RESUMO
Traditional folk treatments for the prevention and management of urinary tract infections (UTIs) and other infectious diseases often include plants and plant extracts that are rich in phenolic compounds. These have been ascribed a variety of activities, including inhibition of bacterial interactions with host cells. Here, we tested a panel of four well-studied phenolic compounds-caffeic acid phenethyl ester (CAPE), resveratrol, catechin, and epigallocatechin gallate-for the effects on host cell adherence and invasion by uropathogenic Escherichia coli (UPEC). These bacteria, which are the leading cause of UTIs, can bind and subsequently invade bladder epithelial cells via an actin-dependent process. Intracellular UPEC reservoirs within the bladder are often protected from antibiotics and host defenses and likely contribute to the development of chronic and recurrent infections. In cell culture-based assays, only resveratrol had a notable negative effect on UPEC adherence to bladder cells. However, both CAPE and resveratrol significantly inhibited UPEC entry into the host cells, coordinate with attenuated phosphorylation of the host actin regulator Focal Adhesion Kinase (FAK or PTK2) and marked increases in the numbers of focal adhesion structures. We further show that the intravesical delivery of resveratrol inhibits UPEC infiltration of the bladder mucosa in a murine UTI model and that resveratrol and CAPE can disrupt the ability of other invasive pathogens to enter host cells. Together, these results highlight the therapeutic potential of molecules like CAPE and resveratrol, which could be used to augment antibiotic treatments by restricting pathogen access to protective intracellular niches.IMPORTANCEUrinary tract infections (UTIs) are exceptionally common and increasingly difficult to treat due to the ongoing rise and spread of antibiotic-resistant pathogens. Furthermore, the primary cause of UTIs, uropathogenic Escherichia coli (UPEC), can avoid antibiotic exposure and many host defenses by invading the epithelial cells that line the bladder surface. Here, we identified two plant-derived phenolic compounds that disrupt activation of the host machinery needed for UPEC entry into bladder cells. One of these compounds, resveratrol, effectively inhibited UPEC invasion of the bladder mucosa in a mouse UTI model, and both phenolic compounds significantly reduced host cell entry by other invasive pathogens. These findings suggest that select phenolic compounds could be used to supplement existing antibacterial therapeutics by denying uropathogens shelter within host cells and tissues and help explain some of the benefits attributed to traditional plant-based medicines.
Assuntos
Infecções por Escherichia coli , Quinase 1 de Adesão Focal , Fenóis , Extratos Vegetais , Infecções Urinárias , Escherichia coli Uropatogênica , Animais , Feminino , Humanos , Camundongos , Aderência Bacteriana/efeitos dos fármacos , Ácidos Cafeicos/farmacologia , Catequina/farmacologia , Catequina/análogos & derivados , Linhagem Celular , Células Epiteliais/microbiologia , Células Epiteliais/efeitos dos fármacos , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Quinase 1 de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/antagonistas & inibidores , Fenóis/farmacologia , Álcool Feniletílico/análogos & derivados , Extratos Vegetais/farmacologia , Resveratrol/farmacologia , Bexiga Urinária/microbiologia , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/patologia , Infecções Urinárias/microbiologia , Infecções Urinárias/tratamento farmacológico , Escherichia coli Uropatogênica/efeitos dos fármacosRESUMO
Renal disease variability in autosomal dominant polycystic kidney disease (ADPKD) is strongly influenced by the gene locus (PKD1 versus PKD2). Recent studies identified nontruncating PKD1 mutations in approximately 30% of patients who underwent comprehensive mutation screening, but the clinical significance of these mutations is not well defined. We examined the genotype-renal function correlation in a prospective cohort of 220 unrelated ADPKD families ascertained through probands with serum creatinine ≤1.4 mg/dl at recruitment. We screened these families for PKD1 and PKD2 mutations and reviewed the clinical outcomes of the probands and affected family members. Height-adjusted total kidney volume (htTKV) was obtained in 161 affected subjects. Multivariate Cox proportional hazard modeling for renal and patient survival was performed in 707 affected probands and family members. Overall, we identified pathogenic mutations in 84.5% of our families, in which the prevalence of PKD1 truncating, PKD1 in-frame insertion/deletion, PKD1 nontruncating, and PKD2 mutations was 38.3%, 4.3%, 27.1%, and 30.3%, respectively. Compared with patients with PKD1 truncating mutations, patients with PKD1 in-frame insertion/deletion, PKD1 nontruncating, or PKD2 mutations have smaller htTKV and reduced risks (hazard ratio [95% confidence interval]) of ESRD (0.35 [0.14 to 0.91], 0.10 [0.05 to 0.18], and 0.03 [0.01 to 0.05], respectively) and death (0.31 [0.11 to 0.87], 0.20 [0.11 to 0.38], and 0.18 [0.11 to 0.31], respectively). Refined genotype-renal disease correlation coupled with targeted next generation sequencing of PKD1 and PKD2 may provide useful clinical prognostication for ADPKD.
