RESUMEN
Noninvasive, cost-effective biomarkers that allow accurate monitoring of graft function are needed in kidney transplantation. Since microRNAs (miRNAs) have emerged as promising disease biomarkers, we sought to establish an miRNA signature in urinary cell pellets comparing kidney transplant patients diagnosed with chronic allograft dysfunction (CAD) with interstitial fibrosis and tubular atrophy and those recipients with normal graft function. Overall, we evaluated 191 samples from 125 deceased donor primary kidney transplant recipients in the discovery, initial validation, and the longitudinal validation studies for noninvasive monitoring of graft function. Of 1733 mature miRNAs studied using microarrays, 22 were found to be differentially expressed between groups. Ontology and pathway analyses showed inflammation as the principal biological function associated with these miRNAs. Twelve selected miRNAs were longitudinally evaluated in urine samples of an independent set of 66 patients, at two time points after kidney transplant. A subset of these miRNAs was found to be differentially expressed between groups early after kidney transplant before histological allograft injury was evident. Thus, a panel of urine miRNAs was identified as potential biomarkers for monitoring graft function and anticipating progression to CAD in kidney transplant patients.
Asunto(s)
Perfilación de la Expresión Génica , Pruebas Genéticas/métodos , Trasplante de Riñón , Riñón/fisiopatología , MicroARNs/orina , Insuficiencia Renal Crónica/diagnóstico , Adulto , Anciano , Atrofia , Biopsia , Estudios de Casos y Controles , Femenino , Fibrosis , Marcadores Genéticos , Humanos , Riñón/patología , Trasplante de Riñón/efectos adversos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Prospectivos , Proteinuria/diagnóstico , Proteinuria/genética , Proteinuria/orina , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/orina , Reproducibilidad de los Resultados , Factores de Tiempo , Resultado del Tratamiento , Urinálisis , Adulto JovenRESUMEN
We show that 3-[4-(4-methoxyphenyl)piperazin-1-yl]piperidin-4-yl biphenyl-4-carboxylate (C10), screened out of a chemical library, selectively kills bacterial persisters that tolerate antibiotic treatment but does not affect normal antibiotic-sensitive cells. C10 led persisters to antibiotic-induced cell death by causing reversion of persisters to antibiotic-sensitive cells. This work is the first demonstration in which the eradication of bacterial persisters is based on single-chemical supplementation. The chemical should be versatile in elucidating the mechanism of persistence.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacosRESUMEN
Acute cellular rejection (ACR) and hepatitis C virus (HCV) recurrence (HCVrec) are common complications after liver transplantation (LT) in HCV patients, who share common clinical and histological features, making a differential diagnosis difficult. Fifty-three liver allograft samples from unique HCV LT recipients were studied using microarrays, including a training set (n = 32) and a validation set (n = 19). Two no-HCV-ACR samples from LT recipients were also included. Probe set intensity values were obtained using the robust multiarray average method (RMA) method. Analysis of variance identified statistically differentially expressed genes (P ≤ 0.005). The limma package was used to fit the mixed-effects models using a restricted maximum likelihood procedure. The last absolute shrinkage and selection operator (LASSO) model was fit with HCVrec versus ACR as the dependent variable predicted. N-fold cross-validation was performed to provide an unbiased estimate of generalization error. A total of 179 probe sets were differentially expressed among groups, with 71 exclusive genes between HCVrec and HCV-ACR. No differences were found within ACR group (HCV-ACR vs. no-HCV-ACR). Supervised clustering analysis displayed two clearly independent groups, and no-HCV-ACR clustered within HCV-ACR. HCVrec-related genes were associated with a cytotoxic T-cell profile, and HCV-ACR-related genes were associated with the inflammatory response. The best-fitting LASSO model classifier accuracy, including 15 genes, has an accuracy of 100% in the training set. N-fold cross-validation accuracy was 78.1%, and sensitivity, specificity and positive and negative predictive values were 50.0%, 90.9%, 71.4% and 80.0%, respectively. Arginase type II (ARG2), ethylmalonic encephalopathy 1 (ETHE1), transmembrane protein 176A (TMEM176A) and TMEM176B genes were significantly confirmed in the validation set. A molecular signature capable of distinguishing HCVrec and ACR in HCV LT recipients was identified and validated.
