOC5CTS-m cells. Growing cells in collagen gels until the cyst stage, we noted that EXOC5-OE cells form mature cysts with single lumens more rapidly than control cysts, whereas Exoc5-KD and EXOC5CTS-m MDCK cells failed to form mature cysts. Adding hepatocyte growth factor to induce tubulogenesis, we observed that EXOC5-OE cell cysts form tubules more efficiently than control MDCK cell cysts, EXOC5CTS-m MDCK cell cysts form significantly fewer tubules than control cell cysts, and Exoc5-KD cysts did not undergo tubulogenesis. Finally, we show that EXOC5 mRNA almost completely rescues the ciliary phenotypes in exoc5-mutant zebrafish, unlike the EXOC5CTS-m mRNA, which could not efficiently rescue the phenotypes. Taken together, these results indicate that the exocyst, acting through the primary cilium, is necessary for renal ciliogenesis, cystogenesis, and tubulogenesis.
Assuntos
Cílios/fisiologia , Cistos/patologia , Túbulos Renais/crescimento & desenvolvimento , Rim/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , DNA Complementar/genética , Cães , Técnicas de Silenciamento de Genes , Humanos , Nefropatias/patologia , Células Madin Darby de Rim Canino , Mutagênese Sítio-Dirigida , Ligação Proteica , Transporte Proteico , RNA Mensageiro/metabolismo , Proteínas de Transporte Vesicular/genética , Peixe-ZebraRESUMO
Although the slit diaphragm proteins in podocytes are uniquely organized to maintain glomerular filtration assembly and function, little is known about the underlying mechanisms that participate in trafficking these proteins to the correct location for development and homeostasis. Identifying these mechanisms will likely provide novel targets for therapeutic intervention to preserve podocyte function following glomerular injury. Analysis of structural variation in cases of human nephrotic syndrome identified rare heterozygous deletions of EXOC4 in two patients. This suggested that disruption of the highly-conserved eight-protein exocyst trafficking complex could have a role in podocyte dysfunction. Indeed, mRNA profiling of injured podocytes identified significant exocyst down-regulation. To test the hypothesis that the exocyst is centrally involved in podocyte development/function, we generated homozygous podocyte-specific Exoc5 (a central exocyst component that interacts with Exoc4) knockout mice that showed massive proteinuria and died within 4 weeks of birth. Histological and ultrastructural analysis of these mice showed severe glomerular defects with increased fibrosis, proteinaceous casts, effaced podocytes, and loss of the slit diaphragm. Immunofluorescence analysis revealed that Neph1 and Nephrin, major slit diaphragm constituents, were mislocalized and/or lost. mRNA profiling of Exoc5 knockdown podocytes showed that vesicular trafficking was the most affected cellular event. Mapping of signaling pathways and Western blot analysis revealed significant up-regulation of the mitogen-activated protein kinase and transforming growth factor-ß pathways in Exoc5 knockdown podocytes and in the glomeruli of podocyte-specific Exoc5 KO mice. Based on these data, we propose that exocyst-based mechanisms regulate Neph1 and Nephrin signaling and trafficking, and thus podocyte development and function.
Assuntos
Deleção de Genes , Glomérulos Renais/patologia , Síndrome Nefrótica/patologia , Podócitos/patologia , Proteínas de Transporte Vesicular/fisiologia , Animais , Apoptose , Movimento Celular , Exocitose , Humanos , Glomérulos Renais/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Síndrome Nefrótica/genética , Fosforilação , Podócitos/metabolismo , Transporte Proteico , Proteinúria/etiologia , Proteinúria/patologia , Transdução de SinaisRESUMO
Definitive diagnosis of glomerular disease requires a kidney biopsy, an invasive procedure that may not be safe or feasible to perform in all patients. We developed a noninvasive, accurate, and economical diagnostic assay with easy commercial adaptability to detect recurrent focal segmental glomerulosclerosis (rFSGS) after kidney transplant. Since FSGS involves podocyte damage and death, our approach involved mRNA profiling of cultured podocytes treated with plasma from patients with rFSGS to identify upregulated genes involved in podocyte damage. For concept validation, three upregulated pro-apoptotic candidate genes (IL1ß, BMF, and IGFBP3) were selected, and their promoter regions were cloned into a luciferase-based reporter vector and transfected into podocytes to generate stable podocyte cell lines. Strikingly, when exposed to rFSGS patient plasma, these cell lines showed increased reporter activity; in contrast, no reporter activity was noted with plasma from patients with non-recurrent FSGS or membranous nephropathy. Area under the receiver operating characteristics curves (AUCs) for models discriminating between rFSGS and other nephropathies (non-recurrent FSGS and membranous nephropathy) and between rFSGS and non-recurrent FSGS ranged from 0.81 to 0.86, respectively. Estimated sensitivities and specificities for the diagnosis of rFSGS were greater than 80% for the IL1ß and BMF cell lines, and were slightly lower for the IGFBP3 cell line. Importantly, the novel approach outlined here for the diagnosis of rFSGS is widely applicable to the design of sensitive and specific diagnostic/prognostic assays for other glomerular diseases.