Assuntos
Estudos de Associação Genética , Mutação , Rim Policístico Autossômico Dominante/genética , Adulto , Feminino , Estudos de Associação Genética/métodos , Humanos , Rim/fisiopatologia , Masculino , Linhagem , Rim Policístico Autossômico Dominante/fisiopatologia , Estudos ProspectivosRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) often results in ESRD but with a highly variable course. Mutations to PKD1 or PKD2 cause ADPKD; both loci have high levels of allelic heterogeneity. We evaluated genotype-phenotype correlations in 1119 patients (945 families) from the HALT Progression of PKD Study and the Consortium of Radiologic Imaging Study of PKD Study. The population was defined as: 77.7% PKD1, 14.7% PKD2, and 7.6% with no mutation detected (NMD). Phenotypic end points were sex, eGFR, height-adjusted total kidney volume (htTKV), and liver cyst volume. Analysis of the eGFR and htTKV measures showed that the PKD1 group had more severe disease than the PKD2 group, whereas the NMD group had a PKD2-like phenotype. In both the PKD1 and PKD2 populations, men had more severe renal disease, but women had larger liver cyst volumes. Compared with nontruncating PKD1 mutations, truncating PKD1 mutations associated with lower eGFR, but the mutation groups were not differentiated by htTKV. PKD1 nontruncating mutations were evaluated for conservation and chemical change and subdivided into strong (mutation strength group 2 [MSG2]) and weak (MSG3) mutation groups. Analysis of eGFR and htTKV measures showed that patients with MSG3 but not MSG2 mutations had significantly milder disease than patients with truncating cases (MSG1), an association especially evident in extreme decile populations. Overall, we have quantified the contribution of genic and PKD1 allelic effects and sex to the ADPKD phenotype. Intrafamilial correlation analysis showed that other factors shared by families influence htTKV, with these additional genetic/environmental factors significantly affecting the ADPKD phenotype.
Assuntos
Mutação , Rim Policístico Autossômico Dominante/genética , Canais de Cátion TRPP/genética , Adulto , Feminino , Previsões , Estudos de Associação Genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , FenótipoRESUMO
The clinical use of conventional ultrasonography (US) in autosomal dominant polycystic kidney disease (ADPKD) is currently limited by reduced diagnostic sensitivity, especially in at-risk subjects younger than 30 years of age. In this single-center prospective study, we compared the diagnostic performance of MRI with that of high-resolution (HR) US in 126 subjects ages 16-40 years born with a 50% risk of ADPKD who underwent both these renal imaging studies and comprehensive PKD1 and PKD2 mutation screening. Concurrently, 45 healthy control subjects without a family history of ADPKD completed the same imaging protocol. We analyzed 110 at-risk subjects whose disease status was unequivocally defined by molecular testing and 45 unaffected healthy control subjects. Using a total of >10 cysts as a test criterion in subjects younger than 30 years of age, we found that MRI provided both a sensitivity and specificity of 100%. Comparison of our results from HR US with those from a previous study of conventional US using the test criterion of a total of three or more cysts found a higher diagnostic sensitivity (approximately 97% versus approximately 82%) with a slightly decreased specificity (approximately 98% versus 100%) in this study. Similar results were obtained in test subjects between the ages of 30 and 40 years old. These results suggest that MRI is highly sensitive and specific for diagnosis of ADPKD. HR US has the potential to rival the diagnostic performance of MRI but is both center- and operator-dependent.
Assuntos
Rim Policístico Autossômico Dominante/diagnóstico por imagem , Adolescente , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Ultrassonografia , Adulto JovemRESUMO
The rate of renal disease progression varies widely among patients with autosomal dominant polycystic kidney disease (ADPKD), necessitating optimal patient selection for enrollment into clinical trials. Patients from the Mayo Clinic Translational PKD Center with ADPKD (n=590) with computed tomography/magnetic resonance images and three or more eGFR measurements over ≥6 months were classified radiologically as typical (n=538) or atypical (n=52). Total kidney volume (TKV) was measured using stereology (TKVs) and ellipsoid equation (TKVe). Typical patients were randomly partitioned into development and internal validation sets and subclassified according to height-adjusted TKV (HtTKV) ranges for age (1A-1E, in increasing order). Consortium for Radiologic Imaging Study of PKD (CRISP) participants (n=173) were used for external validation. TKVe correlated strongly with TKVs, without systematic underestimation or overestimation. A longitudinal mixed regression model to predict eGFR decline showed that log2HtTKV and age significantly interacted with time in typical patients, but not in atypical patients. When 1A-1E classifications were used instead of log2HtTKV, eGFR slopes were significantly different among subclasses and, except for 1A, different from those in healthy kidney donors. The equation derived from the development set predicted eGFR in both validation sets. The frequency of ESRD at 10 years increased from subclass 1A (2.4%) to 1E (66.9%) in the Mayo cohort and from 1C (2.2%) to 1E (22.3%) in the younger CRISP cohort. Class and subclass designations were stable. An easily applied classification of ADPKD based on HtTKV and age should optimize patient selection for enrollment into clinical trials and for treatment when one becomes available.
