RESUMEN
Trimethylamine-N-oxide (TMAO) is a uremic toxin, which has been associated with chronic kidney disease (CKD). Renal tubular epithelial cells play a central role in the pathophysiology of CKD. Megalin is an albumin-binding surface receptor on tubular epithelial cells, which is indispensable for urine protein reabsorption. To date, no studies have investigated the effect of TMAO on megalin expression and the functional properties of human tubular epithelial cells. The aim of this study was first to identify the functional effect of TMAO on human renal proximal tubular cells and second, to unravel the effects of TMAO on megalin-cubilin receptor expression. We found through global gene expression analysis that TMAO was associated with kidney disease. The microarray analysis also showed that megalin expression was suppressed by TMAO, which was also validated at the gene and protein level. High glucose and TMAO was shown to downregulate megalin expression and albumin uptake similarly. We also found that TMAO suppressed megalin expression via PI3K and ERK signaling. Furthermore, we showed that candesartan, dapagliflozin and enalaprilat counteracted the suppressive effect of TMAO on megalin expression. Our results may further help us unravel the role of TMAO in CKD development and to identify new therapeutic targets to counteract TMAOs effects.
Asunto(s)
Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad , Insuficiencia Renal Crónica , Albúminas/metabolismo , Endocitosis , Células Epiteliales/metabolismo , Humanos , Túbulos Renales Proximales/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Sistema de Señalización de MAP Quinasas , Metilaminas , Fosfatidilinositol 3-Quinasas/metabolismo , Insuficiencia Renal Crónica/metabolismoRESUMEN
Trimethylamine N-oxide (TMAO), a product of gut microbiota metabolism, has previously been shown to be implicated in chronic kidney disease. A high TMAO-containing diet has been found to cause tubulointerstitial renal fibrosis in mice. However, today there are no data linking specific molecular pathways with the effect of TMAO on human renal fibrosis. The aim of this study was to investigate the fibrotic effects of TMAO on renal fibroblasts and to elucidate the molecular pathways involved. We found that TMAO promoted renal fibroblast activation and fibroblast proliferation via the PERK/Akt/mTOR pathway, NLRP3, and caspase-1 signaling. We also found that TMAO increased the total collagen production from renal fibroblasts via the PERK/Akt/mTOR pathway. However, TMAO did not induce fibronectin or TGF-ß1 release from renal fibroblasts. We have unraveled that the PERK/Akt/mTOR pathway, NLRP3, and caspase-1 mediates TMAO's fibrotic effect on human renal fibroblasts. Our results can pave the way for future research to further clarify the molecular mechanism behind TMAO's effects and to identify novel therapeutic targets in the context of chronic kidney disease.
Asunto(s)
Caspasa 1/metabolismo , Riñón/patología , Metilaminas/toxicidad , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Transducción de Señal/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Colágeno/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibrosis , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Riñón/citología , Riñón/efectos de los fármacos , Riñón/metabolismo , Modelos Biológicos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , eIF-2 Quinasa/metabolismoRESUMEN
The NLRP3 inflammasome, IL-1ß release and pyroptosis (cell lysis) have recently been proposed to be essential for the progression of urinary tract infection (UTI) and elimination of intracellular bacterial niches. However, the effects of IL-1R antagonist (IL-1RA) on immune responses during UTI, except for its ability to disrupt IL-1ß signalling, are not well understood. The aim of this study was to investigate the role of IL-1RA in UPEC colonization of bladder epithelial cells and the subsequent host inflammatory response. Human bladder epithelial cells (5637) and CRISPR/Cas9 generated NLRP3 and caspase-1 knockdown cells and IL-1RA knockout cells were stimulated with the UPEC isolate CFT073. The results showed that the UPEC virulence factor α-hemolysin is essential for IL-1RA release, and that the inflammasome-associated proteins caspase-1 and NLRP3 affect the release of IL-1RA. IL-1RA deficient cells showed a reduced adherence and invasion by CFT073 compared to wild-type cells, suggesting that IL-1RA may oppose mechanisms that protects against bacterial colonization. A targeted protein analysis of inflammation-related proteins showed that the basal expression of 23 proteins and the UPEC-induced expression of 10 proteins were significantly altered in IL-1RA deficient bladder epithelial cells compared to Cas9 control cells. This suggests that IL-1RA has a broad effect on the inflammatory response in bladder epithelial cells.
