RESUMEN
Diabetic nephropathy is the main cause of end-stage renal disease. MicroRNAs are powerful regulators of the genome, and global expression profiling revealed miR-21 to be among the most highly regulated microRNAs in kidneys of mice with diabetic nephropathy. In kidney biopsies of diabetic patients, miR-21 correlated with tubulointerstitial injury. In situ PCR analysis showed a specific enrichment of miR-21 in glomerular cells. We identified cell division cycle 25a (Cdc25a) and cyclin-dependent kinase 6 (Cdk6) as novel miR-21 targets in mesangial cells. miR-21-mediated repression of Cdc25a and Cdk6 resulted in impaired cell cycle progression and subsequent mesangial cell hypertrophy. miR-21 increased podocyte motility by regulating phosphatase and tensin homolog (Pten). miR-21 antagonism in vitro and in vivo in streptozotocin-induced diabetic mice decreased mesangial expansion, interstitial fibrosis, macrophage infiltration, podocyte loss, albuminuria, and fibrotic- and inflammatory gene expression. In conclusion, miR-21 antagonism rescued various functional and structural parameters in mice with diabetic nephropathy and, thus, might be a viable option in the treatment of patients with diabetic kidney disease.
Asunto(s)
Nefropatías Diabéticas/genética , Silenciador del Gen , MicroARNs/genética , Animales , Puntos de Control del Ciclo Celular/genética , Movimiento Celular , Análisis por Conglomerados , Quinasa 6 Dependiente de la Ciclina/genética , Diabetes Mellitus Experimental , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/terapia , Modelos Animales de Enfermedad , Fibrosis , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Células Mesangiales/metabolismo , Ratones , Podocitos/metabolismo , Interferencia de ARN , Fosfatasas cdc25/genéticaRESUMEN
Chronic renal allograft dysfunction (CAD) is a major limiting factor of long-term graft survival. It is characterized by interstitial fibrosis and tubular atrophy. The underlying pathomechanisms are incompletely understood. MicroRNAs are powerful regulators of gene expression and may have an impact on various diseases by direct mRNA decay or translational inhibition. A murine model of allogenic kidney transplantation was used resulting in CAD at 6 weeks after kidney transplantation. We identified fibrosis-associated miR-21a-5p by whole miRNAome expression analysis to be among the most highly upregulated miRNAs. In vitro in renal fibroblasts, miR-21a-5p was transcriptionally activated by interleukin 6-induced signal transducer and activator of transcription 3. Co-culture of LPS-activated macrophages with renal fibroblasts increased expression levels of miR-21a-5p and markers of fibrosis and inflammation. In addition, mature miR-21a-5p was secreted by macrophages in small vesicles, which were internalized by renal fibroblasts, thereby promoting profibrotic and proinflammatory effects. Notch2 receptor was identified as a potential target of miR-21a-5p and validated by luciferase gene reporter assays. Therapeutic silencing of miR-21a-5p in mice after allogenic kidney transplantation resulted in an amelioration of CAD, as indicated by a reduction in fibrosis development, inflammatory cell influx, tissue injury and BANFF lesion scoring. In a life-supporting model, miR-21a-5p antagonism had beneficial effects on kidney function. miR-21a-5p silencing may therefore be a viable therapeutic option in the treatment of patients following kidney transplantation to halt the development of CAD.
