RESUMO
Chronic kidney disease (CKD) can progress to kidney failure and require dialysis or transplantation, while early diagnosis can alter the course of disease and lead to better outcomes in both pediatric and adult patients. Significant CKD comorbidities include the manifestation of cardiovascular disease, heart failure, coronary disease, and hypertension. The pathogenesis of chronic kidney diseases can present as subtle and especially difficult to distinguish between different glomerular pathologies. Early detection of adult and pediatric CKD and detailed mechanistic understanding of the kidney damage can be helpful in delaying or curtailing disease progression via precise intervention toward diagnosis and prognosis. Clinically, serum creatinine and albumin levels can be indicative of CKD, but often are a lagging indicator only significantly affected once kidney function has severely diminished. The evolution of proteomics and mass spectrometry technologies has begun to provide a powerful research tool in defining these mechanisms and identifying novel biomarkers of CKD. Many of the same challenges and advances in proteomics apply to adult and pediatric patient populations. Additionally, proteomic analysis of adult CKD patients can be transferred directly toward advancing our knowledge of pediatric CKD as well. In this review, we highlight applications of proteomics that have yielded such biomarkers as PLA2R, SEMA3B, and other markers of membranous nephropathy as well as KIM-1, MCP-1, and NGAL in lupus nephritis among other potential diagnostic and prognostic markers. The potential for improving the clinical toolkit toward better treatment of pediatric kidney diseases is significantly aided by current and future development of proteomic applications.
Assuntos
Nefropatias , Insuficiência Renal Crônica , Adulto , Biomarcadores , Criança , Taxa de Filtração Glomerular , Humanos , Nefropatias/diagnóstico , Proteômica , Diálise RenalRESUMO
BACKGROUND: The mechanisms leading to extracellular matrix (ECM) replacement of areas of glomerular capillaries in histologic variants of FSGS are unknown. This study used proteomics to test the hypothesis that glomerular ECM composition in collapsing FSGS (cFSGS) differs from that of other variants. METHODS: ECM proteins in glomeruli from biopsy specimens of patients with FSGS not otherwise specified (FSGS-NOS) or cFSGS and from normal controls were distinguished and quantified using mass spectrometry, verified and localized using immunohistochemistry (IHC) and confocal microscopy, and assessed for gene expression. The analysis also quantified urinary excretion of ECM proteins and peptides. RESULTS: Of 58 ECM proteins that differed in abundance between cFSGS and FSGS-NOS, 41 were more abundant in cFSGS and 17 in FSGS-NOS. IHC showed that glomerular tuft staining for cathepsin B, cathepsin C, and annexin A3 in cFSGS was significantly greater than in other FSGS variants, in minimal change disease, or in membranous nephropathy. Annexin A3 colocalized with cathepsin B and C, claudin-1, phosphorylated ERK1/2, and CD44, but not with synaptopodin, in parietal epithelial cells (PECs) infiltrating cFSGS glomeruli. Transcripts for cathepsins B and C were increased in FSGS glomeruli compared with normal controls, and urinary excretion of both cathepsins was significantly greater in cFSGS compared with FSGS-NOS. Urinary excretion of ECM-derived peptides was enhanced in cFSGS, although in silico analysis did not identify enhanced excretion of peptides derived from cathepsin B or C. CONCLUSIONS: ECM differences suggest that glomerular sclerosis in cFSGS differs from that in other FSGS variants. Infiltration of activated PECs may disrupt ECM remodeling in cFSGS. These cells and their cathepsins may be therapeutic targets.
Assuntos
Proteínas da Matriz Extracelular/análise , Glomerulosclerose Segmentar e Focal/metabolismo , Glomérulos Renais/metabolismo , Proteômica/métodos , Catepsinas/fisiologia , Células Epiteliais/fisiologia , Humanos , Imuno-Histoquímica , Glomérulos Renais/química , Microscopia ConfocalRESUMO
Na+/H+ exchange regulatory cofactor (NHERF)-1, a scaffolding protein, anchors multiple membrane proteins in renal proximal tubules. Cultured proximal tubule cells deficient in Nherf1 and proximal tubules from Nherf1-deficient mice exhibit aberrant trafficking. Nherf1-deficient cells also exhibit an altered transcription pattern and worse survival. These observations suggest that NHERF1 loss increases susceptibility to acute kidney injury (AKI). Male and female wild-type C57BL/6J and Nherf1 knockout mice were treated with saline or cisplatin (20 mg/kg dose i.p.) to induce AKI and were euthanized after 72 hours. Blood and urine were collected for assessments of blood urea nitrogen and neutrophil gelatinase-associated lipocalin, respectively. Kidneys were harvested for histology (hematoxylin and eosin, periodic acid-Schiff) and terminal deoxynucleotidyl transferase dUTP nick end labeling assay, Kim1 mRNA assessment, and Western blot analysis for cleaved caspase 3. Cisplatin treatment was associated with significantly greater severity of AKI in knockout compared with wild-type mice, as demonstrated by semiquantitative injury score (2.8 versus 1.89, P < 0.001), blood urea nitrogen (151.8 ± 17.2 mg/dL versus 97.8 ± 10.1 mg/dL, P < 0.05), and neutrophil gelatinase-associated lipocalin urine protein (55.6 ± 21.3 µg/mL versus 2.7 ± 0.53 µg/mL, P < 0.05). Apoptosis markers were significantly increased in cisplatin-treated Nherf1 knockout and wild-type mice compared to respective controls. These data suggest that NHERF1 loss increases susceptibility to AKI.