Assuntos
Bioensaio/métodos , Glomerulosclerose Segmentar e Focal/diagnóstico , Podócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular , Diagnóstico Diferencial , Perfilação da Expressão Gênica , Genes Reporter , Glomerulosclerose Segmentar e Focal/sangue , Glomerulosclerose Segmentar e Focal/complicações , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Falência Renal Crônica/etiologia , Falência Renal Crônica/cirurgia , Transplante de Rim , Luciferases/genética , Plasma/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , RNA-Seq , Curva ROC , RecidivaRESUMO
Podocytes have limited ability to recover from injury. Here, we demonstrate that increased mitochondrial biogenesis, to meet the metabolic and energy demand of a cell, accelerates podocyte recovery from injury. Analysis of events induced during podocyte injury and recovery showed marked upregulation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a transcriptional co-activator of mitochondrial biogenesis, and key components of the mitochondrial electron transport chain. To evaluate our hypothesis that increasing mitochondrial biogenesis enhanced podocyte recovery from injury, we treated injured podocytes with formoterol, a potent, specific, and long-acting ß2-adrenergic receptor agonist that induces mitochondrial biogenesis in vitro and in vivo. Formoterol increased mitochondrial biogenesis and restored mitochondrial morphology and the injury-induced changes to the organization of the actin cytoskeleton in podocytes. Importantly, ß2-adrenergic receptors were found to be present on podocyte membranes. Their knockdown attenuated formoterol-induced mitochondrial biogenesis. To determine the potential clinical relevance of these findings, mouse models of acute nephrotoxic serum nephritis and chronic (Adriamycin [doxorubicin]) glomerulopathy were used. Mice were treated with formoterol post-injury when glomerular dysfunction was established. Strikingly, formoterol accelerated the recovery of glomerular function by reducing proteinuria and ameliorating kidney pathology. Furthermore, formoterol treatment reduced cellular apoptosis and increased the expression of the mitochondrial biogenesis marker PGC-1α and multiple electron transport chain proteins. Thus, our results support ß2-adrenergic receptors as novel therapeutic targets and formoterol as a therapeutic compound for treating podocytopathies.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Fumarato de Formoterol/farmacologia , Glomerulonefrite/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Podócitos/efeitos dos fármacos , Agonistas de Receptores Adrenérgicos beta 2/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Doxorrubicina/toxicidade , Fumarato de Formoterol/uso terapêutico , Técnicas de Silenciamento de Genes , Glomerulonefrite/induzido quimicamente , Glomerulonefrite/patologia , Humanos , Camundongos , Mitocôndrias/metabolismo , Biogênese de Organelas , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Podócitos/citologia , Podócitos/patologia , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Transdução de SinaisRESUMO
Transforming growth factor-ß (TGF-ß) is known to play a critical role in the pathogenesis of many progressive podocyte diseases. However, the molecular mechanisms regulating TGF-ß signaling in podocytes remain unclear. Using a podocyte-specific myosin (Myo)1c knockout, we demonstrate whether Myo1c is critical for TGF-ß-signaling in podocyte disease pathogenesis. Specifically, podocyte-specific Myo1c knockout mice were resistant to fibrotic injury induced by Adriamycin or nephrotoxic serum. Further, loss of Myo1c also protected from injury in the TGF-ß-dependent unilateral ureteral obstruction mouse model of renal interstitial fibrosis. Mechanistic analyses showed that loss of Myo1c significantly blunted TGF-ß signaling through downregulation of canonical and non-canonical TGF-ß pathways. Interestingly, nuclear rather than the cytoplasmic Myo1c was found to play a central role in controlling TGF-ß signaling through transcriptional regulation. Differential expression analysis of nuclear Myo1c-associated gene promoters showed that nuclear Myo1c targeted the TGF-ß responsive gene growth differentiation factor (GDF)-15 and directly bound to the GDF-15 promoter. Importantly, GDF15 was found to be involved in podocyte pathogenesis, where GDF15 was upregulated in glomeruli of patients with focal segmental glomerulosclerosis. Thus, Myo1c-mediated regulation of TGF-ß-responsive genes is central to the pathogenesis of podocyte injury. Hence, inhibiting this process may have clinical application in treating podocytopathies.