Asunto(s)
Perfilación de la Expresión Génica , Rechazo de Injerto/genética , Hepatitis C/genética , Trasplante de Hígado , Complicaciones Posoperatorias/genética , Enfermedad Aguda , Análisis por Conglomerados , Diagnóstico Diferencial , Rechazo de Injerto/diagnóstico , Hepatitis C/diagnóstico , Humanos , Hígado/metabolismo , Hígado/patología , Hígado/cirugía , Análisis de Secuencia por Matrices de Oligonucleótidos , Complicaciones Posoperatorias/diagnóstico , Recurrencia , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y EspecificidadRESUMEN
Robust biomarkers are needed to identify donor kidneys with poor quality associated with inferior early and longer-term outcome. The occurrence of delayed graft function (DGF) is most often used as a clinical outcome marker to capture poor kidney quality. Gene expression profiles of 92 preimplantation biopsies were evaluated in relation to DGF and estimated glomerular filtration rate (eGFR) to identify preoperative gene transcript changes associated with short-term function. Patients were stratified into those who required dialysis during the first week (DGF group) versus those without (noDGF group) and subclassified according to 1-month eGFR of >45 mL/min (eGFR(hi)) versus eGFR of ≤45 mL/min (eGFR(lo)). The groups and subgroups were compared in relation to clinical donor and recipient variables and transcriptome-associated biological pathways. A validation set was used to confirm target genes. Donor and recipient characteristics were similar between the DGF versus noDGF groups. A total of 206 probe sets were significant between groups (P < 0.01), but the gene functional analyses failed to identify any significantly affected pathways. However, the subclassification of the DGF and noDGF groups identified 283 probe sets to be significant among groups and associated with biological pathways. Kidneys that developed postoperative DGF and sustained an impaired 1-month function (DGF(lo) group) showed a transcriptome profile of significant immune activation already preimplant. In addition, these kidneys maintained a poorer transplant function throughout the first-year posttransplant. In conclusion, DGF is a poor marker for organ quality and transplant outcome. In contrast, preimplant gene expression profiles identify "poor quality" grafts and may eventually improve organ allocation.
Asunto(s)
Funcionamiento Retardado del Injerto/genética , Perfilación de la Expresión Génica/métodos , Trasplante de Riñón/fisiología , Riñón/metabolismo , Riñón/fisiopatología , Adolescente , Adulto , Anciano , Biomarcadores/metabolismo , Biopsia , Estudios de Cohortes , Creatinina/sangre , Funcionamiento Retardado del Injerto/sangre , Demografía , Femenino , Regulación de la Expresión Génica , Tasa de Filtración Glomerular/fisiología , Humanos , Riñón/patología , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Transducción de Señal/genética , Resultado del Tratamiento , Adulto JovenRESUMEN
Allograft gene expression analysis may provide insights into the mechanisms involved in liver damage during hepatitis C virus recurrence (HCVrec) after orthotopic liver transplantation (OLT) and allow the identification of patients who have a higher risk of developing severe disease. Forty-three OLT recipients with hepatitis C virus (HCV) were evaluated. Genomewide gene expression analysis was performed with formalin-fixed, paraffin-embedded (FFPE) liver biopsy samples obtained from 21 OLT recipients with HCV at the time of clinical HCVrec, which was defined as increased alanine aminotransferase levels and detectable HCV RNA levels in serum. Patients were classified into 3 groups according to the severity of the fibrosis in the liver biopsies at 36 months post-OLT : group 1 (G1) for mild fibrosis (F0-F1), group 2 for moderate fibrosis (F2), and group 3 (G3) for severe fibrosis (F3-F4). No significant differences were observed between the groups with respect to donor age, histology during HCVrec, treated episodes of acute cellular rejection, or immunosuppression therapy. The results were validated in the remaining 22 OLT recipients with HCV using quantitative real-time polymerase chain reaction. Fifty-seven beadtypes showed significantly different expression (P < 0.001) between the groups during HCVrec. In G3, the gene expression of interleukin-28RA (IL-28RA), IL-28, and angiotensin-converting enzyme was up-regulated. Samples from G1 and G3 were used to determine whether a multigenetic classifier could be derived to predict the group class. The final model included the intercept and 9 bead types. Pairwise scatter plots of these 9 bead types revealed that G1 and G3 were well separated with respect to each gene. Our analysis has demonstrated the utility of a set of molecular markers indicating HCVrec severity early after OLT.