Assuntos
Falência Renal Crônica/diagnóstico , Rim Policístico Autossômico Dominante/diagnóstico , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Ensaios Clínicos como Assunto , Progressão da Doença , Feminino , Taxa de Filtração Glomerular , Humanos , Processamento de Imagem Assistida por Computador , Rim/patologia , Falência Renal Crônica/mortalidade , Falência Renal Crônica/patologia , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Seleção de Pacientes , Rim Policístico Autossômico Dominante/mortalidade , Rim Policístico Autossômico Dominante/patologia , Tomografia Computadorizada por Raios XRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of ESRD. Affected individuals inherit a defective copy of either PKD1 or PKD2, which encode polycystin-1 (PC1) or polycystin-2 (PC2), respectively. PC1 and PC2 are secreted on urinary exosome-like vesicles (ELVs) (100-nm diameter vesicles), in which PC1 is present in a cleaved form and may be complexed with PC2. Here, label-free quantitative proteomic studies of urine ELVs in an initial discovery cohort (13 individuals with PKD1 mutations and 18 normal controls) revealed that of 2008 ELV proteins, 9 (0.32%) were expressed at significantly different levels in samples from individuals with PKD1 mutations compared to controls (P<0.03). In samples from individuals with PKD1 mutations, levels of PC1 and PC2 were reduced to 54% (P<0.02) and 53% (P<0.001), respectively. Transmembrane protein 2 (TMEM2), a protein with homology to fibrocystin, was 2.1-fold higher in individuals with PKD1 mutations (P<0.03). The PC1/TMEM2 ratio correlated inversely with height-adjusted total kidney volume in the discovery cohort, and the ratio of PC1/TMEM2 or PC2/TMEM2 could be used to distinguish individuals with PKD1 mutations from controls in a confirmation cohort. In summary, results of this study suggest that a test measuring the urine exosomal PC1/TMEM2 or PC2/TMEM2 ratio may have utility in diagnosis and monitoring of polycystic kidney disease. Future studies will focus on increasing sample size and confirming these studies. The data were deposited in the ProteomeXchange (identifier PXD001075).
Assuntos
Exossomos/metabolismo , Mutação , Rim Policístico Autossômico Dominante/metabolismo , Canais de Cátion TRPP/metabolismo , Adulto , Biomarcadores/metabolismo , Western Blotting , Estudos de Casos e Controles , Estudos de Viabilidade , Feminino , Predisposição Genética para Doença , Humanos , Falência Renal Crônica/etiologia , Falência Renal Crônica/fisiopatologia , Masculino , Rim Policístico Autossômico Dominante/complicações , Rim Policístico Autossômico Dominante/diagnóstico , Rim Policístico Autossômico Dominante/genética , Proteômica/métodos , Valores de Referência , Sensibilidade e Especificidade , Canais de Cátion TRPP/genética , Urinálise , Adulto JovemRESUMO
BACKGROUND: Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common form of Polycystic Kidney Disease (PKD) and occurs at a frequency of 1/800 to 1/1000 affecting all ethnic groups worldwide. ADPKD shows significant intrafamilial phenotypic variability in the rate of disease progression and extra-renal manifestations, which suggests the involvement of heritable modifier genes. Here we show that the PKD1 gene can act as a disease causing and a disease modifier gene in ADPKD patients. METHODS: Clinical evaluation of a family with ADPKD was performed to diagnose and assess disease progression in each individual. PKD1 was genotyped in each individual by targeted sequencing. RESULTS: Targeted screening analysis showed that the patients with ADPKD in the family had the PKD1: p.Q2243X nonsense mutation. A more severe disease phenotype, in terms of estimated Glomerular Filtration Rate (eGFR) and total kidney volume, was observed in two patients where in addition to the mutation, they carried a novel PKD1 variant (p.H1769Y). Other patients from the same family carrying only the (p.Q2243X) mutation showed milder disease manifestations. CONCLUSION: ADPKD shows significant intrafamilial phenotypic variability that is generally attributed to other modifier genes. In this rare case, we have shown that a variant at PKD1, in trans with the PKD1 mutation, can also act as a modifier gene in ADPKD patients. Understanding the molecular mechanism through which the gene exerts its disease modifying role may aid our understanding of the pathogenesis of ADPKD.