Assuntos
Injúria Renal Aguda/metabolismo , Cisplatino/efeitos adversos , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/genética , Injúria Renal Aguda/patologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 3/metabolismo , Cisplatino/farmacologia , Suscetibilidade a Doenças , Feminino , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Lipocalina-2/metabolismo , Masculino , Camundongos , Camundongos Knockout , Fosfoproteínas/genética , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
RATIONALE: Endothelial progenitor cells (EPCs) respond to stromal cell-derived factor 1 (SDF-1) through chemokine receptors CXCR7 and CXCR4. Whether SDF-1 receptors involves in diabetes mellitus-induced EPCs dysfunction remains unknown. OBJECTIVE: To determine the role of SDF-1 receptors in diabetic EPCs dysfunction. METHODS AND RESULTS: CXCR7 expression, but not CXCR4 was reduced in EPCs from db/db mice, which coincided with impaired tube formation. Knockdown of CXCR7 impaired tube formation of EPCs from normal mice, whereas upregulation of CXCR7 rescued angiogenic function of EPCs from db/db mice. In normal EPCs treated with oxidized low-density lipoprotein or high glucose also reduced CXCR7 expression, impaired tube formation, and increased oxidative stress and apoptosis. The damaging effects of oxidized low-density lipoprotein or high glucose were markedly reduced by SDF-1 pretreatment in EPCs transduced with CXCR7 lentivirus but not in EPCs transduced with control lentivirus. Most importantly, EPCs transduced with CXCR7 lentivirus were superior to EPCs transduced with control lentivirus for therapy of ischemic limbs in db/db mice. Mechanistic studies demonstrated that oxidized low-density lipoprotein or high glucose inhibited protein kinase B and glycogen synthase kinase-3ß phosphorylation, nuclear export of Fyn and nuclear localization of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), blunting Nrf2 downstream target genes heme oxygenase-1, NAD(P)H dehydrogenase (quinone 1) and catalase, and inducing an increase in EPC oxidative stress. This destructive cascade was blocked by SDF-1 treatment in EPCs transduced with CXCR7 lentivirus. Furthermore, inhibition of phosphatidylinositol 3-kinase/protein kinase B prevented SDF-1/CXCR7-mediated Nrf2 activation and blocked angiogenic repair. Moreover, Nrf2 knockdown almost completely abolished the protective effects of SDF-1/CXCR7 on EPC function in vitro and in vivo. CONCLUSIONS: Elevated expression of CXCR7 enhances EPC resistance to diabetes mellitus-induced oxidative damage and improves therapeutic efficacy of EPCs in treating diabetic limb ischemia. The benefits of CXCR7 are mediated predominantly by a protein kinase B/glycogen synthase kinase-3ß/Fyn pathway via increased activity of Nrf2.
Assuntos
Diabetes Mellitus/metabolismo , Células Progenitoras Endoteliais/fisiologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Isquemia/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Receptores CXCR/biossíntese , Animais , Células Cultivadas , Diabetes Mellitus/patologia , Técnicas de Silenciamento de Genes , Células HEK293 , Membro Posterior/irrigação sanguínea , Membro Posterior/metabolismo , Membro Posterior/patologia , Humanos , Isquemia/patologia , Masculino , Camundongos , Camundongos Transgênicos , Fator 2 Relacionado a NF-E2/metabolismo , Neovascularização Fisiológica/fisiologia , Estresse Oxidativo/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Acute glomerulonephritis is characterized by rapid glomerular neutrophil recruitment, proteinuria, and glomerular hypercellularity. The current study tested the hypothesis that the release of neutrophil granule contents plays a role in both the loss of filtration barrier leading to proteinuria and the increase in glomerular cells. Inhibition of neutrophil exocytosis with a peptide inhibitor prevented proteinuria and attenuated podocyte and endothelial cell injury but had no effect on glomerular hypercellularity in an experimental acute glomerulonephritis model in mice. Cultivation of podocytes with neutrophil granule contents disrupted cytoskeletal organization, an in vitro model for podocyte effacement and loss of filtration barrier. Activated, cultured podocytes released cytokines that stimulated neutrophil chemotaxis, primed respiratory burst activity, and stimulated neutrophil exocytosis. We conclude that crosstalk between podocytes and neutrophils contributes to disruption of the glomerular filtration barrier in acute glomerulonephritis. Neutrophil granule products induce podocyte injury but do not participate in the proliferative response of intrinsic glomerular cells.