Assuntos
Fator 15 de Diferenciação de Crescimento/genética , Nefropatias/patologia , Miosina Tipo I/metabolismo , Podócitos/patologia , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/metabolismo , Animais , Modelos Animais de Doenças , Doxorrubicina/toxicidade , Feminino , Fibrose , Regulação da Expressão Gênica , Humanos , Nefropatias/induzido quimicamente , Masculino , Camundongos , Camundongos Knockout , Miosina Tipo I/genética , Podócitos/efeitos dos fármacos , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
Steroid-resistant nephrotic syndrome is a frequent cause of chronic kidney disease almost inevitably progressing to end-stage renal disease. More than 58 monogenic causes of SRNS have been discovered and majority of known steroid-resistant nephrotic syndrome causing genes are predominantly expressed in glomerular podocytes, placing them at the center of disease pathogenesis. Herein, we describe two unrelated families with steroid-resistant nephrotic syndrome with homozygous mutations in the KIRREL1 gene. One mutation showed high frequency in the European population (minor allele frequency 0.0011) and this patient achieved complete remission following treatment, but later progressed to chronic kidney disease. We found that mutant KIRREL1 proteins failed to localize to the podocyte cell membrane, indicating defective trafficking and impaired podocytes function. Thus, the KIRREL1 gene product has an important role in modulating the integrity of the slit diaphragm and maintaining glomerular filtration function.
Assuntos
Resistência a Medicamentos/genética , Glucocorticoides/farmacologia , Proteínas de Membrana/genética , Síndrome Nefrótica/genética , Insuficiência Renal Crônica/genética , Adolescente , Idade de Início , Linhagem Celular , Criança , Pré-Escolar , Consanguinidade , Análise Mutacional de DNA , Progressão da Doença , Feminino , Seguimentos , Frequência do Gene , Membrana Basal Glomerular/patologia , Membrana Basal Glomerular/ultraestrutura , Glucocorticoides/uso terapêutico , Homozigoto , Humanos , Masculino , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Mutação , Síndrome Nefrótica/tratamento farmacológico , Síndrome Nefrótica/patologia , Linhagem , Podócitos , Insuficiência Renal Crônica/patologia , Sequenciamento do ExomaRESUMO
Beta2-adrenergic receptor (ß2 -AR) is a G-protein-coupled adrenergic receptor family member, whose clinical significance has been extensively investigated in lung, cardiovascular and muscular diseases, but its role in kidney biology remains understudied. In this review, we discuss some of the recent studies, where the effect of agonist/antagonist-mediated activation/inhibition of ß2 -AR on disease pathogenesis process was studied, and highlighted the role of ß2 -AR in kidney biology. The expression of ß2 -AR has been noted in many kidney subunits including proximal tubules, glomeruli and podocytes. In vivo studies have shown that in cultured proximal tubules ß2 -AR is involved in Na-ATPase activity and transcellular Na-transport through protein kinase-C activation; whereas in cultured podocytes, it was associated with depolarization of the membrane. The animal studies further revealed that ß2 -AR activation by short-acting ß2 agonists attenuated monocyte activation, pro-inflammatory and pro-fibrotic responses through ß-arrestin2 dependent NF-kB inactivation in diabetic kidney disease; in contrast, activation by long-acting ß2 agonists restored mitochondrial and renal function in the acute kidney injury mice models through PGC-1α dependent mitochondrial biogenesis. In conclusion, the activation of ß2 -AR may present a rapidly developing therapeutic target for renal diseases.
