RESUMEN
Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disease, characterized by cyst formation and growth. Hyperproliferation is a major contributor to cyst growth. At the nexus of regulating proliferation, is 4E-BP1. We demonstrate that ADPKD mouse and rat models, ADPKD patient renal biopsies and PKD1-/- cells exhibited hyperphosphorylated 4E-BP1, a biomarker of increased translation and proliferation. We hypothesized that expression of constitutively active 4E-BP1 constructs (4E-BP1F113A and 4E-BP1R13AF113A) would decrease proliferation and reduce cyst expansion. Utilizing the Pkd1RC/RC mouse, we determined the effect of 4E-BP1F113A on PKD. Unexpectedly, 4E-BP1F113A resulted in increased cyst burden and suppressed apoptosis markers, increased anti-apoptotic Bcl-2 protein and increased mitochondrial proteins. Exogenous 4E-BP1 enhanced proliferation, decreased apoptosis, increased anti-apoptotic Bcl-2 protein, impaired NADPH oxidoreductase activity, increased mitochondrial proteins and increased superoxide production in PKD patient-derived renal epithelial cells. Reduced 4E-BP1 expression suppressed proliferation, restored apoptosis and improved cellular metabolism. These findings provide insight into how cyst-lining cells respond to 4E-BP1.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Enfermedades Renales Poliquísticas/metabolismo , Riñón Poliquístico Autosómico Dominante/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , NADH NADPH Oxidorreductasas/metabolismo , Fosforilación , Enfermedades Renales Poliquísticas/patología , Riñón Poliquístico Autosómico Dominante/patología , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/metabolismo , Ratas , Canales Catiónicos TRPP/metabolismoRESUMEN
Autosomal dominant polycystic kidney disease (PKD) is characterized by cyst formation and growth, which are partially driven by abnormal proliferation of tubular cells. Proproliferative mechanistic target of rapamycin (mTOR) complexes 1 and 2 (mTORC1 and mTORC2) are activated in the kidneys of mice with PKD. Sirolimus indirectly inhibits mTORC1. Novel mTOR kinase inhibitors directly inhibit mTOR kinase, resulting in the inhibition of mTORC1 and mTORC2. The aim of the present study was to determine the effects of sirolimus versus the mTOR kinase inhibitor torin2 on cyst growth and kidney function in the Pkd1 p.R3277C (Pkd1RC/RC) mouse model, a hypomorphic Pkd1 model orthologous to the human condition, and to determine the effects of sirolimus versus torin2 on mTORC1 and mTORC2 signaling in PKD1-/- cells and in the kidneys of Pkd1RC/RC mice. In vitro, both inhibitors reduced mTORC1 and mTORC2 phosphorylated substrates and negatively impacted cellular metabolic activity, as measured by MTT assay. Pkd1RC/RC mice were treated with sirolimus or torin2 from 50 to 120 days of age. Torin2 was as effective as sirolimus in decreasing cyst growth and improving loss of kidney function. Both sirolimus and torin2 decreased phosphorylated S6 protein, phosphorylated eukaryotic translation initiation factor 4E-binding protein 1, phosphorylated Akt, and proliferation in Pkd1RC/RC kidneys. In conclusion, torin2 and sirolimus were equally effective in decreasing cyst burden and improving kidney function and mediated comparable effects on mTORC1 and mTORC2 signaling and proliferation in the Pkd1RC/RC kidney.
Asunto(s)
Túbulos Renales/efectos de los fármacos , Mutación , Naftiridinas/farmacología , Riñón Poliquístico Autosómico Dominante/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Canales Catiónicos TRPP/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Túbulos Renales/enzimología , Túbulos Renales/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Fosforilación , Riñón Poliquístico Autosómico Dominante/enzimología , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/fisiopatología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Cisplatin is a widely used chemotherapeutic agent used to treat solid tumours, such as ovarian, head and neck, and testicular germ cell. A known complication of cisplatin administration is acute kidney injury (AKI). The development of effective tumour interventions with reduced nephrotoxicity relies heavily on understanding the molecular pathophysiology of cisplatin-induced AKI. Rodent models have provided mechanistic insight into the pathophysiology of cisplatin-induced AKI. In the subsequent review, we provide a detailed discussion of recent advances in the cisplatin-induced AKI phenotype, principal mechanistic findings of injury and therapy, and pre-clinical use of AKI rodent models. Cisplatin-induced AKI murine models faithfully develop gross manifestations of clinical AKI such as decreased kidney function, increased expression of tubular injury biomarkers, and tubular injury evident by histology. Pathways involved in AKI include apoptosis, necrosis, inflammation, and increased oxidative stress, ultimately providing a translational platform for testing the therapeutic efficacy of potential interventions. This review provides a discussion of the foundation laid by cisplatin-induced AKI rodent models for our current understanding of AKI molecular pathophysiology.