Assuntos
Predisposição Genética para Doença/epidemiologia , Mutação , Rim Policístico Autossômico Dominante/diagnóstico , Rim Policístico Autossômico Dominante/genética , Canais de Cátion TRPP/genética , Adulto , Estudos de Coortes , Feminino , Variação Genética , Heterozigoto , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Linhagem , Rim Policístico Autossômico Dominante/epidemiologia , Valor Preditivo dos TestesRESUMO
BACKGROUND: Glomerular diseases are potentially fatal, requiring aggressive interventions and close monitoring. Urine is a readily-accessible body fluid enriched in molecular signatures from the kidney and therefore particularly suited for routine clinical analysis as well as development of non-invasive biomarkers for glomerular diseases. METHODS: The Nephrotic Syndrome Study Network (NEPTUNE; ClinicalTrials.gov Identifier NCT01209000) is a North American multicenter collaborative consortium established to develop a translational research infrastructure for nephrotic syndrome. This includes standardized urine collections across all participating centers for the purpose of discovering non-invasive biomarkers for patients with nephrotic syndrome due to minimal change disease, focal segmental glomerulosclerosis, and membranous nephropathy. Here we describe the organization and methods of urine procurement and banking procedures in NEPTUNE. RESULTS: We discuss the rationale for urine collection and storage conditions, and demonstrate the performance of three experimental analytes (neutrophil gelatinase-associated lipocalin [NGAL], retinol binding globulin, and alpha-1 microglobulin) under these conditions with and without urine preservatives (thymol, toluene, and boric acid). We also demonstrate the quality of RNA and protein collected from the urine cellular pellet and exosomes. CONCLUSIONS: The urine collection protocol in NEPTUNE allows robust detection of a wide range of proteins and RNAs from urine supernatant and pellets collected longitudinally from each patient over 5 years. Combined with the detailed clinical and histopathologic data, this provides a unique resource for exploration and validation of new or accepted markers of glomerular diseases. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT01209000.
Assuntos
Bancos de Espécimes Biológicos/organização & administração , Síndrome Nefrótica/diagnóstico , Síndrome Nefrótica/urina , Proteinúria/urina , Preservação de Tecido/métodos , Coleta de Urina/métodos , Biomarcadores/sangue , Feminino , Humanos , Masculino , Proteinúria/diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estados UnidosRESUMO
Mutations to PKD1 and PKD2 are associated with autosomal dominant polycystic kidney disease (ADPKD). The absence of apparent PKD1/PKD2 linkage in five published European or North American families with ADPKD suggested a third locus, designated PKD3. Here we re-evaluated these families by updating clinical information, re-sampling where possible, and mutation screening for PKD1/PKD2. In the French-Canadian family, we identified PKD1: p.D3782_V3783insD, with misdiagnoses in two individuals and sample contamination explaining the lack of linkage. In the Portuguese family, PKD1: p.G3818A segregated with the disease in 10 individuals in three generations with likely misdiagnosis in one individual, sample contamination, and use of distant microsatellite markers explaining the linkage discrepancy. The mutation PKD2: c.213delC was found in the Bulgarian family, with linkage failure attributed to false positive diagnoses in two individuals. An affected son, but not the mother, in the Italian family had the nonsense mutation PKD1: p.R4228X, which appeared de novo in the son, with simple cysts probably explaining the mother's phenotype. No likely mutation was found in the Spanish family, but the phenotype was atypical with kidney atrophy in one case. Thus, re-analysis does not support the existence of a PKD3 in ADPKD. False positive diagnoses by ultrasound in all resolved families shows the value of mutation screening, but not linkage, to understand families with discrepant data.