Assuntos
Citoesqueleto de Actina/metabolismo , Doença Antimembrana Basal Glomerular/metabolismo , Comunicação Celular , Exocitose , Taxa de Filtração Glomerular , Neutrófilos/metabolismo , Podócitos/metabolismo , Proteinúria/metabolismo , Citoesqueleto de Actina/patologia , Animais , Doença Antimembrana Basal Glomerular/patologia , Doença Antimembrana Basal Glomerular/fisiopatologia , Doença Antimembrana Basal Glomerular/prevenção & controle , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Exocitose/efeitos dos fármacos , Feminino , Produtos do Gene tat/farmacologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Ativação de Neutrófilo , Infiltração de Neutrófilos , Neutrófilos/efeitos dos fármacos , Podócitos/patologia , Proteinúria/patologia , Proteinúria/fisiopatologia , Proteinúria/prevenção & controle , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Explosão Respiratória , Proteínas SNARE/farmacologiaRESUMO
Transcription factor NF-κB regulates expression of numerous genes that control inflammation and is activated in glomerular cells in glomerulonephritis (GN). We previously identified genetic variants for a NF-κB regulatory, ubiquitin-binding protein ABIN1 as risk factors for GN in systemic autoimmunity. The goal was to define glomerular inflammatory events controlled by ABIN1 function in GN. Nephrotoxic serum nephritis was induced in wild-type (WT) and ubiquitin-binding deficient ABIN1[D485N] mice, and renal pathophysiology and glomerular inflammatory phenotypes were assessed. Proteinuria was also measured in ABIN1[D485N] mice transplanted with WT mouse bone marrow. Inflammatory activation of ABIN1[D472N] (D485N homolog) cultured human-derived podocytes, and interaction with primary human neutrophils were also assessed. Disruption of ABIN1 function exacerbated proteinuria, podocyte injury, glomerular NF-κB activity, glomerular expression of inflammatory mediators, and glomerular recruitment and retention of neutrophils in antibody-mediated nephritis. Transplantation of WT bone marrow did not prevent the increased proteinuria in ABIN1[D845N] mice. Tumor necrosis factor-stimulated enhanced expression and secretion of NF-κB-targeted proinflammatory mediators in ABIN1[D472N] cultured podocytes compared with WT cells. Supernatants from ABIN1[D472N] podocytes accelerated chemotaxis of human neutrophils, and ABIN1[D472N] podocytes displayed a greater susceptibility to injurious morphologic findings induced by neutrophil granule contents. These studies define a novel role for ABIN1 dysfunction and NF-κB in mediating GN through proinflammatory activation of podocytes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Glomerulonefrite/patologia , NF-kappa B/metabolismo , Podócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Glomerulonefrite/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos MutantesRESUMO
Dopamine decreases Na-K-ATPase (NKA) activity by PKC-dependent phosphorylation and endocytosis of the NKA α1. Dopamine-mediated regulation of NKA is impaired in aging and some forms of hypertension. Using opossum (OK) proximal tubule cells (PTCs), we demonstrated that sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) associates with NKA α1 and dopamine-1 receptor (D1R). This association is required for the dopamine-mediated regulation of NKA. In OK cells, dopamine decreases NHERF-1 association with NKA α1 but increases its association with D1R. However, it is not known whether NHERF-1 plays a role in dopamine-mediated NKA regulation in animal models of hypertension. We hypothesized that defective dopamine-mediated regulation of NKA results from the decrease in NHERF-1 expression in rat renal PTCs isolated from animal models of hypertension [spontaneously hypertensive rats (SHRs) and aged F344 rats]. To test this hypothesis, we isolated and cultured renal PTCs from 22-mo-old F344 rats and their controls, normotensive 4-mo-old F344 rats, and SHRs and their controls, normotensive Wistar-Kyoto (WKY) rats. The results demonstrate that in both hypertensive models (SHR and aged F344), NHERF-1 expression, dopamine-mediated phosphorylation of NKA, and ouabain-inhibitable K+ transport are reduced. Transfection of NHERF-1 into PTCs from aged F344 and SHRs restored dopamine-mediated inhibition of NKA. These results suggest that decreased renal NHERF-1 expression contributes to the impaired dopamine-mediated inhibition of NKA in PTCs from animal models of hypertension.