Asunto(s)
Células Epiteliales/inmunología , Infecciones por Escherichia coli/inmunología , Inflamasomas/inmunología , Proteína Antagonista del Receptor de Interleucina 1/inmunología , Vejiga Urinaria/inmunología , Infecciones Urinarias/inmunología , Escherichia coli Uropatógena/inmunología , Línea Celular , Células Epiteliales/microbiología , Infecciones por Escherichia coli/patología , Humanos , Vejiga Urinaria/microbiología , Vejiga Urinaria/patología , Infecciones Urinarias/microbiología , Infecciones Urinarias/patologíaRESUMEN
BACKGROUND: Increased resistance to antimicrobial agents is a characteristic of many bacteria growing in biofilms on for example indwelling urinary catheters or in intracellular bacterial reservoirs. Biofilm-related infections caused by multidrug-resistant bacteria, such as extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae, are a major challenge. The aim of this study was to investigate if a carbon monoxide-releasing molecule (CORM-2) has antibacterial effects against ESBL-producing uropathogenic E. coli (UPEC) in the biofilm mode of growth and following colonization of host bladder epithelial cells. RESULTS: The effect of CORM-2 was examined on bacteria grown within an established biofilm (biofilm formed for 24 h on plastic surface) by a live/dead viability staining assay. CORM-2 (500 µM) exposure for 24 h killed approximately 60 % of the ESBL-producing UPEC isolate. A non-ESBL-producing UPEC isolate and the E. coli K-12 strain TG1 were also sensitive to CORM-2 exposure when grown in biofilms. The antibacterial effect of CORM-2 on planktonic bacteria was reduced and delayed in the stationary growth phase compared to the exponential growth phase. In human bladder epithelial cell colonization experiments, CORM-2 exposure for 4 h significantly reduced the bacterial counts of an ESBL-producing UPEC isolate. CONCLUSION: This study shows that CORM-2 has antibacterial properties against multidrug-resistant UPEC under biofilm-like conditions and following host cell colonization, which motivate further studies of its therapeutic potential.
Asunto(s)
Biopelículas/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Infecciones por Escherichia coli/microbiología , Compuestos Organometálicos/farmacología , Escherichia coli Uropatógena/fisiología , Carga Bacteriana/efectos de los fármacos , Línea Celular , Células Epiteliales/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Vejiga Urinaria/citología , Escherichia coli Uropatógena/efectos de los fármacos , Escherichia coli Uropatógena/aislamiento & purificaciónRESUMEN
Inadequate and delayed antibiotic treatment of extended spectrum beta-lactamase (ESBL)-producing isolates have been associated with increased mortality of affected patients. The purpose of this study was to compare the host response of human renal epithelial cells and polymorphonuclear leucocyte (PMN) cells when infected by ESBL-producing uropathogenic Escherichia coli (UPEC) isolates in the presence or absence of ineffective antibiotics. The renal epithelial cell line A498 and PMN cells were stimulated with ESBL-producing UPEC isolates in the presence or absence of three different antibiotics (trimetoprim, ceftibuten and ciprofloxacin). Host cell responses were evaluated as release of cytokines (IL-6, IL-8), reactive oxygen species (ROS), ATP and endotoxins. Bacterial morphology and PMN phagocytosis were evaluated by microscopy. In the presence of ceftibuten, 2 out of 3 examined ESBL-isolates changed their morphology into a filamentous form. The presence of ceftibuten enhanced IL-6, IL-8 and ROS-production from host cells, but only from cells stimulated by the filamentous isolates. The bacterial supernatant and not the filamentous bacteria per se was responsible for the increased release of IL-6, IL-8 and ROS. Increased endotoxin and ATP levels were found in the bacterial supernatants from filamentous isolates. Apyrase decreased IL-6 secretion from A498 cells and polymyxin B abolished the increased ROS-production from PMN cells. PMN were able to inhibit the bacterial growth of some ESBL-isolates in the presence of ceftibuten. In conclusion, antibiotic-induced filamentation of ESBL-producing UPEC isolates and the associated release of ATP and endotoxins can alter the host cell response in the urinary tract.