Asunto(s)
Aloinjertos/patología , Rechazo de Injerto/genética , Trasplante de Riñón/efectos adversos , Riñón/patología , MicroARNs/metabolismo , Receptor Notch2/genética , Animales , Biomarcadores/metabolismo , Enfermedad Crónica , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Fibroblastos , Fibrosis , Perfilación de la Expresión Génica , Supervivencia de Injerto/genética , Humanos , Macrófagos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Oligonucleótidos/genética , Receptor Notch2/metabolismo , Trasplante Homólogo/efectos adversos , Regulación hacia ArribaRESUMEN
BACKGROUND: MiRNA-181c, miRNA-633 and miRNA-922 have been reported to be deregulated in multiple sclerosis. OBJECTIVES: To investigate the association between miRNA-181c, miRNA-633 and miRNA-922 and conversion from clinically isolated syndrome (CIS) to relapsing-remitting multiple sclerosis (RRMS); and to compare microRNAs in cerebrospinal fluid (CSF) and serum with regard to dysfunction of the blood-CSF barrier. METHODS: CSF and serum miRNA-181c, miRNA-633 and miRNA-922 were retrospectively determined by quantitative real-time polymerase chain reaction in CIS patients with (CIS-RRMS) and without (CIS-CIS) conversion to RRMS within 1 year. RESULTS: Thirty of 58 CIS patients developed RRMS. Cerebrospinal fluid miRNA-922, serum miRNA-922 and cerebrospinal fluid miRNA-181c were significantly higher in CIS-RRMS compared to CIS-CIS (P=0.027, P=0.048, P=0.029, respectively). High levels of cerebrospinal fluid miRNA-181c were independently associated with conversion from CIS to RRMS in multivariate Cox regression analysis (hazard ratio 2.99, 95% confidence interval 1.41-6.34, P=0.005). A combination of high cerebrospinal fluid miRNA-181c, younger age and more than nine lesions on magnetic resonance imaging showed the highest specificity (96%) and positive predictive value (94%) for conversion from CIS to RRMS. MiRNA-181c was higher in serum than in cerebrospinal fluid (P <0.001), while miRNA-633 and miRNA-922 were no different in cerebrospinal fluid and serum. Cerebrospinal fluid/serum albumin quotients did not correlate with microRNAs in cerebrospinal fluid (all P>0.711). CONCLUSIONS: Cerebrospinal fluid miRNA-181c might serve as a biomarker for early conversion to RRMS. Moreover, our data suggest an intrathecal origin of microRNAs detected in the cerebrospinal fluid.
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Enfermedades Desmielinizantes/líquido cefalorraquídeo , MicroARNs/líquido cefalorraquídeo , Esclerosis Múltiple Recurrente-Remitente/líquido cefalorraquídeo , Adulto , Distribución de Chi-Cuadrado , Enfermedades Desmielinizantes/sangre , Enfermedades Desmielinizantes/diagnóstico , Enfermedades Desmielinizantes/genética , Progresión de la Enfermedad , Femenino , Marcadores Genéticos , Alemania , Humanos , Estimación de Kaplan-Meier , Masculino , MicroARNs/sangre , MicroARNs/genética , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/sangre , Esclerosis Múltiple Recurrente-Remitente/diagnóstico , Esclerosis Múltiple Recurrente-Remitente/genética , Análisis Multivariante , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Factores de Riesgo , Factores de Tiempo , Adulto JovenRESUMEN
AIMS: Osteopontin (OPN) is a multifunctional cytokine critically involved in cardiac fibrosis. However, the underlying mechanisms are unresolved. Non-coding RNAs are powerful regulators of gene expression and thus might mediate this process. METHODS AND RESULTS: OPN and miR-21 were significantly increased in cardiac biopsies of patients with myocardial fibrosis. Ang II infusion via osmotic minipumps led to specific miRNA regulations with miR-21 being strongly induced in wild-type (WT) but not OPN knockout (KO) mice. This was associated with enhanced cardiac collagen content, myofibroblast activation, ERK-MAP kinase as well as AKT signalling pathway activation and a reduced expression of Phosphatase and Tensin Homologue (PTEN) as well as SMAD7 in WT but not OPN KO mice. In contrast, cardiotropic AAV9-mediated overexpression of OPN in vivo further enhanced cardiac fibrosis. In vitro, Ang II induced expression of miR-21 in WT cardiac fibroblasts, while miR-21 levels were unchanged in OPN KO fibroblasts. As pri-miR-21 was also increased by Ang II, we studied potential involved upstream regulators; Electrophoretic Mobility Shift and Chromatin Immunoprecipitation analyses confirmed activation of the miR-21 upstream-transcription factor AP-1 by Ang II. Recombinant OPN directly activated miR-21, enhanced fibrosis, and activated the phosphoinositide 3-kinase pathway. Locked nucleic acid-mediated miR-21 silencing ameliorated cardiac fibrosis development in vivo. CONCLUSION: In cardiac fibrosis related to Ang II, miR-21 is transcriptionally activated and targets PTEN/SMAD7 resulting in increased fibroblast survival. OPN KO animals are protected from miR-21 increase and fibrosis development due to impaired AP-1 activation and fibroblast activation.