Assuntos
Hipertensão/genética , Túbulos Renais Proximais/metabolismo , Fosfoproteínas/biossíntese , Trocadores de Sódio-Hidrogênio/biossíntese , ATPase Trocadora de Sódio-Potássio/biossíntese , Animais , Pressão Sanguínea/genética , Linhagem Celular , Modelos Animais de Doenças , Dopamina/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Hipertensão/metabolismo , Hipertensão/patologia , Rim/metabolismo , Rim/patologia , Túbulos Renais Proximais/patologia , Masculino , Fosfoproteínas/genética , Ratos , Ratos Endogâmicos SHR , Transdução de Sinais/genética , Trocadores de Sódio-Hidrogênio/genética , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
Abnormal extracellular matrix (ECM) remodeling is a prominent feature of many glomerular diseases and is a final common pathway of glomerular injury. However, changes in ECM composition accompanying disease-related remodeling are unknown. The physical properties of ECM create challenges for characterization of composition using standard protein extraction techniques, as the insoluble components of ECM are frequently discarded and many ECM proteins are in low abundance compared to other cell proteins. Prior proteomic studies defining normal ECM composition used a large number of glomeruli isolated from human kidneys retrieved for transplantation or by nephrectomy for cancer. Here we examined the ability to identify ECM proteins by mass spectrometry using glomerular sections compatible with those available from standard renal biopsy specimens. Proteins were classified as ECM by comparison to the Matrisome database and previously identified glomerular ECM proteins. Optimal ECM protein identification resulted from sequential decellularization and protein extraction of 100 human glomerular sections isolated by laser capture microdissection from either frozen or formalin-fixed, paraffin-embedded tissue. In total, 147 ECM proteins were identified, including the majority of structural and GBM proteins previously identified along with a number of matrix and glomerular basement membrane proteins not previously associated with glomeruli. Thus, our study demonstrates the feasibility of proteomic analysis of glomerular ECM from retrieved glomerular sections isolated from renal biopsy tissue and expands the list of known ECM proteins in glomeruli.
Assuntos
Proteínas da Matriz Extracelular/análise , Matriz Extracelular/química , Membrana Basal Glomerular/química , Nefropatias/metabolismo , Microdissecção e Captura a Laser , Proteômica/métodos , Biomarcadores/análise , Biópsia , Bases de Dados de Proteínas , Matriz Extracelular/patologia , Estudos de Viabilidade , Fixadores , Formaldeído , Secções Congeladas , Membrana Basal Glomerular/patologia , Humanos , Nefropatias/diagnóstico , Espectrometria de Massas , Inclusão em Parafina , Valor Preditivo dos Testes , Mapas de Interação de Proteínas , Reprodutibilidade dos Testes , Fixação de Tecidos/métodosRESUMO
BACKGROUND: A targeted analysis of the 50kDa C-terminal fragment of insulin-response element binding protein-1 (IRE-BP1) activation of target genes through the insulin receptor substrate receptor/PI-3 kinase/Akt pathway has been demonstrated for the insulin growth factor-1 receptor. The broader effects of 50kDa C-terminal IRE-BP1 fragment over-expression on protein abundance in pancreatic islet beta cells have not been determined. RESULTS: Liquid-chromatography coupled to tandem mass spectrometry (LC-MS/MS) analyses of replicate lysates of pancreatic islets isolated from background strain animals and transgenic animals, overexpressing IRE-BP1 in pancreatic islet beta cells, demonstrated statistically significant increases in the expression of proteins involved in protein synthesis, endoplasmic reticulum (ER) stress and scaffolding proteins important for protein kinase C signaling; some of which were confirmed by immunoblot analyses. Bioinformatic analysis of protein expression network patterns suggested IRE-BP1 over-expression leads to protein expression patterns indicative of activation of functional protein networks utilized for protein post-translational modification, protein folding, and protein synthesis. Co-immunoprecipitation experiments demonstrate a novel interaction between two differentially regulated proteins receptor for activated protein kinase C (RACK1) and translationally controlled tumor protein (TCTP). CONCLUSIONS: Proteomic analysis of IRE-BP1 over-expression in pancreatic islet beta cells suggest IRE-BP1 (a) directly or indirectly through establishing hyperglycemia results in increased expression of ribosomal proteins and markers of ER stress and (b) leads to the enhanced and previously un-described interaction of RACK1 and TCTP. SIGNIFICANCE: This study identified C-terminal 50kDa domain of IRE-BP1 over-expression results in increased markers of ER-stress and a novel interaction between the scaffolding proteins RACK1 and TCTP.
Assuntos
Biomarcadores Tumorais/metabolismo , Biomarcadores/metabolismo , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Proteína 1 Reguladora do Ferro/metabolismo , Ilhotas Pancreáticas/metabolismo , Neuropeptídeos/metabolismo , Animais , Estresse do Retículo Endoplasmático/fisiologia , Hiperglicemia , Insulina/metabolismo , Camundongos , Proteína Quinase C/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteômica/métodos , Receptores de Quinase C Ativada , Elementos de Resposta/fisiologia , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
Microbotryum lychnidis-dioicae is a member of a species complex infecting host plants in the Caryophyllaceae. It is used as a model system in many areas of research, but attempts to make this organism tractable for reverse genetic approaches have not been fruitful. Here, we exploited the recently obtained genome sequence and transcriptome analysis to inform our design of constructs for use in Agrobacterium-mediated transformation techniques currently available for other fungi. Reproducible transformation was demonstrated at the genomic, transcriptional and functional levels. Moreover, these initial proof-of-principle experiments provide evidence that supports the findings from initial global transcriptome analysis regarding expression from the respective promoters under different growth conditions of the fungus. The technique thus provides for the first time the ability to stably introduce transgenes and over-express target M. lychnidis-dioicae genes.