Assuntos
Injúria Renal Aguda/metabolismo , Nefropatias Diabéticas/metabolismo , Rim/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/patologia , Injúria Renal Aguda/fisiopatologia , Agonistas de Receptores Adrenérgicos beta 2/uso terapêutico , Animais , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/fisiopatologia , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Rim/fisiopatologia , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Fármacos Renais/uso terapêutico , Transdução de SinaisRESUMO
Podocytes have a unique structure that supports glomerular filtration function, and many glomerular diseases result in loss of this structure, leading to podocyte dysfunction and ESRD (end stage renal disease). These structural and functional changes involve a complex set of molecular and cellular mechanisms that remain poorly understood. To understand the molecular signature of podocyte injury, we performed transcriptome analysis of cultured human podocytes injured either with PAN (puromycin aminonucleoside) or doxorubicin/adriamycin (ADR). The pathway analysis through DE (differential expression) and gene-enrichment analysis of the injured podocytes showed Tumor protein p53 (P53) as one of the major signaling pathways that was significantly upregulated upon podocyte injury. Accordingly, P53 expression was also up-regulated in the glomeruli of nephrotoxic serum (NTS) and ADR-injured mice. To further confirm these observations, cultured podocytes were treated with the P53 inhibitor pifithrin-α, which showed significant protection from ADR-induced actin cytoskeleton damage. In conclusion, signaling pathways that are involved in podocyte pathogenesis and can be therapeutically targeted were identified by high-throughput transcriptomic analysis of injured podocytes.
Assuntos
Doxorrubicina/efeitos adversos , Regulação da Expressão Gênica/efeitos dos fármacos , Nefropatias/metabolismo , Podócitos/metabolismo , Puromicina Aminonucleosídeo/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Animais , Doxorrubicina/farmacologia , Humanos , Nefropatias/induzido quimicamente , Nefropatias/patologia , Camundongos , Fosforilação/efeitos dos fármacos , Podócitos/patologia , Puromicina Aminonucleosídeo/farmacologiaRESUMO
We previously have shown that the highly conserved eight-protein exocyst trafficking complex is required for ciliogenesis in kidney tubule cells. We hypothesized here that ciliogenic programs are conserved across organs and species. To determine whether renal primary ciliogenic programs are conserved in the eye, and to characterize the function and mechanisms by which the exocyst regulates eye development in zebrafish, we focused on exoc5, a central component of the exocyst complex, by analyzing both exoc5 zebrafish mutants, and photoreceptor-specific Exoc5 knock-out mice. Two separate exoc5 mutant zebrafish lines phenocopied exoc5 morphants and, strikingly, exhibited a virtual absence of photoreceptors, along with abnormal retinal development and cell death. Because the zebrafish mutant was a global knockout, we also observed defects in several ciliated organs, including the brain (hydrocephalus), heart (cardiac edema), and kidney (disordered and shorter cilia). exoc5 knockout increased phosphorylation of the regulatory protein Mob1, consistent with Hippo pathway activation. exoc5 mutant zebrafish rescue with human EXOC5 mRNA completely reversed the mutant phenotype. We accomplished photoreceptor-specific knockout of Exoc5 with our Exoc5 fl/fl mouse line crossed with a rhodopsin-Cre driver line. In Exoc5 photoreceptor-specific knock-out mice, the photoreceptor outer segment structure was severely impaired at 4 weeks of age, although a full-field electroretinogram indicated a visual response was still present. However, by 6 weeks, visual responses were eliminated. In summary, we show that ciliogenesis programs are conserved in the kidneys and eyes of zebrafish and mice and that the exocyst is necessary for photoreceptor ciliogenesis and retinal development, most likely by trafficking cilia and outer-segment proteins.