Asunto(s)
Lesión Renal Aguda/inducido químicamente , Antineoplásicos/efectos adversos , Cisplatino/efectos adversos , Lesión Renal Aguda/patología , Lesión Renal Aguda/fisiopatología , Lesión Renal Aguda/terapia , Animales , Antineoplásicos/uso terapéutico , Cisplatino/uso terapéutico , Modelos Animales de Enfermedad , Humanos , Riñón/efectos de los fármacos , Riñón/patología , Riñón/fisiopatología , Neoplasias/tratamiento farmacológicoRESUMEN
HIV-1-specific T-cell responses in exposed seronegative subjects suggest that a viral breach of the exposure site is more common than current transmission rates would suggest and that host immunity can extinguish subsequent infection foci. The Preexposure Prophylaxis Initiative (iPrEx) chemoprophylaxis trial provided an opportunity to rigorously investigate these responses in a case-control immunology study; 84 preinfection peripheral blood mononuclear cell samples from individuals enrolled in the iPrEx trial who later seroconverted were matched with 480 samples from enrolled subjects who remained seronegative from both the placebo and active treatment arms. T-cell responses to HIV-1 Gag, Protease, Integrase, Reverse Transcriptase, Vif, and Nef antigens were quantified for all subjects in an IFN-γ enzyme-linked immunospot (ELISpot) assay. IFN-γ responses varied in magnitude and frequency across subjects. A positive response was more prevalent in those who remained persistently HIV-1-negative for Gag (P = 0.007), Integrase (P < 0.001), Vif (P < 0.001), and Nef (P < 0.001). When correlated with outcomes in the iPrEx trial, Vif- and Integrase-specific T-cell responses were associated with reduced HIV-1 infection risk [hazard ratio (HR) = 0.36, 95% confidence interval (95% CI) = 0.19-0.66 and HR = 0.52, 95% CI = 0.28-0.96, respectively]. Antigen-specific responses were independent of emtricitabine/tenofovir disoproxil fumarate use. IFN-γ secretion in the ELISpot was confirmed using multiparametric flow cytometry and largely attributed to effector memory CD4+ or CD8+ T cells. Our results show that HIV-1-specific T-cell immunity can be detected in exposed but uninfected individuals and that these T-cell responses can differentiate individuals according to infection outcomes.
Asunto(s)
Infecciones por VIH/inmunología , VIH-1/inmunología , Inmunidad Celular/inmunología , Leucocitos Mononucleares/inmunología , Adulto , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Infecciones por VIH/sangre , Infecciones por VIH/virología , Seropositividad para VIH/inmunología , VIH-1/metabolismo , VIH-1/fisiología , Proteínas del Virus de la Inmunodeficiencia Humana/inmunología , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Leucocitos Mononucleares/metabolismo , Modelos Logísticos , Masculino , Análisis Multivariante , Adulto JovenRESUMEN
Polycystic kidney disease (PKD) involves progressive hepatorenal cyst expansion and fibrosis, frequently leading to end-stage renal disease. Increased vasopressin and cAMP signaling, dysregulated calcium homeostasis, and hypertension play major roles in PKD progression. The guanylyl cyclase A agonist, B-type natriuretic peptide (BNP), stimulates cGMP and shows anti-fibrotic, anti-hypertensive, and vasopressin-suppressive effects, potentially counteracting PKD pathogenesis. Here, we assessed the impacts of guanylyl cyclase A activation on PKD progression in a rat model of PKD. Sustained BNP production significantly reduced kidney weight, renal cystic indexes and fibrosis, in concert with suppressed hepatic cystogenesis in vivo. In vitro, BNP decreased cystic epithelial cell proliferation, suppressed fibrotic gene expression, and increased intracellular calcium. Together, our data demonstrate multifaceted effects of sustained activation of guanylyl cyclase A on polycystic kidney and liver disease. Thus, targeting the guanylyl cyclase A-cGMP axis may provide a novel therapeutic strategy for hepatorenal fibrocystic diseases.