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Hepatitis C/diagnóstico , Cirrosis Hepática/patología , Cirrosis Hepática/virología , Trasplante de Hígado/métodos , Adulto , Biopsia , Progresión de la Enfermedad , Femenino , Genoma , Hepacivirus/metabolismo , Hepatitis C/patología , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunosupresores/uso terapéutico , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Control de Calidad , Recurrencia , TranscriptomaRESUMEN
BACKGROUND: Severe liver steatosis is a known risk factor for increased ischemia-reperfusion injury (IRI) and poor outcomes after liver transplantation (LT). This study aimed to identify steatosis-related molecular mechanisms associated with IRI exacerbation after LT. METHODS: Paired graft biopsies (n = 60) were collected before implantation (L1) and 90 minutes after reperfusion (L2). The LT recipients (n = 30) were classified by graft macrosteatosis: without steatosis (WS) of 5% or less (n = 13) and with steatosis (S) of 25% or greater (n = 17). Plasma samples were collected at L1, L2, and 1 day after LT (postoperative [POD]1) for cytokines evaluation. Tissue RNA was isolated for gene expression microarrays. Probeset summaries were obtained using robust multiarray average algorithm. Pairwise comparisons were fit using 2-sample t test. P values 0.01 or less were significant (false discovery rate <5%). Molecular pathway analyses were conducted using Ingenuity Pathway Analysis tool. RESULTS: Significantly differentially expressed genes were identified for WS and S grafts after reperfusion. Comprehensive comparison analysis of molecular profiles revealed significant association of S grafts molecular profile with innate immune response activation, macrophage production of nitric oxide and reactive oxygen species, IL-6, IL-8, IL-10 signaling activation, recruitment of granulocytes, and accumulation of myeloid cells. Postreperfusion histological patterns of S grafts revealed neutrophilic infiltration surrounding fat accumulation. Circulating proinflammatory cytokines after reperfusion and 24 hours after LT concurred with intragraft-deregulated molecular pathways. All tested cytokines were significantly increased in plasma of S grafts recipients after reperfusion when compared with WS group at same time. CONCLUSIONS: Increases of graft steatosis exacerbate IRI by exacerbation of innate immune response after LT. Preemptive strategies should consider it for safety usage of steatotic livers.
Asunto(s)
Biomarcadores/metabolismo , Hígado Graso/cirugía , Inmunidad Innata/inmunología , Trasplante de Hígado , Hígado/patología , Daño por Reperfusión/etiología , Donantes de Tejidos , Biopsia , Citocinas/biosíntesis , Citocinas/genética , Hígado Graso/complicaciones , Hígado Graso/patología , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Daño por Reperfusión/inmunología , Daño por Reperfusión/patologíaRESUMEN
Despite improvement on outcomes post liver transplantation (LT), complications such as HCV recurrence (HCV-rec) and acute cellular rejection (ACR) continue to be a challenge for transplant physicians. Accurate diagnostic tools to better dissect between those complications post-LT are crucial for prompt and correct diagnosis and treatment. It is well known that the overlapping features of clinical and histo-pathological characteristics between these conditions turn difficult the appropriate differential diagnosis. Recently, new technological advances had supported the field of biomarker discovery in many diseases. Disease biomarkers capable to differentiate ACR versus HCV-rec post-LT is a long waited task in the transplant community. This editorial describes and discusses potential biomarkers of disease differentiation including recent reports in the field of genomics, proteomics, immunohistochemistry among other technologies.