Assuntos
Cílios/metabolismo , Exocitose , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Células Fotorreceptoras de Vertebrados/patologia , Retina/patologia , Proteínas de Transporte Vesicular/deficiência , Proteínas de Transporte Vesicular/metabolismo , Peixe-ZebraRESUMO
Podocyte dysfunction and loss is an early event and a hallmark of proteinuric kidney diseases. A podocyte's normal function is maintained via its unique cellular architecture that relies on an intracellular network of filaments, including filamentous actin (F-actin) and microtubules, that provides mechanical support. Damage to this filamentous network leads to changes in cellular morphology and results in podocyte injury, dysfunction, and death. Conversely, stabilization of this network protects podocytes and ameliorates proteinuria. This suggests that stabilization of podocyte architecture via its filamentous network could be a key therapeutic strategy for proteinuric kidney diseases. However, development of podocyte-directed therapeutics, especially those that target the cell's filamentous network, is still lacking, partly because of unavailability of appropriate cellular assays for use in a drug discovery environment. Here, we describe a new high-content screening-based methodology and its implementation on podocytes to identify paullone derivatives as a novel group of podocyte-protective compounds. We find that three compounds, i.e., kenpaullone, 1-azakenpaullone, and alsterpaullone, dose dependently protect podocytes from puromycin aminonucleoside (PAN)-mediated injury in vitro by reducing PAN-induced changes in both the filamentous actin and microtubules, with alsterpaullone providing maximal protection. Mechanistic studies further show that alsterpaullone suppressed PAN-induced activation of signaling downstream of GSK3ß and p38 mitogen-activated protein kinase. In vivo it reduced ADR-induced glomerular injury in a zebrafish model. Together, these results identify paullone derivatives as novel podocyte-protective agents for future therapeutic development.
Assuntos
Benzazepinas/farmacologia , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala , Indóis/farmacologia , Podócitos/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Fármacos Renais/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/patologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Doxorrubicina , Glicogênio Sintase Quinase 3 beta/metabolismo , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Nefropatias/patologia , Camundongos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/patologia , Podócitos/metabolismo , Podócitos/patologia , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Aminopeptidase A (APA) is expressed in glomerular podocytes and tubular epithelia and metabolizes angiotensin II (AngII), a peptide known to promote glomerulosclerosis. In this study, we tested whether APA expression changes in response to progressive nephron loss or whether APA exerts a protective role against glomerular damage and during AngII-mediated hypertensive kidney injury. At advanced stages of FSGS, fawn-hooded hypertensive rat kidneys exhibited distinctly increased APA staining in areas of intact glomerular capillary loops. Moreover, BALB/c APA-knockout (KO) mice injected with a nephrotoxic serum showed persistent glomerular hyalinosis and albuminuria 96 hours after injection, whereas wild-type controls achieved virtually full recovery. We then tested the effect of 4-week infusion of AngII (400 ng/kg per minute) in APA-KO and wild-type mice. Although we observed no significant difference in achieved systolic BP, AngII-treated APA-KO mice developed a significant rise in albuminuria not observed in AngII-treated wild-type mice along with increased segmental and global sclerosis and/or collapse of juxtamedullary glomeruli, microcystic tubular dilation, and tubulointerstitial fibrosis. In parallel, AngII treatment significantly increased the kidney AngII content and attenuated the expression of podocyte nephrin in APA-KO mice but not in wild-type controls. These data show that deficiency of APA increases susceptibility to glomerular injury in BALB/c mice. The augmented AngII-mediated kidney injury observed in association with increased intrarenal AngII accumulation in the absence of APA suggests a protective metabolizing role of APA in AngII-mediated glomerular diseases.
Assuntos
Glutamil Aminopeptidase/deficiência , Nefropatias/enzimologia , Nefropatias/etiologia , Glomérulos Renais , Animais , Suscetibilidade a Doenças , Masculino , Camundongos , Camundongos Knockout , RatosRESUMO
Asymmetric disposition of Fab arms in the structures solved for the broadly neutralizing monoclonal antibody (nmAb) IgG1 b12 raised the question of whether the unusual shape observed for b12 is common for all IgG1 mAbs or if there is a difference in the overall shape of nmAbs versus non-nmAbs. We compared small angle x-ray scattering (SAXS) data-based models and limited proteolysis profiles of some IgG1 mAbs known to be having and lacking HIV-1 neutralizing potency. In non-nmAbs, the Fab arms were found to be symmetrically disposed in space relative to central Fc, but in most nmAbs, the Fab arms were asymmetrically disposed, as seen for IgG1 b12. The only exceptions were 2G12 and 4E10, where both Fab arms were closed above Fc, suggesting some Fab-Fc and/or Fab-Fab interaction in the nmAbs that constrained extension of the Fab-Fc linker. Interestingly, these observations were correlated with differential proteolysis profiles of the mAbs by papain. Under conditions when papain could cut both Fab arms of non-nmAbs, only one Fab arm could be removed from neutralizing ones (except for 2G12 and 4E10). Chromatography and small angle x-ray scattering results of papain-digested products revealed that 1) the Fab-Fc or Fab-Fab interactions in unliganded mAbs are retained in digested products, and 2) whereas anti-gp120 non-nmAbs could bind two gp120 molecules, nmAbs could bind only one gp120. Additional experiments showed that except for 2G12 and 4E10, unopen shapes of nmAbs remain uninfluenced by ionic strength but can be reversibly opened by low pH of buffer accompanied by loss of ligand binding ability.