Assuntos
Loci Gênicos , Mutação , Rim Policístico Autossômico Dominante/genética , Canais de Cátion TRPP/genética , Adolescente , Adulto , Idoso , Canadá , Criança , Análise Mutacional de DNA , Erros de Diagnóstico , Europa (Continente) , Reações Falso-Positivas , Feminino , Ligação Genética , Predisposição Genética para Doença , Testes Genéticos/métodos , Haplótipos , Hereditariedade , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Rim Policístico Autossômico Dominante/diagnóstico por imagem , Valor Preditivo dos Testes , Ultrassonografia , Adulto JovemRESUMO
Heterozygous mutations in PKD1 or PKD2, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively, cause autosomal dominant PKD (ADPKD), whereas mutations in PKHD1, which encodes fibrocystin/polyductin (FPC), cause autosomal recessive PKD (ARPKD). However, the relationship between these proteins and the pathogenesis of PKD remains unclear. To model PKD in human cells, we established induced pluripotent stem (iPS) cell lines from fibroblasts of three ADPKD and two ARPKD patients. Genetic sequencing revealed unique heterozygous mutations in PKD1 of the parental ADPKD fibroblasts but no pathogenic mutations in PKD2. Undifferentiated PKD iPS cells, control iPS cells, and embryonic stem cells elaborated primary cilia and expressed PC1, PC2, and FPC at similar levels, and PKD and control iPS cells exhibited comparable rates of proliferation, apoptosis, and ciliogenesis. However, ADPKD iPS cells as well as somatic epithelial cells and hepatoblasts/biliary precursors differentiated from these cells expressed lower levels of PC2 at the cilium. Additional sequencing confirmed the retention of PKD1 heterozygous mutations in iPS cell lines from two patients but identified possible loss of heterozygosity in iPS cell lines from one patient. Furthermore, ectopic expression of wild-type PC1 in ADPKD iPS-derived hepatoblasts rescued ciliary PC2 protein expression levels, and overexpression of PC1 but not a carboxy-terminal truncation mutant increased ciliary PC2 expression levels in mouse kidney cells. Taken together, these results suggest that PC1 regulates ciliary PC2 protein expression levels and support the use of PKD iPS cells for investigating disease pathophysiology.
Assuntos
Células-Tronco Pluripotentes Induzidas/patologia , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Recessivo/genética , Canais de Cátion TRPP/genética , Adulto , Estudos de Casos e Controles , Linhagem Celular , Feminino , Humanos , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Recessivo/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Canais de Cátion TRPP/metabolismoRESUMO
Meckel syndrome (MKS) is an embryonic lethal, autosomal recessive disorder characterized by polycystic kidney disease, central nervous system defects, polydactyly and liver fibrosis. This disorder is thought to be associated with defects in primary cilia; therefore, it is classed as a ciliopathy. To date, six genes have been commonly associated with MKS (MKS1, TMEM67, TMEM216, CEP290, CC2D2A and RPGRIP1L). However, mutation screening of these genes revealed two mutated alleles in only just over half of our MKS cohort (46 families), suggesting an even greater level of genetic heterogeneity. To explore the full genetic complexity of MKS, we performed exon-enriched next-generation sequencing of 31 ciliopathy genes in 12 MKS pedigrees using RainDance microdroplet-PCR enrichment and IlluminaGAIIx next-generation sequencing. In family M456, we detected a splice-donor site change in a novel MKS gene, B9D1. The B9D1 protein is structurally similar to MKS1 and has been shown to be of importance for ciliogenesis in Caenorhabditis elegans. Reverse transcriptase-PCR analysis of fetal RNA revealed, hemizygously, a single smaller mRNA product with a frameshifting exclusion of B9D1 exon 4. ArrayCGH showed that the second mutation was a 1.713 Mb de novo deletion completely deleting the B9D1 allele. Immunofluorescence analysis highlighted a significantly lower level of ciliated patient cells compared to controls, confirming a role for B9D1 in ciliogenesis. The fetus inherited an additional likely pathogenic novel missense change to a second MKS gene, CEP290; p.R2210C, suggesting oligogenic inheritance in this disorder.
Assuntos
Transtornos da Motilidade Ciliar/genética , Encefalocele/genética , Éxons/genética , Doenças Renais Policísticas/genética , Proteínas/genética , Deleção de Sequência/genética , Sequência de Aminoácidos , Sequência de Bases , Cílios/genética , Cílios/patologia , Transtornos da Motilidade Ciliar/metabolismo , Transtornos da Motilidade Ciliar/patologia , Proteínas do Citoesqueleto , Encefalocele/metabolismo , Encefalocele/patologia , Feminino , Feto , Fibroblastos/metabolismo , Ordem dos Genes , Humanos , Espaço Intracelular/metabolismo , Masculino , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Linhagem , Fenótipo , Doenças Renais Policísticas/metabolismo , Doenças Renais Policísticas/patologia , Polimorfismo de Nucleotídeo Único/genética , Transporte Proteico/genética , Retinose Pigmentar , Alinhamento de SequênciaRESUMO
Mutations in two large multi-exon genes, PKD1 and PKD2, cause autosomal dominant polycystic kidney disease (ADPKD). The duplication of PKD1 exons 1-32 as six pseudogenes on chromosome 16, the high level of allelic heterogeneity, and the cost of Sanger sequencing complicate mutation analysis, which can aid diagnostics of ADPKD. We developed and validated a strategy to analyze both the PKD1 and PKD2 genes using next-generation sequencing by pooling long-range PCR amplicons and multiplexing bar-coded libraries. We used this approach to characterize a cohort of 230 patients with ADPKD. This process detected definitely and likely pathogenic variants in 115 (63%) of 183 patients with typical ADPKD. In addition, we identified atypical mutations, a gene conversion, and one missed mutation resulting from allele dropout, and we characterized the pattern of deep intronic variation for both genes. In summary, this strategy involving next-generation sequencing is a model for future genetic characterization of large ADPKD populations.