Asunto(s)
Quistes/patología , Riñón/patología , Hepatopatías/patología , Hígado/patología , Péptido Natriurético Encefálico/metabolismo , Riñón Poliquístico Autosómico Recesivo/patología , Receptores del Factor Natriurético Atrial/metabolismo , Animales , Proliferación Celular , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Quistes/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Fibrosis , Vectores Genéticos/genética , Humanos , Hipertensión/patología , Hepatopatías/genética , Masculino , Péptido Natriurético Encefálico/genética , Parvovirinae/genética , Riñón Poliquístico Autosómico Recesivo/genética , Ratas , Ratas Sprague-Dawley , Receptores del Factor Natriurético Atrial/agonistas , Transducción de Señal , Vasopresinas/metabolismoRESUMEN
Adeno-associated viruses (AAV) have evolved to exploit the dynamic reorganization of host cell machinery during co-infection by adenoviruses and other helper viruses. In the absence of helper viruses, host factors such as the proteasome and DNA damage response machinery have been shown to effectively inhibit AAV transduction by restricting processes ranging from nuclear entry to second-strand DNA synthesis. To identify host factors that might affect other key steps in AAV infection, we screened an siRNA library that revealed several candidate genes including the PHD finger-like domain protein 5A (PHF5A), a U2 snRNP-associated protein. Disruption of PHF5A expression selectively enhanced transgene expression from AAV by increasing transcript levels and appears to influence a step after second-strand synthesis in a serotype and cell type-independent manner. Genetic disruption of U2 snRNP and associated proteins, such as SF3B1 and U2AF1, also increased expression from AAV vector, suggesting the critical role of U2 snRNP spliceosome complex in this host-mediated restriction. Notably, adenoviral co-infection and U2 snRNP inhibition appeared to target a common pathway in increasing expression from AAV vectors. Moreover, pharmacological inhibition of U2 snRNP by meayamycin B, a potent SF3B1 inhibitor, substantially enhanced AAV vector transduction of clinically relevant cell types. Further analysis suggested that U2 snRNP proteins suppress AAV vector transgene expression through direct recognition of intact AAV capsids. In summary, we identify U2 snRNP and associated splicing factors, which are known to be affected during adenoviral infection, as novel host restriction factors that effectively limit AAV transgene expression. Concurrently, we postulate that pharmacological/genetic manipulation of components of the spliceosomal machinery might enable more effective gene transfer modalities with recombinant AAV vectors.
Asunto(s)
Proteínas Portadoras/metabolismo , Dependovirus/genética , Interacciones Huésped-Parásitos/fisiología , Empalmosomas/metabolismo , Transducción Genética , Línea Celular , Dependovirus/patogenicidad , Vectores Genéticos , Biblioteca Genómica , Humanos , Immunoblotting , Inmunoprecipitación , Microscopía Confocal , ARN Interferente Pequeño , Proteínas de Unión al ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , TransactivadoresRESUMEN
Sema4D, also known as CD100, is a constitutively expressed immune semaphorin on T cells and NK cells. CD100 has important immune regulatory functions that improve antigen-specific priming by antigen-presenting cells, and can also act as a costimulatory molecule on T cells. We investigated the consequence of HIV-1 infection on CD100 expression by T cells, and whether CD100 expression signifies functionally competent effector cells. CD100 expression on T cells from healthy individuals was compared with HIV-1-infected subjects including elite controllers, noncontrollers, and patients receiving antiretroviral therapy. The frequency and fluorescence intensity of CD100 on CD8(+) and CD4(+) T cells were decreased during HIV-1 infection. Furthermore, the absolute number of CD100-expressing CD8(+) T cells was positively associated with the magnitude of HIV-1-specific T-cell responses. CD8(+) T cells lacking CD100 expression were functionally impaired and present in increased numbers in HIV-1-infected individuals. The number of CD100(-)CD8(+) T cells positively correlated with T-cell immunosenescence, immune activation, and viral load. Loss of CD100 expression appears to result from direct antigen stimulation, as in vitro cytokine exposure and viral replication did not significantly impact CD100 expression. These data suggest that loss of CD100 expression probably plays an important role in dysfunctional immunity in HIV-1 infection.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , Semaforinas/deficiencia , Células Presentadoras de Antígenos , Antígenos CD , Terapia Antirretroviral Altamente Activa , Linfocitos T CD8-positivos/metabolismo , Estudios de Casos y Controles , Estudios Transversales , Citocinas/metabolismo , Citometría de Flujo , Infecciones por VIH/tratamiento farmacológico , Humanos , Activación de Linfocitos , Carga Viral , Replicación ViralRESUMEN
Measles, mumps, and rubella are viral diseases that may adversely affect nonimmune pregnant women and their fetuses/neonates. Prevention of these diseases and their complications can be achieved through measles-mumps-rubella (MMR) vaccination before pregnancy. The vaccine is contraindicated during pregnancy, because it contains live, attenuated viruses that pose a theoretical risk to the fetus. However, accidental receipt of MMR vaccination is not known to cause maternal/fetal complications. MMR immunization is recommended to nonimmune obstetric patients upon completion or termination of pregnancy.