Assuntos
Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , HIV-1/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Ligantes , Modelos Moleculares , Papaína/metabolismo , Conformação Proteica , ProteóliseRESUMO
Podocytes are specialized epithelial cells that are critical components of the glomerular filtration barrier, and their dysfunction leads to proteinuria and renal failure. Therefore, preserving podocyte function is therapeutically significant. In this study, we identified Neph1 signaling as a therapeutic target that upon inhibition prevented podocyte damage from a glomerular injury-inducing agent puromycin aminonucleoside (PAN). To specifically inhibit Neph1 signaling, we used a protein transduction approach, where the cytoplasmic domain of Neph1 (Neph1CD) tagged with a protein transduction domain trans-activator of transcription was transduced in cultured podocytes prior to treatment with PAN. The PAN-induced Neph1 phosphorylation was significantly reduced in Neph1CD-transduced cells; in addition, these cells were resistant to PAN-induced cytoskeletal damage. The biochemical analysis using subfractionation studies showed that unlike control cells Neph1 was retained in the lipid raft fractions in the transduced cells following treatment with PAN, indicating that transduction of Neph1CD in podocytes prevented PAN-induced mislocalization of Neph1. In accordance, the immunofluorescence analysis further suggested that Neph1CD-transduced cells had increased ability to retain endogenous Neph1 at the membrane in response to PAN-induced injury. Similar results were obtained when angiotensin was used as an injury-inducing agent. Consistent with these observations, maintaining high levels of Neph1 at the membrane using a podocyte cell line overexpressing chimeric Neph1 increased the ability of podocytes to resist PAN-induced injury and PAN-induced albumin leakage. Using a zebrafish in vivo PAN and adriamycin injury models, we further demonstrated the ability of transduced Neph1CD to preserve glomerular function. Collectively, these results support the conclusion that inhibiting Neph1 signaling is therapeutically significant in preventing podocyte damage from glomerular injury.
Assuntos
Membrana Basal Glomerular/lesões , Membrana Basal Glomerular/metabolismo , Proteínas de Membrana/metabolismo , Podócitos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Antimetabólitos Antineoplásicos/efeitos adversos , Antimetabólitos Antineoplásicos/farmacologia , Linhagem Celular , Membrana Basal Glomerular/patologia , Humanos , Microdomínios da Membrana/genética , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/patologia , Proteínas de Membrana/genética , Fosforilação/genética , Podócitos/patologia , Puromicina Aminonucleosídeo/efeitos adversos , Puromicina Aminonucleosídeo/farmacologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
It has been suggested that soluble urokinase receptor (suPAR) is a causative circulating factor for and a biomarker of focal and segmental glomerulosclerosis (FSGS). Here we undertook validation of these assumptions in both mouse and human models. Injection of recombinant suPAR in wild-type mice did not induce proteinuria within 24 h. Moreover, a disease phenotype was not seen in an inducible transgenic mouse model that maintained elevated suPAR concentrations for 6 weeks. Plasma and urine suPAR concentrations were evaluated as clinical biomarkers in 241 patients with glomerular disease from the prospective, longitudinal multicenter observational NEPTUNE cohort. The serum suPAR concentration at baseline inversely correlated with estimated glomerular filtration rate (eGFR) and the urine suPAR/creatinine ratio positively correlated with the urine protein/creatinine ratio. After adjusting for eGFR and urine protein, neither the serum nor urine suPAR level was an independent predictor of FSGS histopathology. A multivariable mixed-effects model of longitudinal data evaluated the association between the change in serum suPAR concentration from baseline with eGFR. After adjusting for baseline suPAR concentration, age, gender, proteinuria, and time, the change in suPAR from baseline was associated with eGFR, but this association was not different for patients with FSGS as compared with other diagnoses. Thus these results do not support a pathological role for suPAR in FSGS.