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Rechazo de Injerto/diagnóstico , Hepatitis C/cirugía , Trasplante de Hígado/efectos adversos , Técnicas de Diagnóstico Molecular , Enfermedad Aguda , Biomarcadores/análisis , Diagnóstico Diferencial , Marcadores Genéticos , Rechazo de Injerto/genética , Rechazo de Injerto/inmunología , Hepatitis C/diagnóstico , Hepatitis C/genética , Hepatitis C/virología , Humanos , Técnicas de Diagnóstico Molecular/métodos , Valor Predictivo de las Pruebas , Recurrencia , Resultado del TratamientoRESUMEN
BACKGROUND: Ischemia-reperfusion (I/R) injury is a multifactorial phenomenon that occurs during the transplant event and frequently compromises early graft function after liver transplantation (LT). Current comprehension of molecular mechanisms and regulation processes of I/R injury lacks clarity. MicroRNA (miRNA) regulation results critical in several biological processes. METHODS: This study evaluated gene expression and miRNA expression profiles using microarrays in 34 graft biopsies collected at preimplantation (L1) and at 90 min postreperfusion (L2) from consecutives deceased-donor LT recipients. miRNA profiles were first analyzed. Data integration analysis (gene expression/miRNA expression) aimed to identify potential target genes for each identified miRNA from the L1/L2 differential gene expression profile. RESULTS: Pairwise comparison analyses identified 40 miRNAs and 3168 significantly differentially expressed genes at postreperfusion time compared with preimplantation time. Pathway analysis of miRNAs associated these profiles with antiapoptosis, inhibition of cellular proliferation, and proinflammatory processes. Target analysis identified an miRNA-associated molecular profile of 2172 genes involved in cellular growth and proliferation modulation by cell cycle regulation, cell death and survival, and proinflammatory and anti-inflammatory processes. miRNA-independent genes involved proinflammatory molecules. CONCLUSION: We identified a miRNA profile involved in posttranscriptional regulatory mechanisms in I/R injury post-LT. A better understanding of these molecular processes involved in I/R may contribute to develop new strategies to minimize graft injury.
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Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Rechazo de Injerto/genética , Trasplante de Hígado , MicroARNs/genética , Daño por Reperfusión/genética , Adulto , Anciano , Biopsia , Femenino , Estudios de Seguimiento , Rechazo de Injerto/metabolismo , Rechazo de Injerto/patología , Humanos , Masculino , MicroARNs/biosíntesis , Persona de Mediana Edad , Periodo Posoperatorio , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Transcripción Genética , Adulto JovenRESUMEN
UNLABELLED: The increased disparity between organ supply and need has led to the use of extended criteria donors and donation after cardiac death donors with other comorbidities. METHODS: We have examined the preimplantation transcriptome of 112 kidney transplant recipient samples from 100 deceased-donor kidneys by microarray profiling. Subject groups were segregated based on estimated glomerular filtration rate (eGFR) at 1 month after transplantation: the GFR-high group (n=74) included patients with eGFR 45 mL/min per 1.73 m(2), whereas the GFR-low group (n=35) included patients with eGFR 45 mL/min or less per 1.73 m(2). RESULTS: Gene expression profiling identified higher expression of 160 probe sets (140 genes) in the GFR-low group, whereas expression of 37 probe sets (33 genes) was higher in the GFR-high group (P<0.01, false discovery rate <0.2). Four genes (CCL5, CXCR4, ITGB2, and EGF) were selected based on fold change and P value and further validated using an independent set of samples. A random forest analysis identified three of these genes (CCL5, CXCR4, and ITGB2) as important predictors of graft function after transplantation. CONCLUSIONS: Inclusion of pretransplantation molecular gene expression profiles in donor quality assessment systems may provide the necessary information for better donor organ selection and function prediction. These biomarkers would further allow a more objective and complete assessment of procured renal allografts at pretransplantation time.