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Vacuna contra el Sarampión-Parotiditis-Rubéola/administración & dosificación , Sarampión/prevención & control , Paperas/prevención & control , Complicaciones Infecciosas del Embarazo/prevención & control , Rubéola (Sarampión Alemán)/prevención & control , Contraindicaciones , Femenino , Promoción de la Salud , Humanos , Inmunoglobulinas/uso terapéutico , Factores Inmunológicos/uso terapéutico , Transmisión Vertical de Enfermedad Infecciosa/prevención & control , Sarampión/complicaciones , Sarampión/diagnóstico , Sarampión/tratamiento farmacológico , Sarampión/epidemiología , Paperas/complicaciones , Paperas/diagnóstico , Paperas/epidemiología , Embarazo , Complicaciones Infecciosas del Embarazo/etiología , Rubéola (Sarampión Alemán)/diagnóstico , Rubéola (Sarampión Alemán)/epidemiología , Poblaciones VulnerablesRESUMEN
Many surgical models are used to study kidney and other diseases in mice, yet the effects of the surgical procedure itself on the kidney and other tissues have not been elucidated. In the present study, we found that both sham surgery and unilateral nephrectomy (UNX), which is used as a model of renal compensatory hypertrophy, in mice resulted in increased mammalian target of rapamycin complex 1/2 (mTORC1/2) in the remaining kidney. mTORC1 is known to regulate lysosomal biogenesis and autophagy. Genes associated with lysosomal biogenesis and function were decreased in sham surgery and UNX kidneys. In both sham surgery and UNX, there was suppressed autophagic flux in the kidney as indicated by the lack of an increase in LC3-II or autophagosomes seen on immunoblot, IF and EM after bafilomycin A1 administration and a concomitant increase in p62, a marker of autophagic cargo. There was a massive increase in pro-inflammatory cytokines, which are known to activate ERK1/2, in the serum after sham surgery and UNX. There was a large increase in ERK1/2 in sham surgery and UNX kidneys, which was blocked by the MEK1/2 inhibitor, trametinib. Trametinib also resulted in a significant decrease in p62. In summary, there was an intense systemic inflammatory response, an ERK-mediated increase in p62 and suppressed autophagic flux in the kidney after sham surgery and UNX. It is important that researchers are aware that changes in systemic pro-inflammatory cytokines, ERK1/2 and autophagy can be caused by sham surgery as well as the kidney injury/disease itself.
Asunto(s)
Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia , Enfermedades Renales/metabolismo , Riñón/cirugía , Nefrectomía/efectos adversos , Animales , Proteínas Relacionadas con la Autofagia/genética , Línea Celular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/etiología , Enfermedades Renales/genética , Enfermedades Renales/patología , Lisosomas/genética , Lisosomas/metabolismo , Lisosomas/patología , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/genética , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Metabolómica , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder characterized by kidney cyst growth often resulting in end-stage renal disease. There is growing attention on understanding the role of impaired autophagy in ADPKD. Trehalose (TRE) has been shown to increase both protein stability and aggregate clearance and induce autophagy in neurodegenerative diseases. TRE treatment in wild type mice compared to vehicle resulted in increased expression in the kidney of Atg12-5 complex and increased Rab9a, autophagy-related proteins that play a role in the formation of autophagosomes. Thus, the aim of the study was to determine the effect of TRE on cyst growth and autophagy-related proteins, in the hypomorphic Pkd1RC/RC mouse model of ADPKD. Pkd1RC/RC mice were treated 2% TRE in water from days 50 to 120 of age. TRE did not slow cyst growth or improve kidney function or affect proliferation and apoptosis in Pkd1RC/RC kidneys. In Pkd1RC/RC vs. wild type kidneys, expression of the Atg12-5 complex was inhibited by TRE resulting in increased free Atg12 and TRE was unable to rescue the deficiency of the Atg12-5 complex. Rab9a was decreased in Pkd1RC/RC vs. wild type kidneys and unaffected by TRE. The TRE-induced increase in p62, a marker of autophagic cargo, that was seen in normal kidneys was blocked in Pkd1RC/RC kidneys. In summary, the autophagy phenotype in Pkd1RC/RC kidneys was characterized by decreases in crucial autophagy-related proteins (Atg12-5 complex, Atg5, Atg16L1), decreased Rab9a and increased mTORC1 (pS6S240/244, pmTORS2448) proteins. TRE increased Atg12-5 complex, Rab9a and p62 in normal kidneys, but was unable to rescue the deficiency in autophagy proteins or suppress mTORC1 in Pkd1RC/RC kidneys and did not protect against cyst growth.