Asunto(s)
Antibacterianos/farmacología , Cefalosporinas/farmacología , Infecciones por Escherichia coli/metabolismo , Escherichia coli Uropatógena/efectos de los fármacos , Escherichia coli Uropatógena/crecimiento & desarrollo , beta-Lactamasas/metabolismo , Adenosina Trifosfato/metabolismo , Ceftibuteno , Línea Celular , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Escherichia coli Uropatógena/enzimología , Escherichia coli Uropatógena/genética , beta-Lactamasas/genéticaRESUMEN
BACKGROUND: Nitric oxide (NO) is produced as part of the host immune response to bacterial infections, including urinary tract infections. The enzyme flavohemoglobin, coded by the hmp gene, is involved in protecting bacterial cells from the toxic effects of NO and represents a potentially interesting target for development of novel treatment concepts against resistant uropathogenic bacteria. The aim of the present study was to investigate if the in vitro antibacterial effects of NO can be enhanced by pharmacological modulation of the enzyme flavohemoglobin. RESULTS: Four clinical isolates of multidrug-resistant extended-spectrum ß-lactamase (ESBL)-producing uropathogenic E. coli were included in the study. It was shown that the NO-donor substance DETA/NO, but not inactivated DETA/NO, caused an initial growth inhibition with regrowth noted after 8 h of exposure. An hmp-deficient strain showed a prolonged growth inhibition in response to DETA/NO compared to the wild type. The imidazole antibiotic miconazole, that has been shown to inhibit bacterial flavohemoglobin activity, prolonged the DETA/NO-evoked growth inhibition. When miconazole was combined with polymyxin B nonapeptide (PMBN), in order to increase the bacterial wall permeability, DETA/NO caused a prolonged bacteriostatic response that lasted for up to 24 h. CONCLUSION: An NO-donor in combination with miconazole and PMBN showed enhanced antimicrobial effects and proved effective against multidrug-resistant ESBL-producing uropathogenic E. coli.
Asunto(s)
Antibacterianos/farmacología , Dihidropteridina Reductasa/metabolismo , Proteínas de Escherichia coli/metabolismo , Hemoproteínas/metabolismo , Miconazol/farmacología , NADH NADPH Oxidorreductasas/metabolismo , Óxido Nítrico/farmacología , Polimixina B/análogos & derivados , Escherichia coli Uropatógena/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Pruebas de Sensibilidad Microbiana , Nanopartículas/metabolismo , Polimixina B/farmacología , Escherichia coli Uropatógena/enzimología , Escherichia coli Uropatógena/crecimiento & desarrollo , beta-Lactamasas/metabolismoRESUMEN
Carbon monoxide (CO) releasing molecules (CO-RMs) have been shown to inhibit growth of commensal Escherichia coli (E. coli). In the present study we examined the effect of CORM-2 on uropathogenic E. coli (UPEC) that produces extended-spectrum ß-lactamase (ESBL). Viability experiments showed that CORM-2 inhibited the growth of several different ESBL-producing UPEC isolates and that 500 µM CORM-2 had a bactericidal effect within 4 h. The bactericidal effect of CORM-2 was significantly more pronounced than the effect of the antibiotic nitrofurantoin. CORM-2 demonstrated a low level of cytotoxicity in eukaryotic cells (human bladder epithelial cell line 5637) at the concentrations and time-points where the antibacterial effect was obtained. Real-time RT-PCR studies of different virulence genes showed that the expression of capsule group II kpsMT II and serum resistance traT was reduced and that some genes encoding iron acquisition systems were altered by CORM-2. Our results demonstrate that CORM-2 has a fast bactericidal effect against multiresistant ESBL-producing UPEC isolates, and also identify some putative UPEC virulence factors as targets for CORM-2. CO-RMs may be candidate drugs for further studies in the field of finding new therapeutic approaches for treatment of uropathogenic ESBLproducing E. coli.