Assuntos
Mutação , Rim Policístico Autossômico Dominante/genética , Análise de Sequência de DNA/métodos , Canais de Cátion TRPP/genética , Processamento Eletrônico de Dados , Humanos , Reação em Cadeia da PolimeraseRESUMO
Traditional folk treatments for the prevention and management of urinary tract infections (UTIs) and other infectious diseases often include plants and plant extracts that are rich in phenolic and polyphenolic compounds. These have been ascribed a variety of activities, including inhibition of bacterial interactions with host cells. Here we tested a panel of four well-studied phenolic compounds - caffeic acid phenethyl ester (CAPE), resveratrol, catechin, and epigallocatechin gallate - for effects on host cell adherence and invasion by uropathogenic Escherichia coli (UPEC). These bacteria, which are the leading cause of UTIs, can bind and subsequently invade bladder epithelial cells via an actin-dependent process. Intracellular UPEC reservoirs within the bladder are often protected from antibiotics and host defenses, and likely contribute to the development of chronic and recurrent infections. Using cell culture-based assays, we found that only resveratrol had a notable negative effect on UPEC adherence to bladder cells. However, both CAPE and resveratrol significantly inhibited UPEC entry into the host cells, coordinate with attenuated phosphorylation of the host actin regulator Focal Adhesion Kinase (FAK, or PTK2) and marked increases in the numbers of focal adhesion structures. We further show that the intravesical delivery of resveratrol inhibits UPEC infiltration of the bladder mucosa in a murine UTI model, and that resveratrol and CAPE can disrupt the ability of other invasive pathogens to enter host cells. Together, these results highlight the therapeutic potential of molecules like CAPE and resveratrol, which could be used to augment antibiotic treatments by restricting pathogen access to protective intracellular niches.
RESUMO
A 39-year-old woman with autosomal dominant polycystic kidney disease (ADPKD) presented with acromegaly and a pituitary macroadenoma. There was a family history of this renal disorder. She had undergone surgery for pituitary adenoma 6 years prior. Physical examination disclosed bitemporal hemianopsia and elevation of both basal growth hormone (GH) 106 ng/mL (normal 0-5) and insulin-like growth factor (IGF-1) 811 ng/mL (normal 48-255) blood levels. A magnetic resonance imaging scan disclosed a 3.0 cm sellar and suprasellar mass with both optic chiasm compression and left cavernous sinus invasion. Pathologic, cytogenetic, molecular and in silico analysis was undertaken. Histologic, immunohistochemical and ultrastructural studies of the lesion disclosed a sparsely granulated somatotroph adenoma. Standard chromosome analysis on the blood sample showed no abnormality. Sequence analysis of the coding regions of PKD1 and PKD2 employing DNA from both peripheral leukocytes and the tumor revealed the most common PKD1 mutation, 5014_5015delAG. Analysis of the entire SSTR5 gene disclosed the variant c.142C>A (p.L48M, rs4988483) in the heterozygous state in both blood and tumor, while no pathogenic mutations were noted in the MEN1, AIP, p27Kip1 and SSTR2 genes. To our knowledge, this is the fourth reported case of a GH-producing pituitary adenoma associated with ADPKD, but the first subjected to extensive morphological, ultrastructural, cytogenetic and molecular studies. The physical proximity of the PKD1 and SSTR5 genes on chromosome 16 suggests a causal relationship between ADPKD and somatotroph adenoma.