Asunto(s)
Riñón Poliquístico Autosómico Dominante/tratamiento farmacológico , Proteína Quinasa C/metabolismo , Trehalosa/farmacología , Animales , Autofagia/efectos de los fármacos , Proteína 12 Relacionada con la Autofagia/metabolismo , Proteína 5 Relacionada con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Cardiac hypertrophy is common in autosomal dominant polycystic kidney disease (ADPKD) patients. We found increased heart weight in Pkd1RC/RC and Pkd2WS25/+ mouse models of ADPKD. As there is a link between increased heart weight and mammalian target of rapamycin (mTOR), the aim of the study was to determine mTOR complex 1 and 2 signaling proteins in the heart in the Pkd1RC/RC mouse model of PKD. In 70 day old Pkd1RC/RC hearts, on immunoblot analysis, there was a large increase in p-AMPKThr172, a known autophagy inducer, and an increase in p-AktSer473 and p-AktThr308, but no increase in other mTORC1/2 proteins (p-S6Ser240/244, p-mTORSer2448). In 150 day old Pkd1RC/RC hearts, there was an increase in mTORC1 (p-S6Ser240/244) and mTOR-related proteins (p-AktThr308, p-GSK3ßSer9, p-AMPKThr172). As the mTOR pathway is the master regulator of autophagy, autophagy proteins were measured. There was an increase in p-Beclin-1 (BECN1), an autophagy regulator and activating molecule in Beclin-1-regulated autophagy (AMBRA1), a regulator of Beclin that play a role in autophagosome formation, an early stage of autophagy. There was a defect in the later stage of autophagy, the fusion of the autophagosome with the lysosome, known as autophagic flux, as evidenced by the lack of an increase in LC3-II, a marker of autophagosomes, with the lysosomal inhibitor bafilomycin, in both 70 day old and 150 day old hearts. To determine the role of autophagy in causing increased heart weight, Pkd1RC/RC were treated with 2-deoxyglucose (2-DG) or Tat-Beclin1 peptide, agents known to induce autophagy. 2-DG treatment from 150 to 350 days of age, a time period when increased heart weight developed, did not reduce the increased heart weight. Unexpectedly, Tat-Beclin 1 peptide treatment from 70 to 120 days of age resulted in increased heart weight. In summary, there is suppressed autophagic flux in the heart at an early age in Pkd1RC/RC mice. Increased mTOR signaling in older mice is associated suppressed autophagic flux. There was a large increase in p-AMPKThr172, a known autophagy inducer, in both young and old mice. 2-DG treatment did not impact increased heart weight and Tat-Beclin1 peptide increased heart weight.
Asunto(s)
Cardiomegalia/metabolismo , Riñón Poliquístico Autosómico Dominante , Serina-Treonina Quinasas TOR/fisiología , Animales , Autofagia , Modelos Animales de Enfermedad , Ratones , Riñón Poliquístico Autosómico Dominante/metabolismo , Riñón Poliquístico Autosómico Dominante/patologíaRESUMEN
In a clinically-relevant model of 4 week, low-dose cisplatin-induced AKI, mice were injected subcutaneously with non small cell lung cancer (NSCLC) cells that harbor an activating Kirsten rat sarcoma viral oncogene homolog (KRAS)G12V mutation. Phospho extracellular signal-regulated kinase1/2 (pERK1/2) expression in kidney and tumors was decreased by the MEK1/2 inhibitors, U0126 and trametinib, that potently inhibit pERK1/2. U0126 resulted in a significant improvement in kidney function, acute tubular necrosis (ATN) and tubular cell apoptosis in mice with AKI. Genes that were significantly decreased by U0126 were heat shock protein 1, cyclin-dependent kinase 4 (CDK4) and stratifin (14-3-3σ). U0126 resulted in a significant decrease in tumor weight and volume and significantly increased the chemotherapeutic effect of cisplatin. Trametinib, a MEK1/2 inhibitor that is FDA-approved for the treatment of cancer, did not result in functional protection against AKI or worse AKI, but dramatically decreased tumor growth more than cisplatin. Smaller tumors in cisplatin or MEK1/2 inhibitor-treated mice were not related to changes in microtubule-associated proteins 1A/1B light chain 3B (LC3-II), p62, cleaved caspase-3, granzyme B, or programmed death-ligand 1 (PD-L1). In summary, despite ERK inhibition by both U0126 and trametinib, only U0126 protected against AKI suggesting that the protection against AKI by U0126 was due to an off-target effect independent of ERK inhibition. The effect of U0126 to decrease AKI may be mediated by inhibition of heat shock protein 1, CDK4 or stratifin (14-3-3σ). Trametinib was more effective than cisplatin in decreasing tumor growth, but unlike cisplatin, trametinib did not cause AKI.