Assuntos
Basidiomycota/genética , Técnicas de Transferência de Genes , Genoma Fúngico/genética , Doenças das Plantas/microbiologia , Plantas/microbiologia , Transformação Genética/genética , Mapeamento Cromossômico , Perfilação da Expressão Gênica , Genômica , Transcriptoma/genéticaRESUMO
Dimorphic transitions between yeast-like and filamentous forms occur in many fungi and are often associated with pathogenesis. One of the cues for such a dimorphic switch is the availability of nutrients. Under conditions of nitrogen limitation, fungal cells (such as those of Saccharomyces cerevisiae and Ustilago maydis) switch from budding to pseudohyphal or filamentous growth. Ammonium transporters (AMTs) are responsible for uptake and, in some cases, for sensing the availability of ammonium, a preferred nitrogen source. Homodimer and/or heterodimer formation may be required for regulating the activity of the AMTs. To investigate the potential interactions of Ump1 and Ump2, the AMTs of the maize pathogen U. maydis, we first used the split-ubiquitin system, followed by a modified split-YFP (yellow fluorescent protein) system, to validate the interactions in vivo. This analysis showed the formation of homo- and hetero-oligomers by Ump1 and Ump2. We also demonstrated the interaction of the high-affinity ammonium transporter, Ump2, with the Rho1 GTPase, a central protein in signaling, with roles in controlling polarized growth. This is the first demonstration in eukaryotes of the physical interaction in vivo of an ammonium transporter with the signaling protein Rho1. Moreover, the Ump proteins interact with Rho1 during the growth of cells in low ammonium concentrations, a condition required for the expression of the Umps. Based on these results and the genetic evidence for the interaction of Ump2 with both Rho1 and Rac1, another small GTPase, we propose a model for the role of these interactions in controlling filamentation, a fundamental aspect of development and pathogenesis in U. maydis.
Assuntos
Proteínas Fúngicas/genética , Transporte de Íons/genética , Transdução de Sinais , Ustilago/genética , Sequência de Aminoácidos , AMP Cíclico , Regulação Fúngica da Expressão Gênica/genética , Hifas/genética , Hifas/crescimento & desenvolvimento , Chaperonas Moleculares/genética , Nitrogênio/metabolismo , Saccharomyces cerevisiae/genética , Ustilago/crescimento & desenvolvimento , Zea mays/microbiologiaRESUMO
The mechanisms by which aldosterone increases Na(+), K(+) ATPase and sodium channel activity in cortical collecting duct and distal nephron have been extensively studied. Recent investigations demonstrate that aldosterone increases Na-H exchanger-3 (NHE-3) activity, bicarbonate transport, and H(+) ATPase in proximal tubules. However, the role of aldosterone in regulation of Na(+), K(+) ATPase in proximal tubules is unknown. We hypothesize that aldosterone increases Na(+), K(+) ATPase activity in proximal tubules through activation of the mineralocorticoid receptor (MR). Immunohistochemistry of kidney sections from human, rat, and mouse kidneys revealed that the MR is expressed in the cytosol of tubules staining positively for Lotus tetragonolobus agglutinin and type IIa sodium-phosphate cotransporter (NpT2a), confirming proximal tubule localization. Adrenalectomy in Sprague-Dawley rats decreased expression of MR, ENaC α, Na(+), K(+) ATPase α1, and NHE-1 in all tubules, while supplementation with aldosterone restored expression of above proteins. In human kidney proximal tubule (HKC11) cells, treatment with aldosterone resulted in translocation of MR to the nucleus and phosphorylation of SGK-1. Treatment with aldosterone also increased Na(+), K(+) ATPase-mediated (86)Rb uptake and expression of Na(+), K(+) ATPase α1 subunits in HKC11 cells. The effects of aldosterone on Na(+), K(+) ATPase-mediated (86)Rb uptake were prevented by spironolactone, a competitive inhibitor of aldosterone for the MR, and partially by Mifepristone, a glucocorticoid receptor (GR) inhibitor. These results suggest that aldosterone regulates Na(+), K(+) ATPase in renal proximal tubule cells through an MR-dependent mechanism.
Assuntos
Trifosfato de Adenosina/metabolismo , Aldosterona/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Receptores de Mineralocorticoides/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Western Blotting , Membrana Celular , Células Cultivadas , Humanos , Hidrólise , Técnicas Imunoenzimáticas , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Masculino , Camundongos , Ratos , Ratos Sprague-DawleyRESUMO
Microbotryum lychnidis-dioicae is an obligate fungal species colonizing the plant host, Silene latifolia. The fungus synthesizes and secretes effector proteins into the plant host during infection to manipulate the host for completion of the fungal lifecycle. The goal of this study was to continue functional characterization of such M. lychnidis-dioicae effectors. Here, we identified three putative effectors and their putative host-plant target proteins. MVLG_02245 is highly upregulated in M. lychnidis-dioicae during infection; yeast two-hybrid analysis suggests it targets a tubulin α-1 chain protein ortholog in the host, Silene latifolia. A potential plant protein interacting with MVLG_06175 was identified as CASP-like protein 2C1 (CASPL2C1), which facilitates the polymerization of the Casparian strip at the endodermal cells. Proteins interacting with MVLG_05122 were identified as CSN5a or 5b, involved in protein turnover. Fluorescently labelled MVLG_06175 and MVLG_05122 were expressed in the heterologous plant, Arabidopsis thaliana. MVLG_06175 formed clustered granules at the tips of trichomes on leaves and in root caps, while MVLG_05122 formed a band structure at the base of leaf trichomes. Plants expressing MVLG_05122 alone were more resistant to infection with Fusarium oxysporum. These results indicate that the fungus might affect the formation of the Casparian strip in the roots and the development of trichomes during infection as well as alter plant innate immunity.