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Angiotensina II/fisiología , MicroARNs/genética , Miocardio/patología , Osteopontina/fisiología , Adenoviridae , Anciano , Animales , Supervivencia Celular , Células Cultivadas , Colágeno/metabolismo , Femenino , Fibrosis/genética , Silenciador del Gen , Vectores Genéticos/administración & dosificación , Humanos , Técnicas In Vitro , Masculino , Ratones Noqueados , MicroARNs/metabolismo , Miofibroblastos/fisiología , Osteopontina/farmacología , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Recombinantes/farmacología , Factores de TranscripciónRESUMEN
BACKGROUND: Long noncoding RNAs (lncRNAs) are novel intracellular noncoding ribonucleotides regulating the genome and proteome. They are detectable in the blood of patients with acute kidney injury. We tested whether lncRNAs are present in urine and may serve as new predictors of outcome in renal transplant patients with acute rejection. METHODS: A global lncRNA expression analysis was performed with RNA from urine of patients with acute T cell-mediated renal allograft rejection and control transplant patients. Deregulated lncRNAs were confirmed in kidney biopsies and urine in a validation cohort of 62 patients with acute rejection, 10 of them after successful antirejection therapy, and 31 control transplant patients. RESULTS: A global screen revealed several lncRNAs to be deregulated in urine of patients with acute rejection. Three intergenic lncRNAs, LNC-MYH13-3:1, RP11-395P13.3-001, and RP11-354P17.15-001, were most strongly altered. These were validated in the whole cohort of patients. RP11-395P13.3-001 and RP11-354P17.15-001 were upregulated in patients with acute rejection compared with controls. Only levels of RP11-354P17.15-001 normalized in patients with acute rejection after successful antirejection therapy. RP11-354P17.15-001 was associated with higher decline in glomerular filtration rate 1 year after transplantation. In vitro, in tubular epithelial cells, all lncRNAs were enriched by interleukin-6 treatment, but only RP11-395P13.3-001 and RP11-354P17.15-001 increased in cell culture supernatant, indicating that these lncRNAs might be secreted under inflammatory conditions. CONCLUSIONS: lncRNAs are strongly altered in urine of patients with acute rejection. Urinary RP11-354P17.15-001 may serve as a novel biomarker of acute kidney rejection, identifying patients with acute rejection and predicting loss of kidney function.