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Lesión Renal Aguda/tratamiento farmacológico , Cisplatino/uso terapéutico , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Animales , Apoptosis/efectos de los fármacos , Nitrógeno de la Urea Sanguínea , Butadienos/farmacología , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Riñón/efectos de los fármacos , Riñón/lesiones , Riñón/patología , Lipocalina 2/metabolismo , Neoplasias Pulmonares/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones Endogámicos C57BL , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas de Neoplasias/metabolismo , Nitrilos/farmacología , Piridonas/farmacología , Pirimidinonas/farmacología , Carga Tumoral/efectos de los fármacosRESUMEN
BACKGROUND: Elabela (ELA) is a recently identified apelin receptor agonist essential for cardiac development, but its biology and therapeutic potential are unclear. In humans, ELA transcripts are detected in embryonic stem cells, induced pluripotent stem cells, kidney, heart and blood vessels. ELA through the apelin (APJ) receptor promotes angiogenesis in vitro, relaxes murine aortic blood vessels and attenuates high blood pressure in vivo. The APJ receptor when bound to its original ligand, apelin, exerts peripheral vasodilatory and positive inotropic effects, conferring cardioprotection in vivo. METHODS: This study initially assessed endogenous ELA expression in normal and diseased rats and then characterized the effects of long-term ELA gene delivery by adeno-associated virus serotype 9 (AAV9) vectors on cardiorenal function in Dahl salt-sensitive rats (DS) on a high-salt diet over 3 months. RESULTS: Endogenous ELA was predominantly expressed in the kidneys, especially in the renal collecting duct cells and was not affected by disease. Rat ELA was overexpressed in the heart via AAV9 vector by a single intravenous injection. ELA-treated animals showed delayed onset of blood pressure elevation. Prior to high-salt diet, a reduction in the fractional sodium and chloride excretion was observed in rats given the AAV9-ELA vector. After three months on a high-salt diet, ELA preserved glomerular architecture, decreased renal fibrosis and suppressed expression of fibrosis-associated genes in the kidneys. CONCLUSION: ELA is constitutively expressed in renal collecting ducts in rats. Sustained AAV-ELA expression may offer a potential long-term therapy for hypertension and renal remodeling.
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Receptores de Apelina/agonistas , Proteínas Portadoras/genética , Terapia Genética/métodos , Hipertensión/terapia , Cloruro de Sodio Dietético/efectos adversos , Animales , Presión Sanguínea/genética , Proteínas Portadoras/metabolismo , Dependovirus/genética , Expresión Génica , Hipertensión/etiología , Hipertensión/genética , Túbulos Renales Colectores/metabolismo , Masculino , Ratas Endogámicas Dahl , Ratas WistarRESUMEN
Sexual dimorphisms are recognized in cardiovascular conditions such as hypertension, stroke, thrombosis and vasculitis. B-type natriuretic peptide (BNP) is a guanylyl cyclase A (GC-A) agonist. The anti-hypertensive, vasodilatory, anti-fibrotic, and anti-hypertrophic properties of BNP are well established in male animal models. Although circulating BNP levels are higher in women, when compared to age-matched men, the cardiovascular protective propensity of BNP in females is poorly understood. We assessed the cardiovascular consequences of BNP deletion in genetically null (Nppb-/-) female rat lines. Throughout the study, blood pressure (BP) remained uninfluenced by genotype, and cardiorenal consequences of BNP knock out remained minor. Unexpectedly, approximately 60% of Nppb-/- females developed mesenteric polyarteritis-nodosa (PAN)-like vasculitis in their life span, some as early as 4 months of age. Mesenteric lesions involved intense arterial remodeling, progressive inflammation, occluded lumens, and less frequently intestinal necrosis and multiple visceral arterial aneurysms. Cumulative pathologies resulted in a significant decline in survival of the Nppb-/- female. This study highlights BNP's vasoprotective propensity, bringing to light a possible sex specific difference in the cardiovascular protection provided by BNP. Defects in the BNP/GC-A/cGMP pathway may play a role in arteriopathies in women, while GC-A agonists may provide effective therapy for arteritis.
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Arterias Mesentéricas/metabolismo , Péptido Natriurético Encefálico/deficiencia , Remodelación Vascular , Vasculitis/metabolismo , Animales , Presión Sanguínea , Femenino , Humanos , Hipertensión/genética , Hipertensión/fisiopatología , Masculino , Arterias Mesentéricas/patología , Arterias Mesentéricas/fisiopatología , Péptido Natriurético Encefálico/genética , Poliarteritis Nudosa/genética , Poliarteritis Nudosa/metabolismo , Ratas Endogámicas Dahl , Factores Sexuales , Factores de Tiempo , Vasculitis/genéticaRESUMEN
UNLABELLED: Human induced pluripotent stem cells (iPSCs) and derived progeny provide invaluable regenerative platforms, yet their clinical translation has been compromised by their biosafety concern. Here, we assessed the safety of transplanting patient-derived iPSC-generated pancreatic endoderm/progenitor cells. Transplantation of progenitors from iPSCs reprogrammed by lentiviral vectors (LV-iPSCs) led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immunodeficient mice. Moreover, removal of primary tumors from LV-iPSC progeny-transplanted hosts generated secondary and metastatic tumors. Combined transgene-free (TGF) reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplantation, ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo. The incidence of tumor formation in TGF-iPSCs was titratable, depending on the oncogenic load, with reintegration of the cMYC expressing vector abolishing tumor-free transplantation. Thus, transgene-free cMYC-independent reprogramming and elimination of residual pluripotent cells are mandatory steps in achieving transplantation of iPSC progeny for customized and safe islet regeneration in vivo. SIGNIFICANCE: Pluripotent stem cell therapy for diabetes relies on the safety as well as the quality of derived insulin-producing cells. Data from this study highlight prominent tumorigenic risks of induced pluripotent stem cell (iPSC) products, especially when reprogrammed with integrating vectors. Two major underlying mechanisms in iPSC tumorigenicity are residual pluripotent cells and cMYC overload by vector integration. This study also demonstrated that combined transgene-free reprogramming and enzymatic dissociation allows teratoma-free transplantation of iPSC progeny in the mouse model in testing the tumorigenicity of iPSC products. Further safety assessment and improvement in iPSC specification into a mature ß cell phenotype would lead to safe islet replacement therapy for diabetes.