RESUMO
This study was to investigate whether sulforaphane (SFN) can prevent diabetic cardiomyopathy. Type 1 diabetes was induced in FVB mice by multiple intraperitoneal injections with low-dose streptozotocin. Hyperglycemic and age-matched control mice were treated with or without SFN at 0.5mg/kg daily in five days of each week for 3 months and then kept until 6 months. At 3 and 6 months of diabetes, blood pressure and cardiac function were assessed. Cardiac fibrosis, inflammation, and oxidative damage were assessed by Western blot, real-time qPCR, and histopathological examination. SFN significantly prevented diabetes-induced high blood pressure and cardiac dysfunction at both 3 and 6 months, and also prevented diabetes-induced cardiac hypertrophy (increased the ratio of heart weight to tibia length and the expression of atrial natriuretic peptide mRNA and protein) and fibrosis (increased the accumulation of collagen and expression of connective tissue growth factor and tissue growth factor-ß). SFN also almost completely prevented diabetes-induced cardiac oxidative damage (increased accumulation of 3-nitrotyrosine and 4-hydroxynonenal) and inflammation (increased tumor necrotic factor-α and plasminogen activator inhibitor 1 expression). SFN up-regulated NFE2-related factor 2 (Nrf2) expression and transcription activity that was reflected by increased Nrf2 nuclear accumulation and phosphorylation as well as the mRNA and protein expression of Nrf2 downstream antioxidants. Furthermore, in cultured H9c2 cardiac cells silencing Nrf2 gene with its siRNA abolished the SFN's prevention of high glucose-induced fibrotic response. These results suggest that diabetes-induced cardiomyopathy can be prevented by SFN, which was associated with the up-regulated Nrf2 expression and transcription function.
Assuntos
Diabetes Mellitus Experimental/complicações , Cardiomiopatias Diabéticas/prevenção & controle , Hipertrofia Ventricular Esquerda/prevenção & controle , Fator 2 Relacionado a NF-E2/metabolismo , Tiocianatos/uso terapêutico , Ativação Transcricional , Animais , Catalase/metabolismo , Linhagem Celular , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/fisiopatologia , Avaliação Pré-Clínica de Medicamentos , Fibrose , Heme Oxigenase-1/metabolismo , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/metabolismo , Isotiocianatos , Peroxidação de Lipídeos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Ratos , Serpina E2/metabolismo , Sulfóxidos , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Tiocianatos/farmacologia , Transcrição Gênica , Regulação para CimaRESUMO
The acute inhibitory effects of parathyroid hormone (PTH) on proximal tubule Na(+)-K(+)-ATPase (Na-K) and sodium-dependent phosphate (NaPi) transport have been extensively studied, while little is known about the chronic effects of PTH. Patients with primary hyperparathyroidism, a condition characterized by chronic elevations in PTH, exhibit persistent hypophosphatemia but not significant evidence of salt wasting. We postulate that chronic PTH stimulation results in differential desensitization of PTH responses. To address this hypothesis, we compared the effects of chronic PTH stimulation on Na-P(i) cotransporter (Npt2a) expression and Na-K activity and expression in Sprague Dawley rats, transgenic mice featuring parathyroid-specific cyclin D1 overexpression (PTH-D1), and proximal tubule cell culture models. We demonstrated a progressive decrease in brush-border membrane (BBM) expression of Npt2a from rats treated with PTH for 6 h or 4 days, while Na-K expression and activity in the basolateral membranes (BLM) exhibited an initial decrease followed by recovery to control levels by 4 days. Npt2a protein expression in PTH-D1 mice was decreased relative to control animals, whereas levels of Na-K, NHERF-1, and PTH receptor remained unchanged. In PTH-D1 mice, NpT2a mRNA expression was reduced by 50% relative to control mice. In opossum kidney proximal tubule cells, PTH decreased Npt2a mRNA levels. Both actinomycin D and cycloheximide treatment prevented the PTH-mediated decrease in Npt2a mRNA, suggesting that the PTH response requires transcription and translation. These findings suggest that responses to chronic PTH exposure are selectively regulated at a posttranscriptional level. The persistence of the phosphaturic response to PTH occurs through posttranscriptional mechanisms.