Asunto(s)
Antibacterianos/farmacología , Compuestos Organometálicos/farmacología , Escherichia coli Uropatógena/efectos de los fármacos , beta-Lactamasas/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Adulto , Proteínas de la Membrana Bacteriana Externa/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo/química , ADN Bacteriano/genética , Farmacorresistencia Bacteriana Múltiple , Células Epiteliales/efectos de los fármacos , Proteínas de Escherichia coli/metabolismo , Femenino , Regulación Bacteriana de la Expresión Génica , Humanos , Proteínas de Transporte de Membrana/metabolismo , Viabilidad Microbiana/efectos de los fármacos , Persona de Mediana Edad , Nitrofurantoína/farmacología , Vejiga Urinaria/citología , Orina/microbiología , Escherichia coli Uropatógena/enzimología , Factores de Virulencia/metabolismoRESUMEN
The recent recognition of receptor-mediated ATP signalling as a pathway of epithelial pro-inflammatory cytokine release challenges the ubiquitous role of the TLR4 pathway during urinary tract infection. The aim of this study was to compare cellular responses of renal epithelial cells infected with uropathogenic Escherichia coli (UPEC) strain IA2 to stimulation with ATP-γ-S. A498 cells were infected or stimulated in the presence or absence of apyrase, that degrades extracellular ATP, or after siRNA-mediated knockdown of ATP-responding P2Y2 receptors. Cellular IL-8 release and global gene expression were analysed. Both IA2 and A498 cells per se released ATP, which increased during infection. IA2 and ATP-γ-S caused a â¼5-fold increase in cellular release of IL-8 and stimulations performed in the presence of apyrase or after siRNA knockdown of P2Y2 receptors resulted in attenuation of IA2-mediated IL-8 release. Microarray results show that both IA2 and ATP-γ-S induced marked changes in gene expression of renal cells. Thirty-six genes were in common between both stimuli, and many of these are key genes belonging to classical response pathways of bacterial infection. Functional analysis shows that 88 biological function-annotated cellular pathways were identical between IA2 and ATP-γ-S stimuli. Results show that UPEC-induced release of IL-8 is dependent on P2Y2 signalling and that cellular responses elicited by UPEC and ATP-γ-S have many identical features. This indicates that renal epithelial responses elicited by bacteria could be mediated by bacteria- or host-derived ATP, thus defining a key role of ATP during infection.
Asunto(s)
Adenosina Trifosfato/fisiología , Células Epiteliales/fisiología , Redes Reguladoras de Genes/fisiología , Interleucina-8/biosíntesis , Escherichia coli Uropatógena/fisiología , Línea Celular , Células Epiteliales/microbiología , Regulación de la Expresión Génica , Humanos , Interleucina-8/genética , Riñón/citología , Riñón/microbiología , Riñón/fisiología , Receptores Purinérgicos P2Y2/deficienciaRESUMEN
BACKGROUND: Infections caused by extended spectrum beta-lactamases (ESBL)-producing bacteria have been emerging worldwide and the majority of ESBL-producing E. coli strains are isolated from patients with urinary tracts infections. The purpose of this study was to compare the host-response mechanisms in human polymorphonucleated leukocytes (PMN) and renal epithelial cells when stimulated by ESBL- or non-ESBL-producing uropathogenic E. coli (UPEC) isolates. The host-pathogen interaction of these ESBL-producing strains in the urinary tract is not well studied. RESULTS: The ability of ESBL strains to evoke ROS-production from PMN cells was significantly higher than that of the non-ESBL strains. The growth of ESBL strains was slightly suppressed in the presence of PMN compared to non-ESBL strains after 30 min and 2h, but the opposite was observed after 5 and 6h. The number of migrating PMN was significantly higher in response to ESBL strains compared to non-ESBL strains. Stimulation of A498 cells with ESBL strains elicited lower production of IL-6 and IL-8 compared to non-ESBL strains. CONCLUSION: Significant differences in host-response mechanisms were identified when host cells were stimulated by ESBL- or non-ESBL producing strains. The obtained results on the early interactions of ESBL-producing strains with the host immune system may provide valuable information for management of these infections.