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Diabetes Mellitus Tipo 1/cirugía , Diabetes Mellitus Tipo 2/cirugía , Células Madre Pluripotentes Inducidas/trasplante , Trasplante de Islotes Pancreáticos/métodos , Islotes Pancreáticos/cirugía , Queratinocitos/trasplante , Regeneración , Teratocarcinoma/prevención & control , Adulto , Anciano , Animales , Diferenciación Celular , Células Cultivadas , Técnicas de Reprogramación Celular , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/diagnóstico , Regulación Neoplásica de la Expresión Génica , Vectores Genéticos , Xenoinjertos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Trasplante de Islotes Pancreáticos/efectos adversos , Queratinocitos/metabolismo , Queratinocitos/patología , Lentivirus/genética , Masculino , Ratones SCID , Fenotipo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Teratocarcinoma/genética , Teratocarcinoma/metabolismo , Teratocarcinoma/patología , TransfecciónRESUMEN
Altered myocardial structure and function, secondary to chronically elevated blood pressure, are leading causes of heart failure and death. B-type natriuretic peptide (BNP), a guanylyl cyclase A agonist, is a cardiac hormone integral to cardiovascular regulation. Studies have demonstrated a causal relationship between reduced production or impaired BNP release and the development of human hypertension. However, the consequences of BNP insufficiency on blood pressure and hypertension-associated complications remain poorly understood. Therefore, the goal of this study was to create and characterize a novel model of BNP deficiency to investigate the effects of BNP absence on cardiac and renal structure, function, and survival. Genetic BNP deletion was generated in Dahl salt-sensitive rats. Compared with age-matched controls, BNP knockout rats demonstrated adult-onset hypertension. Increased left ventricular mass with hypertrophy and substantially augmented hypertrophy signaling pathway genes, developed in young adult knockout rats, which preceded hypertension. Prolonged hypertension led to increased cardiac stiffness, cardiac fibrosis, and thrombi formation. Significant elongation of the QT interval was detected at 9 months in knockout rats. Progressive nephropathy was also noted with proteinuria, fibrosis, and glomerular alterations in BNP knockout rats. End-organ damage contributed to a significant decline in overall survival. Systemic BNP overexpression reversed the phenotype of genetic BNP deletion. Our results demonstrate the critical role of BNP defect in the development of systemic hypertension and associated end-organ damage in adulthood.
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Modelos Animales de Enfermedad , Hipertensión/etiología , Péptido Natriurético Encefálico/fisiología , Edad de Inicio , Animales , Adaptabilidad , Muerte Súbita Cardíaca/etiología , Fibrosis , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Hipertensión/genética , Hipertensión/patología , Hipertensión/prevención & control , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/patología , Glomérulos Renales/patología , Síndrome de QT Prolongado/etiología , Contracción Miocárdica/genética , Miocardio/patología , Péptido Natriurético Encefálico/deficiencia , Péptido Natriurético Encefálico/genética , Fenotipo , Ratas , Ratas Endogámicas Dahl , Proteínas Recombinantes de Fusión/metabolismo , Insuficiencia Renal Crónica/etiología , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Diabetes engenders the loss of pancreatic ß-cell mass and/or function, resulting in insulin deficiency relative to the metabolic needs of the body. Diabetic care has traditionally relied on pharmacotherapy, exemplified by insulin replacement to target peripheral actions of the hormone. With growing understanding of the pathogenesis of diabetic disease, alternative approaches aiming at repair and restoration of failing ß-cell function are increasingly considered as complements to current diabetes therapy regimens. To this end, emphasis is placed on transplantation of exogenous pancreas/islets or artificial islets, enhanced proliferation and maturation of endogenous ß cells, prevention of ß-cell loss, or fortified renewal of ß-like-cell populations from stem cell pools and non-ß-cell sources. In light of emerging clinical experiences with human embryonic stem cells and approval of the first in-human trial with induced pluripotent stem cells, in this study we highlight advances in ß-cell regeneration strategies with a focus on pluripotent stem cell platforms in the context of translational applications.