Assuntos
Hipofosfatemia/genética , Túbulos Renais Proximais/fisiologia , Hormônio Paratireóideo/metabolismo , Estabilidade de RNA/fisiologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Animais , Células Cultivadas , Ciclina D1/genética , Ciclina D1/metabolismo , Modelos Animais de Doenças , Hipofosfatemia/metabolismo , Córtex Renal/citologia , Córtex Renal/fisiologia , Túbulos Renais Proximais/citologia , Camundongos , Camundongos Transgênicos , Gambás , Hormônio Paratireóideo/farmacologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/fisiologia , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismoRESUMO
The role of exocytosis in the human neutrophil respiratory burst was determined using a fusion protein (TAT-SNAP-23) containing the HIV transactivator of transcription (TAT) cell-penetrating sequence and the N-terminal SNARE domain of synaptosome-associated protein-23 (SNAP-23). This agent inhibited stimulated exocytosis of secretory vesicles and gelatinase and specific granules but not azurophil granules. GST pulldown showed that TAT-SNAP-23 bound to the combination of vesicle-associated membrane protein-2 and syntaxin-4 but not to either individually. TAT-SNAP-23 reduced phagocytosis-stimulated hydrogen peroxide production by 60% without affecting phagocytosis or generation of HOCl within phagosomes. TAT-SNAP-23 had no effect on fMLF-stimulated superoxide release but significantly inhibited priming of this response by TNF-α and platelet-activating factor. Pretreatment with TAT-SNAP-23 inhibited the increase in plasma membrane expression of gp91(phox) in TNF-α-primed neutrophils, whereas TNF-α activation of ERK1/2 and p38 MAPK was not affected. The data demonstrate that neutrophil granule exocytosis contributes to phagocytosis-induced respiratory burst activity and plays a critical role in priming of the respiratory burst by increasing expression of membrane components of the NADPH oxidase.
Assuntos
Grânulos Citoplasmáticos/imunologia , Exocitose/imunologia , Ativação de Neutrófilo/imunologia , Explosão Respiratória/imunologia , Apoptose/genética , Apoptose/imunologia , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/metabolismo , Exocitose/genética , Produtos do Gene tat/antagonistas & inibidores , Produtos do Gene tat/genética , Produtos do Gene tat/metabolismo , HIV-1/imunologia , Humanos , Ativação de Neutrófilo/genética , Fagocitose/genética , Fagocitose/imunologia , Fator de Ativação de Plaquetas/fisiologia , Estrutura Terciária de Proteína/genética , Proteínas Qb-SNARE/antagonistas & inibidores , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/antagonistas & inibidores , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/metabolismo , Explosão Respiratória/genética , Proteínas SNARE/antagonistas & inibidores , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Notch proteins (Notch 1-4) are a family of trans-membrane cell surface receptors that are converted into transcriptional regulators when activated by interactions with cell surface ligands on adjacent cells. Ligand-binding stimulates proteolytic cleavage of the trans-membrane domain, releasing an active intracellular domain (ICD) that translocates to the nucleus and impacts transcription. In transit, the ICD may interact with regulatory proteins that modulate the expression and transcriptional activity. We have found that Notch4(ICD) expression is enhanced in the tubule cells of fibrotic kidneys from diabetic mice and humans and identified Notch4(ICD) interacting proteins that could be pertinent to normal and pathological functions. Using proteomic techniques, several components of the Elongin C complex were identified as candidate Notch4(ICD) interactors. Elongin C complexes can function as ubiquitin ligases capable of regulating proteasomal degradation of specific protein substrates. Our studies indicate that ectopic Elongin C expression stimulates Notch4(ICD) degradation and inhibits its transcriptional activity in human kidney tubule HK11 cells. Blocking Elongin C mediated degradation by MG132 indicates the potential for ubiquitin-mediated Elongin C regulation of Notch4(ICD). Functional interaction of Notch4(ICD) and Elongin C provides novel insight into regulation of Notch signaling in epithelial cell biology and disease.
Assuntos
Túbulos Renais/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Notch/metabolismo , Fatores de Transcrição/fisiologia , Animais , Células Cultivadas , Elonguina , Fibrose/genética , Fibrose/metabolismo , Regulação da Expressão Gênica , Humanos , Túbulos Renais/patologia , Túbulos Renais/fisiologia , Camundongos , Camundongos Transgênicos , Ligação Proteica/genética , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/fisiologia , Estabilidade Proteica , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Receptor Notch4 , Receptores Notch/química , Receptores Notch/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Fator de Crescimento Transformador beta/metabolismoRESUMO
The main determinant of pathogenicity in Ustilago maydis is the b-mating locus, where establishment of heterozygosity is sufficient to cause galls/tumors on maize plants. However, matings between haploids where one partner contains a mutation, in e.g., the smu1 gene, encoding a Ste20-like PAK kinase, often show reduced mating and pathogenicity compared to wild type. Here we show that similarly, diploids lacking one copy of smu1, are reduced in production of aerial hyphae, but do not show significantly-reduced virulence. Haplo-insufficiency was also observed for additional genes. UmPde1 is a cyclic phosphodiesterase involved in cAMP turnover as part of the cAMP-dependent PKA pathway. Hsl7 plays a role in cell length and in the filamentous response to low ammonium in haploid cells. Diploids deleted for one copy of either the pde1 or hsl7 genes had reduced or increased production of aerial hyphae, respectively, and both were severely impaired in virulence compared to wild type diploids. rho1 and pdc1 are two genes essential for cell viability in haploids. These genes also displayed haplo-insufficiency for pathogenesis. rho1/Δrho1 diploid cells were defective in pheromone production and detection, aerial hyphae induction, and were avirulent. In contrast, pdc1/Δpdc1 diploid cells only failed to produce tumors when applied to maize whorls. We predict the haplo-insufficiency of most of these signaling components is due to stoichiometric imbalance of the respective gene products with their interacting partners, thereby impairing virulence-induction mechanism(s). Further investigation of the bases for such haplo-insufficiency as well as of additional genes displaying this phenotype will provide important insights into fundamental aspects of development in this organism as well as inter-nuclear communication and genetic control.