Assuntos
Acromegalia/complicações , Acromegalia/genética , Adenoma Hipofisário Secretor de Hormônio do Crescimento/complicações , Adenoma Hipofisário Secretor de Hormônio do Crescimento/genética , Neoplasias Hipofisárias/complicações , Rim Policístico Autossômico Dominante/complicações , Rim Policístico Autossômico Dominante/genética , Receptores de Somatostatina/genética , Canais de Cátion TRPP/genética , Acromegalia/patologia , Adenoma/complicações , Adenoma/genética , Adulto , Sequência de Bases , Feminino , Humanos , Hipófise/patologia , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/patologia , Polimorfismo GenéticoRESUMO
Uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, typically express filamentous adhesive organelles called type 1 pili that mediate both bacterial attachment to and invasion of bladder urothelial cells. Several host proteins have previously been identified as receptors for type 1 pili, but none have been conclusively shown to promote UPEC entry into host bladder cells. Using overlay assays with FimH, the purified type 1 pilus adhesin, and mass spectroscopy, we have identified beta1 and alpha3 integrins as key host receptors for UPEC. FimH recognizes N-linked oligosaccharides on these receptors, which are expressed throughout the urothelium. In a bladder cell culture system, beta1 and alpha3 integrin receptors co-localize with invading type 1-piliated bacteria and F-actin. FimH-mediated bacterial invasion of host bladder cells is inhibited by beta1 and alpha3 integrin-specific antibodies and by disruption of the beta1 integrin gene in the GD25 fibroblast cell line. Phosphorylation site mutations within the cytoplasmic tail of beta1 integrin that alter integrin signaling also variably affect UPEC entry into host cells, by either attenuating or boosting invasion frequencies. Furthermore, focal adhesion and Src family kinases, which propagate integrin-linked signaling and downstream cytoskeletal rearrangements, are shown to be required for FimH-dependent bacterial invasion of target host cells. Cumulatively, these results indicate that beta1 and alpha3 integrins are functionally important receptors for type 1 pili-expressing bacteria within the urinary tract and possibly at other sites within the host.
Assuntos
Adesinas de Escherichia coli , Escherichia coli/patogenicidade , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas , Interações Hospedeiro-Patógeno , Integrina alfa3/metabolismo , Integrina beta1/metabolismo , Actinas/metabolismo , Sequência de Bases , Linhagem Celular , Escherichia coli/fisiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Fímbrias/imunologia , Humanos , Integrina alfa3/imunologia , Integrina beta1/imunologia , Espectrometria de Massas , Dados de Sequência Molecular , Mutação , Fosforilação , Bexiga Urinária/citologia , Bexiga Urinária/microbiologia , Infecções Urinárias/microbiologia , Urotélio/metabolismo , Urotélio/microbiologiaRESUMO
The DNA sequences of the actin genes of several fungi were compared and highly conserved regions in the coding sequence were identified. Deoxyoligonucleotide primers were synthesized based on conserved sequence blocks in the 5' and 3' ends of the open reading frame encoding the actin protein. In addition, primers (internal transcribed spacer [ITS] regions 1 and 4) based on conserved regions of the ribosomal RNA (rRNA) genes of fungi were synthesized. Use of the primers in the polymerase chain reaction (PCR) resulted in the amplification of DNA products from the genomes of sugar beet fungal pathogens of a size consistent with the amplification of the actin gene and rRNA gene sequences, respectively, in these fungi. With one primer pair (5FWDACT and MIDREVACT) directed to the actin gene, the major products amplified from the DNA of Aphanomyces cochlioides, Pythium ultimum, Cercospora beticola, Phoma betae, Fusarium oxysporum, and Rhizoctonia solani were of the sizes of 0.9, 0.9, 1.1, 1.1, 1.2 and 1.7 kilobase pairs (kbp), respectively, whereas no product was generated from the DNA of sugar beet (Beta vulgaris L.). Restriction endonuclease digestion of products amplified using 5FWDACT and MIDREVACT permitted the differentiation of A. cochlioides from A. euteiches. Use of ITS1 and ITS4 in PCR reactions employing the same template DNAs and reaction conditions yielded single products of 0.7, 0.8, 0.5, 0.5, 0.6, and 0.7 kbp, respectively, as well as a 0.7-kbp product from DNA of sugar beet. The data indicate that actin and rRNA gene sequences are appropriate targets for the development of PCR-based strategies for distinguishing sugar beet fungal pathogens at the genus level. The presence of A. cochlioides DNA in extracts of diseased sugar beet seedlings was detected using PCR with primers 5FWDACT and MIDRE-VACT.