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Diabetes Mellitus/terapia , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/trasplante , Células Secretoras de Insulina , Medicina Regenerativa/métodos , Trasplante de Células Madre , Animales , Diabetes Mellitus/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismoRESUMEN
BACKGROUND: Hypertension is a highly prevalent disease associated with cardiovascular morbidity and mortality. Recent studies suggest that patients with hypertension also have a deficiency of certain cardiac peptides. Previously we demonstrated that a single intravenous injection of the myocardium-tropic adeno-associated virus (AAV) 9-based vector encoding for proBNP prevented the development of hypertensive heart disease (HHD) in spontaneously hypertensive rats (SHRs). The current study was designed to determine the duration of cardiac transduction after a single AAV9 injection and to determine whether cardiac BNP overexpression can delay the progression of previously established HHD, and improve survival in aged SHRs with overt HHD. METHODS AND RESULTS: To evaluate the duration of cardiac transduction induced by the AAV9 vector, we used four week old SHRs. Effective long-term selective cardiac transduction was determined by luciferase expression. A single intravenous administration of a luciferase-expressing AAV9 vector resulted in efficient cardiac gene delivery for up to 18-months. In aged SHRs (9-months of age), echocardiographic studies demonstrated progression of HHD in untreated controls, while AAV9-BNP vector treatment arrested the deterioration of cardiac function at six months post-injection (15-months of age). Aged SHRs with established overt HHD were further monitored to investigate survival. A single intravenous injection of the AAV9-vector encoding rat proBNP was associated with significantly prolonged survival in the treated SHRs (613?38 days, up to 669 days) compared to the untreated rats (480±69 days, up to 545 days)(p<0.05). CONCLUSIONS: A single intravenous injection of AAV9 vector elicited prolonged cardiac transduction (up to 18 months post-injection). AAV9 induced cardiac BNP overexpression prevented development of congestive heart failure, and significantly prolonged the survival of aged SHRs with previously established overt HHD. These findings support the beneficial effects of chronic supplementation of BNP in a frequent and highly morbid condition such as HHD.
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Terapia Genética/métodos , Cardiopatías/prevención & control , Hipertensión/complicaciones , Péptido Natriurético Encefálico/administración & dosificación , Adenoviridae , Animales , Vectores Genéticos , Cardiopatías/etiología , Masculino , Péptido Natriurético Encefálico/genética , Ratas , Ratas Endogámicas SHR , Transducción GenéticaRESUMEN
All-cause mortality from cardiovascular disease is declining in the USA. However, there remains a significant difference in risk factors for disease and in mortality between men and women. For example, prevalence and outcomes for heart failure with preserved ejection fraction differ between men and women. The reasons for these differences are multifactorial, but reflect, in part, an incomplete understanding of sex differences in the etiology of cardiovascular diseases and a failure to account for sex differences in pre-clinical studies including those designed to develop new diagnostic and treatment modalities. This review focuses on the underlying physiology of these sex differences and provides evidence that inclusion of female animals in pre-clinical studies of heart failure and in development of imaging modalities to assess cardiac function might provide new information from which one could develop sex-specific diagnostic criteria and approaches to treatment.
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Disparidades en el Estado de Salud , Disparidades en Atención de Salud , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/terapia , Anciano , Animales , Modelos Animales de Enfermedad , Femenino , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/mortalidad , Insuficiencia Cardíaca/fisiopatología , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Medición de Riesgo , Factores de Riesgo , Factores Sexuales , Volumen Sistólico , Resultado del Tratamiento , Remodelación VentricularRESUMEN
CD8+ T cells are important for HIV-1 virus control, but are also a major contributing factor that drives HIV-1 virus sequence evolution. Although HIV-1 cytotoxic T cell (CTL) escape mutations are a common aspect during HIV-1 infection, less is known about the importance of T cell pressure in reversing HIV-1 virus back to a consensus sequences. In this study we aimed to assess the frequency with which reversion of transmitted mutations in T cell epitopes were associated with T cell responses to the mutation. This study included 14 HIV-1 transmission pairs consisting of a 'source' (virus-donor) and a 'recipient' (newly infected individual). Non-consensus B sequence amino acids (mutations) in T cell epitopes in HIV-1 gag regions p17, p24, p2 and p7 were identified in each pair and transmission of mutations to the recipient was verified with population viral sequencing. Longitudinal analyses of the recipient's viral sequence were used to identify whether reversion of mutations back to the consensus B sequence occurred. Autologous 12-mer peptides overlapping by 11 were synthesized, representing the sequence region surrounding each reversion and longitudinal analysis of T cell responses to source-derived mutated and reverted epitopes were assessed. We demonstrated that mutations in the source were frequently transmitted to the new host and on an average 17 percent of mutated epitopes reverted to consensus sequence in the recipient. T cell responses to these mutated epitopes were detected in 7 of the 14 recipients in whom reversion occurred. Overall, these findings indicate that transmitted non-consensus B epitopes are frequently immunogenic in HLA-mismatched recipients and new T cell pressures to T cell escape mutations following transmission play a significant role in maintaining consensus HIV-1 sequences.