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Trasplante de Riñón , Donantes de Tejidos , Transcriptoma , Biomarcadores , Tasa de Filtración Glomerular , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Modelos de Riesgos Proporcionales , Transducción de Señal , Trasplante HomólogoRESUMEN
BACKGROUND: The use of expanded criteria donor kidneys (ECD) had been associated with worse outcomes. Whole gene expression of pre-implantation allograft biopsies from deceased donor kidneys (DDKs) was evaluated to compare the effect of pulsatile pump preservation (PPP) vs. cold storage preservation (CSP) on standard and ECD kidneys. METHODOLOGY/PRINCIPAL FINDINGS: 99 pre-implantation DDK biopsies were studied using gene expression with GeneChips. Kidneys transplant recipients were followed post transplantation for 35.8 months (rangeâ=â24-62). The PPP group included 60 biopsies (cold ischemia time (CIT)â=â1,367+/-509 minutes) and the CSP group included 39 biopsies (CITâ=â1,022+/-485 minutes) (P<0.001). Donor age (42.0±14.6 vs. 34.1±14.2 years, Pâ=â0.009) and the percentage of ECD kidneys (PPPâ=â35% vs. CSPâ=â12.8%, Pâ=â0.012) were significantly different between groups. A two-sample t-test was performed, and probe sets having a P<0.001 were considered significant. Probe set level linear models were fit using cold ischemia time and CSP/PPP as independent variables to determine significant probe sets (P<0.001) between groups after adjusting for cold ischemia time. Thus, 43 significant genes were identified (P<0.001). Over-expression of genes associated with inflammation (CD86, CD209, CLEC4, EGFR2, TFF3, among others) was observed in the CSP group. Cell-to-cell signaling and interaction, and antigen presentation were the most important pathways with genes significantly over-expressed in CSP kidneys. When the analysis was restricted to ECD kidneys, genes involved in inflammation were also differentially up-regulated in ECD kidneys undergoing CSP. However, graft survival at the end of the study was similar between groups (Pâ=â0.2). Moreover, the incidence of delayed graft function was not significant between groups. CONCLUSIONS/SIGNIFICANCE: Inflammation was the most important up-regulated pattern associated with pre-implantation biopsies undergoing CSP even when the PPP group has a larger number of ECD kidneys. No significant difference was observed in delayed graft function incidence and graft function post-transplantation. These findings support the use of PPP in ECD donor kidneys.
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Perfilación de la Expresión Génica , Trasplante de Riñón , Riñón/metabolismo , Preservación de Órganos/métodos , Adulto , Anciano , Biopsia , Cadáver , Funcionamiento Retardado del Injerto/epidemiología , Femenino , Regulación de la Expresión Génica , Humanos , Inflamación/genética , Inflamación/inmunología , Riñón/inmunología , Trasplante de Riñón/inmunología , Trasplante de Riñón/métodos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND: Loss of kidney graft function due to interstitial fibrosis (IF) and tubular atrophy (TA) is the most common cause of kidney allograft loss. METHODS: One hundred one allograft tissues (26 samples with IF/TA, 17 normal allografts, and an independent biopsy group collected at 3 month [n=34] posttransplantation) underwent microarray analysis to identify early detection/diagnostic biomarkers of IF/TA. Profiling of 24 allograft biopsies collected at or after 9-month posttransplantation (range 9-18 months) was used for validation. Three-month posttransplantation biopsies were classified as IF/TA nonprogressors (group 1) or progressors (group 2) using graft function and histology at 9-month posttransplantation. RESULTS: We identified 2223 differentially expressed probe sets between IF/TA and normal allograft biopsies using a Bonferroni correction. Genes up-regulated in IF/TA were primarily involved in pathways related to T-cell activation, natural killer cell-mediated cytotoxicity, and programmed cell death. A least absolute shrinkage and selection operator model was derived from the differentially expressed probe sets, resulting in a final model that included 10 probe sets and had 100% training set accuracy. The N-fold crossvalidated error was 2.4% (sensitivity 95.8% and specificity 100%). When 3-month biopsies were tested using the model, all the samples were classified as normal. However, evaluating gene expression of the 3-month biopsies and fitting a new penalized model, 100% sensitivity was observed in classifying the samples as group1 or 2. This model was evaluated in the sample set collected at or after 9-month posttransplantation. CONCLUSIONS: An IF/TA gene expression signature was identified, and it was useful for diagnosis but not prediction. However, gene expression profiles at 3 months might predict IF/TA progression.