Assuntos
Taxa de Filtração Glomerular , Glomerulonefrite/sangue , Glomerulonefrite/urina , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Adolescente , Adulto , Albuminúria/urina , Animais , Biomarcadores/sangue , Biomarcadores/urina , Criança , Creatinina/urina , Feminino , Glomerulonefrite/patologia , Glomerulonefrite por IGA/sangue , Glomerulonefrite por IGA/patologia , Glomerulonefrite por IGA/urina , Glomerulonefrite Membranosa/sangue , Glomerulonefrite Membranosa/patologia , Glomerulonefrite Membranosa/urina , Glomerulosclerose Segmentar e Focal/sangue , Glomerulosclerose Segmentar e Focal/patologia , Glomerulosclerose Segmentar e Focal/urina , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Nefrose Lipoide/sangue , Nefrose Lipoide/patologia , Nefrose Lipoide/urina , Estudos Prospectivos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/administração & dosagem , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Proteínas Recombinantes/farmacologia , Adulto JovemRESUMO
Activation of the slit diaphragm protein nephrin induces actin cytoskeletal remodeling, resulting in lamellipodia formation in podocytes in vitro in a phosphatidylinositol-3 kinase-, focal adhesion kinase-, Cas-, and Crk1/2-dependent fashion. In mice, podocyte-specific deletion of Crk1/2 prevents or attenuates foot process effacement in two models of podocyte injury. This suggests that cellular mechanisms governing lamellipodial protrusion in vitro are similar to those in vivo during foot process effacement. As Crk1/2-null mice developed and aged normally, we tested whether the Crk1/2 paralog, CrkL, functionally complements Crk1/2 in a podocyte-specific context. Podocyte-specific CrkL-null mice, like podocyte-specific Crk1/2-null mice, developed and aged normally but were protected from protamine sulfate-induced foot process effacement. Simultaneous podocyte-specific deletion of Crk1/2 and CrkL resulted in albuminuria detected by 6 weeks postpartum and associated with altered podocyte process architecture. Nephrin-induced lamellipodia formation in podocytes in vitro was CrkL-dependent. CrkL formed a hetero-oligomer with Crk2 and, like Crk2, was recruited to tyrosine phosphorylated nephrin. Thus, Crk1/2 and CrkL are physically linked, functionally complement each other during podocyte foot process spreading, and together are required for developing typical foot process architecture.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Nucleares/metabolismo , Podócitos/metabolismo , Proteínas Proto-Oncogênicas c-crk/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Albuminúria/genética , Albuminúria/metabolismo , Animais , Genótipo , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Camundongos Knockout , Morfogênese , Complexos Multiproteicos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fenótipo , Fosforilação , Podócitos/efeitos dos fármacos , Podócitos/ultraestrutura , Protaminas/toxicidade , Proteínas Proto-Oncogênicas c-crk/deficiência , Proteínas Proto-Oncogênicas c-crk/genética , Pseudópodes/metabolismo , Interferência de RNA , Transdução de Sinais , TransfecçãoRESUMO
BACKGROUND: Podocytes have a remarkable ability to recover from injury; however, little is known about the recovery mechanisms involved in this process. We recently showed that formoterol, a long-acting ß2-adrenergic receptor (ß2-AR) agonist, induced mitochondrial biogenesis (MB) in podocytes and led to renoprotection in mice. However, it is not clear whether this effect was mediated by formoterol acting through the ß2-AR or if it occurred through "off-target" effects. METHODS: We genetically deleted the ß2-AR specifically in murine podocytes and used these mice to determine whether formoterol acting through the podocyte ß2-AR alone is sufficient for recovery of renal filtration function following injury. The podocyte-specific ß2-AR knockout mice (ß2-ARfl/fl/PodCre) were generated by crossing ß2-AR floxed mice with podocin Cre (B6.Cg-Tg(NPHS2-cre)295Lbh/J) mice. These mice were then subjected to both acute and chronic glomerular injury using nephrotoxic serum (NTS) and adriamycin (ADR), respectively. The extent of injury was evaluated by measuring albuminuria and histological and immunostaining analysis of the murine kidney sections. RESULTS: A similar level of injury was observed in ß2-AR knockout and control mice; however, the ß2-ARfl/fl/PodCre mice failed to recover in response to formoterol. Functional evaluation of the ß2-ARfl/fl/PodCre mice following injury plus formoterol showed similar albuminuria and glomerular injury to control mice that were not treated with formoterol. CONCLUSIONS: These results indicate that the podocyte ß2-AR is a critical component of the recovery mechanism and may serve as a novel therapeutic target for treating podocytopathies.