RESUMO
BACKGROUND AND OBJECTIVES: Autosomal dominant polycystic kidney disease (ADPKD) patients have an increased risk for intracranial aneurysms (IAs). The importance of screening for unruptured IAs (UIAs) depends on their risks for growth and rupture. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: ADPKD patients with UIAs found by presymptomatic screening with magnetic resonance angiography (MRA) during 1989 to 2009 were followed initially at 6 months and annually, and less frequently after demonstration of stability. RESULTS: Forty-five saccular aneurysms were detected in 38 patients from 36 families. Most were small (median diameter 3.5 mm) and in the anterior circulation (84%). Median age at diagnosis was 49 years. During cumulative imaging follow-up of 243 years, one de novo UIA was detected and increased in size from 2 to 4.4 mm over 144 months and two UIAs grew from 4.5 to 5.9 mm and 4.7 to 6.2 mm after 69 and 184 months, respectively. Seven patients did not have imaging follow-up. No change was detected in the remaining 28 patients. During cumulative clinical follow-up of 316 years, no aneurysm ruptured. Five patients died from unrelated causes and two were lost to follow-up after 8 and 120 months. Three patients underwent surgical clipping. CONCLUSIONS: Most UIAs detected by presymptomatic screening in ADPKD patients are small and in the anterior circulation. Growth and rupture risks are not higher than those of UIAs in the general population. These data support very selective screening for UIAs in ADPKD patients, and widespread screening is not indicated.
Assuntos
Angiografia Cerebral/métodos , Aneurisma Intracraniano/diagnóstico , Angiografia por Ressonância Magnética , Rim Policístico Autossômico Dominante/complicações , Adulto , Aneurisma Roto/diagnóstico , Aneurisma Roto/etiologia , Aneurisma Roto/prevenção & controle , Doenças Assintomáticas , Análise Mutacional de DNA , Progressão da Doença , Feminino , Seguimentos , Humanos , Aneurisma Intracraniano/etiologia , Aneurisma Intracraniano/cirurgia , Masculino , Pessoa de Meia-Idade , Minnesota , Mutação , Rim Policístico Autossômico Dominante/genética , Valor Preditivo dos Testes , Medição de Risco , Fatores de Risco , Fatores de Tempo , Adulto JovemRESUMO
INTRODUCTION: End-stage renal disease (ESRD) is a major public health problem. Although kidney transplantation is a viable therapeutic option, this therapy is associated with significant limitations, including a shortage of donor organs. Induced pluripotent stem (iPS) cell technology, which allows derivation of patient-specific pluripotent stem cells, could provide a possible alternative modality for kidney replacement therapy for patients with ESRD. METHODS: The feasibility of iPS cell generation from patients with a history of ESRD was investigated using lentiviral vectors expressing pluripotency-associated factors. RESULTS: In the present article we report, for the first time, generation of iPS cells from kidney transplant recipients with a history of autosomal-dominant polycystic kidney disease (ADPKD), systemic lupus erythematosus, or Wilms tumor and ESRD. Lentiviral transduction of OCT4, SOX2, KLF4 and c-MYC, under feeder-free conditions, resulted in reprogramming of skin-derived keratinocytes. Keratinocyte-derived iPS cells exhibited properties of human embryonic stem cells, including morphology, growth properties, expression of pluripotency genes and surface markers, spontaneous differentiation and teratoma formation. All iPS cell clones from the ADPKD patient retained the conserved W3842X mutation in exon 41 of the PKD1 gene. CONCLUSIONS: Our results demonstrate successful iPS cell generation from patients with a history of ESRD, PKD1 gene mutation, or chronic immunosuppression. iPS cells from autosomal kidney diseases, such as ADPKD, would provide unique opportunities to study patient-specific disease pathogenesis in vitro.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Transplante de Rim , Diferenciação Celular , Reprogramação Celular , Fibroblastos/citologia , Vetores Genéticos/metabolismo , Humanos , Queratinócitos/citologia , Falência Renal Crônica/metabolismo , Falência Renal Crônica/terapia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Lentivirus/genética , Mutação , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismoRESUMO
Strains of uropathogenic Escherichia coli (UPEC) can invade terminally differentiated superficial bladder epithelial cells and subsequently multiply, forming large biofilm-like inclusions referred to as pods. In contrast, within immature bladder cells UPEC enter a more quiescent state and often fail to replicate appreciably. As immature bladder epithelial cells undergo terminal differentiation the actin cytoskeleton is radically diminished, a phenomenon that we reasoned could influence the intracellular fate of UPEC. Here we show that UPEC within undifferentiated bladder cells is trafficked into acidic compartments having key features of late endosomes and lysosomes. These UPEC-containing vacuoles are often enmeshed within a network of actin filaments, the disruption of which stimulates intravacuolar growth and efflux of UPEC in cell culture-based studies. In this in vitro model system, release of UPEC into the host cytosol further stimulates intracellular bacterial growth and the rapid development of pod-like inclusions. These inclusions, as well as those observed using an in vivo mouse model, develop in association with cytokeratin intermediate filaments that may act as scaffolding for intracellular biofilm formation. Our data suggest an aetiological basis for recurrent urinary tract infections, linking bladder cell differentiation and the accompanying redistribution of actin microfilaments with the resurgence of UPEC from quiescent intravacuolar reservoirs within the bladder epithelium.