Assuntos
Proteínas Fúngicas/genética , Doenças das Plantas/microbiologia , Ploidias , Ustilago/genética , Ustilago/patogenicidade , Fatores de Virulência/genética , Zea mays/microbiologia , Proteínas Fúngicas/metabolismo , Hifas/crescimento & desenvolvimento , Ustilago/crescimento & desenvolvimento , Virulência , Fatores de Virulência/metabolismoRESUMO
Inflammation and oxidative stress are well established in systemic lupus erythematosus (SLE) and are critical to the pathogenesis of autoimmune diseases. The transcription factor NF-E2 related factor 2 (Nrf2) is a central regulator of cellular anti-oxidative responses, inflammation, and restoration of redox balance. Accumulating reports support an emerging role for the regulation of Nrf2 in SLE. These include findings on the development of lupus-like autoimmune nephritis and altered immune cell populations in mice lacking Nrf2, as well as decreased Nrf2 abundance in the dendritic cells of patients with SLE. Nrf2-inducing agents have been shown to alleviate oxidative and inflammatory stress and reduce tissue injury in SLE mouse models. Since Nrf2 expression can be increased in activated T cells, the precise role of Nrf2 activation in different immune cell types and their function remains to be defined. However, targeting Nrf2 for the treatment of diseases associated with oxidative stress and inflammation, such as SLE, is promising. As investigation of Nrf2-inducing agents in clinical trials grows, defining the signaling and molecular mechanisms of action and downstream effects in response to different Nrf2-inducing agents in specific cells, tissues, and diseases, will be critical for effective clinical use.
RESUMO
Background: Lupus nephritis (LN) is a prevalent and severe complication of systemic lupus erythematosus (SLE). Non-invasive diagnostics are limited, and current therapies have inadequate response rates. Expression of the chemokine Interferon-γ-induced protein 10 (IP-10) is regulated by Interferon-γ signaling and NF-κB, and its molecular activity and enhanced urine concentrations are implicated in LN, but its utility as a diagnostic marker and association with demographic, clinical, or pathologic features is not defined. Methods: 38 LN patients and 11 patients with non-LN glomerular diseases (GD) with active disease were included. Eighteen of the LN patients had achieved remission at one follow-up during the study time. Serum and urine were obtained from these samples, and the IP-10 levels were measured. Results: Serum and urine IP-10 levels are significantly enhanced in LN patients with active disease as compared with normal individuals (serum average 179.7 pg/mL vs. 7.2 pg/mL, p < 0.0001; urine average 28.7 pg/mg vs. 1.6 pg/mg, p = 0.0019) and patients with other forms of glomerular disease (serum average 179.7 pg/mL vs. 84.9 pg/mL, p = 0.0176; urine average 28.7 pg/mg vs. 0.18 pg/mg, p = 0.0011). Urine IP-10 levels are significantly higher in patients with proliferative LN (PLN) than those with membranous LN (MLN) (average 32.8 pg/mg vs. 7.6 pg/mg, p = 0.0155). Urine IP-10 levels are also higher in MLN versus primary membranous nephropathy (MN) (average 7.6 pg/mg vs. 0.2 pg/mg, p = 0.0193). Importantly, serum IP-10 levels remain elevated during active LN and LN remission, but urine IP-10 levels are decreased from active LN to remission in 72% of our patients. Lastly, serum, but not urine IP-10 levels are significantly higher in African American than White American LN patients in active LN (average 227.8 pg/mL vs. 103.4 pg/mL, p = 0.0309) and during LN remission (average 254.6 pg/mL vs. 89.2 pg/mL, p = 0.0399). Conclusions: Our findings suggest that serum and urine IP-10 measurements provide promising tests for monitoring LN activity, differentiation between classifications of LN, and differentiation between LN and other forms of glomerular disease. We also conclude that further assessment of elevated IP-10 levels in the serum and urine of high-risk populations (i.e., African American) could be beneficial in determining why many of these patients have worse outcomes and are non-responsive to standard therapeutics.