Asunto(s)
Lesión Renal Aguda/cirugía , Rechazo de Injerto/diagnóstico , Rechazo de Injerto/orina , ARN Largo no Codificante/orina , Enfermedad Aguda , Lesión Renal Aguda/genética , Lesión Renal Aguda/patología , Adolescente , Adulto , Anciano , Biomarcadores/orina , Células Cultivadas , Estudios de Cohortes , Diagnóstico Precoz , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Regulación de la Expresión Génica , Tasa de Filtración Glomerular , Rechazo de Injerto/genética , Rechazo de Injerto/patología , Humanos , Interleucina-6/farmacología , Riñón/metabolismo , Riñón/patología , Riñón/cirugía , Trasplante de Riñón , Masculino , Persona de Mediana Edad , Pronóstico , ARN Largo no Codificante/metabolismoRESUMEN
BACKGROUND: Long noncoding RNAs (lncRNAs) are novel intracellular noncoding ribonucleotides regulating gene expression. Intriguingly, these RNA transcripts are detectable and stable in the blood of patients with cancer and cardiovascular disease. We tested whether circulating lncRNAs in plasma of critically ill patients with acute kidney injury (AKI) at inception of renal replacement therapy were deregulated and might predict survival. METHODS: We performed a global lncRNA expression analysis using RNA isolated from plasma of patients with AKI, healthy controls, and ischemic disease controls. This global screen revealed several deregulated lncRNAs in plasma samples of patients with AKI. lncRNA-array-based alterations were confirmed in kidney biopsies of patients as well as in plasma of 109 patients with AKI, 30 age-matched healthy controls, and 30 disease controls by quantitative real-time PCR. RESULTS: Circulating concentrations of the novel intronic antisense lncRNA TrAnscript Predicting Survival in AKI (TapSAKI) (P < 0.0001) were detectable in kidney biopsies and upregulated in plasma of patients with AKI. Cox regression and Kaplan-Meier curve analysis revealed TapSAKI as an independent predictor of 28-day survival (P < 0.01). TapSAKI was enriched in tubular epithelial cells subjected to ATP depletion (P = 0.03). CONCLUSIONS: The alteration of circulating concentrations of lncRNAs in patients with AKI supports TapSAKI as a predictor of mortality in this patient cohort.
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Lesión Renal Aguda/sangre , Lesión Renal Aguda/genética , ARN Largo no Codificante/sangre , ARN Largo no Codificante/genética , Lesión Renal Aguda/mortalidad , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , Enfermedad Crítica , Femenino , Perfilación de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Ischemia-reperfusion (I/R) injury of the kidney is a major cause of AKI. MicroRNAs (miRs) are powerful regulators of various diseases. We investigated the role of apoptosis-associated miR-24 in renal I/R injury. miR-24 was upregulated in the kidney after I/R injury of mice and in patients after kidney transplantation. Cell-sorting experiments revealed a specific miR-24 enrichment in renal endothelial and tubular epithelial cells after I/R induction. In vitro, anoxia/hypoxia induced an enrichment of miR-24 in endothelial and tubular epithelial cells. Transient overexpression of miR-24 alone induced apoptosis and altered functional parameters in these cells, whereas silencing of miR-24 ameliorated apoptotic responses and rescued functional parameters in hypoxic conditions. miR-24 effects were mediated through regulation of H2A histone family, member X, and heme oxygenase 1, which were experimentally validated as direct miR-24 targets through luciferase reporter assays. In vitro, adenoviral overexpression of miR-24 targets lacking miR-24 binding sites along with miR-24 precursors rescued various functional parameters in endothelial and tubular epithelial cells. In vivo, silencing of miR-24 in mice before I/R injury resulted in a significant improvement in survival and kidney function, a reduction of apoptosis, improved histologic tubular epithelial injury, and less infiltration of inflammatory cells. miR-24 also regulated heme oxygenase 1 and H2A histone family, member X, in vivo. Overall, these results indicate miR-24 promotes renal ischemic injury by stimulating apoptosis in endothelial and tubular epithelial cell. Therefore, miR-24 inhibition may be a promising future therapeutic option in the treatment of patients with ischemic AKI.