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2 , Doxorrubicina , Fumarato de Formoterol , Camundongos Knockout , Podócitos , Receptores Adrenérgicos beta 2 , Animais , Podócitos/metabolismo , Podócitos/efeitos dos fármacos , Podócitos/patologia , Receptores Adrenérgicos beta 2/metabolismo , Camundongos , Fumarato de Formoterol/farmacologia , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Doxorrubicina/farmacologia , Doxorrubicina/toxicidade , Masculino , Camundongos Endogâmicos C57BL , Albuminúria/metabolismo , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologiaRESUMO
Neph1 is present in podocytes, where it plays a critical role in maintaining the filtration function of the glomerulus, in part through signaling events mediated by its cytoplasmic domain that are involved in actin cytoskeleton organization. To understand the function of this protein, a detailed knowledge of the structure of the Neph1 cytoplasmic domain (Neph1-CD) is required. In this study, the solution structure of this domain was determined by small/wide angle x-ray scattering (SWAXS). Analysis of Neph1-CD by SWAXS suggested that this protein adopts a global shape with a radius of gyration and a maximum linear dimension of 21.3 and 70 Å, respectively. These parameters and the data from circular dichroism experiments were used to construct a structural model of this protein. The His-ZO-1-PDZ1 (first PDZ domain of zonula occludens) domain that binds Neph1-CD was also analyzed by SWAXS, to confirm that it adopts a global structure similar to its crystal structure. We used the SWAXS intensity profile, the structural model of Neph1-CD, and the crystal structure of ZO-1-PDZ1 to construct a structural model of the Neph1-CD·ZO-1-PDZ1 complex. Mapping of the intermolecular interactions suggested that in addition to the C-terminal residues Thr-His-Val, residues Lys-761 and Tyr-762 in Neph1 are also critical for stabilizing the complex. Estimated intensity values from the SWAXS data and in vivo and in vitro pull-down experiments demonstrated loss of binding to ZO-1 when these residues were individually mutated to alanines. Our findings present a structural model that provides novel insights into the molecular structure and function of Neph1-CD.
Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Podócitos/metabolismo , Sítios de Ligação , Proteínas de Membrana/genética , Estrutura Molecular , Domínios PDZ , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Podócitos/química , Ligação Proteica , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo , Proteína da Zônula de Oclusão-1RESUMO
The targeting and organization of podocyte slit diaphragm proteins nephrin and neph1 is critical for development and maintenance of a functional glomerular filtration barrier. Myo1c is a non-muscle myosin motor protein that interacts directly with nephrin and neph1, and mediates their intracellular transport to the podocyte intercellular junction. Here we investigated the necessity of Myo1c in podocyte development using zebrafish as a model system. Immunofluorescence microscopy and in situ RNA hybridization analysis of zebrafish embryos showed that Myo1c is widely expressed in various tissues including the zebrafish glomerulus. Knockdown of the Myo1c gene in zebrafish using antisense morpholino derivatives resulted in an abnormal developmental phenotype that included pericardial edema and dilated renal tubules. Ultrastructural analysis of the glomerulus in Myo1c-depleted zebrafish showed abnormal podocyte morphology and absence of the slit diaphragm. Consistent with these observations, the glomerular filter permeability appeared altered in zebrafish in which Myo1c expression was attenuated. The specificity of Myo1c knockdown was confirmed by a rescue experiment in which co-injection of Myo1c morpholino derivatives with orthologous Myo1c mRNA prepared from mouse cDNA lessened phenotypic abnormalities including edema in Myo1c morphants. Thus, our results demonstrate that Myo1c is necessary for podocyte morphogenesis.