Asunto(s)
Túbulos Renales/metabolismo , Riñón/metabolismo , Riñón/patología , MicroARNs/antagonistas & inhibidores , Daño por Reperfusión/patología , Adulto , Animales , Apoptosis , Sitios de Unión , Células Endoteliales/citología , Endotelio/patología , Células Epiteliales/metabolismo , Femenino , Silenciador del Gen , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1/metabolismo , Histonas/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inflamación/metabolismo , Túbulos Renales/patología , Masculino , Ratones , MicroARNs/genética , Persona de Mediana Edad , Receptores de Lisoesfingolípidos/metabolismo , Receptores de Esfingosina-1-FosfatoRESUMEN
Changes in gene expression during inflammation are in part caused by post-transcriptional mechanisms. A transcriptome-wide screen for changes in ribosome occupancy indicated that the inflammatory cytokine IL-17 activates translation of a group of mRNAs that overlaps partially with those affected similarly by IL-1. Included are mRNAs of IκBζ and of MCPIP1, important regulators of the quality and course of immune and inflammatory responses. Evidence for increased ribosome association of these mRNAs was also obtained in LPS-activated RAW264.7 macrophages and human peripheral blood mononuclear cells. Like IL-1, IL-17 activated translation of IκBζ mRNA by counteracting the function of a translational silencing element in its 3'-UTR defined previously. Translational silencing of MCPIP1 mRNA in unstimulated cells resulted from the combined suppressive activities of its 5'-UTR, which contains upstream open reading frames, and of its 3'-UTR, which silences independently of the 5'-UTR. Only the silencing function of the 3'-UTR was counteracted by IL-17 as well as by IL-1. Translational silencing by the 3'-UTR was dependent on a putative stem-loop-forming region previously associated with rapid degradation of the mRNA. The results suggest that translational control exerted by IL-1 and IL-17 plays an important role in the coordination of an inflammatory reaction.
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Regulación de la Expresión Génica , Interleucina-17/metabolismo , Interleucina-1/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Ribonucleasas/metabolismo , Factores de Transcripción/metabolismo , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Animales , Línea Celular , Citocinas/metabolismo , Silenciador del Gen , Células HeLa , Humanos , Inflamación , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Activación TranscripcionalRESUMEN
2-Arachidonoyl glycerol (2AG) is an endocannabinoid that activates cannabinoid (CB) receptors CB1 and CB2. Monoacylglycerol lipase (MAGL) inactivates 2AG through hydrolysis to arachidonic acid (AA) and glycerol, thus modulating the activity at CB receptors. In the brain, AA released from 2AG by the action of MAGL serves as a substrate for cyclooxygenases which produce pro-inflammatory prostaglandins. Here we report stable-isotope GC-MS and LC-MS/MS assays for the reliable measurement of MAGL activity. The assays utilize deuterium-labeled 2AG (d8-2AG; 10µM) as the MAGL substrate and measure deuterium-labeled AA (d8-AA; range 0-1µM) as the MAGL product. Unlabelled AA (d0-AA, 1µM) serves as the internal standard. d8-AA and d0-AA are extracted from the aqueous buffered incubation mixtures by ethyl acetate. Upon solvent evaporation the residue is reconstituted in the mobile phase prior to LC-MS/MS analysis or in anhydrous acetonitrile for GC-MS analysis. LC-MS/MS analysis is performed in the negative electrospray ionization mode by selected-reaction monitoring the mass transitions [M-H]-â[M-H - CO2]-, i.e., m/z 311âm/z 267 for d8-AA and m/z 303âm/z 259 for d0-AA. Prior to GC-MS analysis d8-AA and d0-AA were converted to their pentafluorobenzyl (PFB) esters by means of PFB-Br. GC-MS analysis is performed in the electron-capture negative-ion chemical ionization mode by selected-ion monitoring the ions [M-PFB]-, i.e., m/z 311 for d8-AA and m/z 303 for d0-AA. The GC-MS and LC-MS/MS assays were cross-validated. Linear regression analysis between the concentration (range, 0-1µM) of d8-AA measured by LC-MS/MS (y) and that by GC-MS (x) revealed a straight line (r2=0.9848) with the regression equation y=0.003+0.898x, indicating a good agreement. In dog liver, we detected MAGL activity that was inhibitable by the MAGL inhibitor JZL-184. Exogenous eicosatetraynoic acid is suitable as internal standard for the quantitative determination of d8-AA produced from d8-2AG by hepatic MAGL activity. The formation of d8-prostaglandin E2 by the consecutive catalytic action of recombinant MAGL on d8-2AG and recombinant cyclooxygenase-2 (COX) on d8-AA was demonstrated by GC-MS/MS.