Asunto(s)
Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Infecciones Urinarias/inmunología , Infecciones Urinarias/microbiología , Escherichia coli Uropatógena/enzimología , beta-Lactamasas/metabolismo , Células Cultivadas , Interacciones Huésped-Patógeno , Humanos , Neutrófilos/inmunología , Neutrófilos/microbiología , Especies Reactivas de Oxígeno/inmunología , Escherichia coli Uropatógena/clasificación , Escherichia coli Uropatógena/inmunología , Escherichia coli Uropatógena/aislamiento & purificación , beta-Lactamasas/genéticaRESUMEN
The NLRP3 inflammasome, estrogen and antimicrobial peptides have all been found to have a vital role in the protection of the bladder urothelium. However, the interdependence between these protective factors during a bladder infection is currently unknown. Our aim was to investigate the role of NLRP3 in the regulation of antimicrobial peptides and estrogen signaling in bladder epithelial cells during a UPEC infection. Human bladder epithelial cells and CRISPR/Cas9-generated NLRP3-deficient cells were stimulated with the UPEC strain CFT073 and estradiol. The gene and protein expression were evaluated with microarray, qRT-PCR, western blot and ELISA. Microarray results showed that the expression of most antimicrobial peptides was reduced in CFT073-infected NLRP3-deficient cells compared to Cas9 control cells. Conditioned medium from NLRP3-deficient cells also lost the ability to suppress CFT073 growth. Moreover, NLRP3-deficient cells had lower basal release of Beta-defensin-1, Beta-defensin-2 and RNase7. The ability of estradiol to induce an increased expression of antimicrobial peptides was also abrogated in NLRP3-deficient cells. The decreased antimicrobial peptide expression might be linked to the observed reduced expression and activity of estradiol receptor beta in NLRP3-deficient cells. This study suggests that NLRP3 may regulate the release and expression of antimicrobial peptides and affect estrogen signaling in bladder epithelial cells.
RESUMEN
PURPOSE: We characterized and identified the uroepithelial P2 receptor responsible for adenosine triphosphate mediated release of the cytokines interleukin-8 and 6. MATERIALS AND METHODS: The human renal epithelial cell line A498 (ATCC™) was cultured and stimulated with different purinergic agonists with or without prior inhibition with different antagonists or signaling pathway inhibitors. Supernatant was analyzed for interleukin-8 and 6 by enzyme-linked immunosorbent assay. P2 receptor mRNA expression was assessed by real-time reverse transcriptase-polymerase chain reaction. The candidate receptor was knocked down with siRNA technology. Interleukin-8 and 6 responses were measured after purinergic stimulation of knocked down cells. RESULTS: ATP and ATP-γ-S (Roche Diagnostics, Mannheim, Germany) were equipotent as inducers of interleukin-8 and 6 release. Agonist profile experiments using different P2 receptor agonists indicated that P2Y(2) was the main contributor to this release, although P2Y(11) and P2X(7) activation could not be excluded. Signaling pathway experiments showed that interleukin-8 release involved phospholipase C and inositol trisphosphate mediated signaling, indicating a P2Y receptor subtype. Antagonist experiments indicated P2Y(2) as the responsible receptor. Gene expression analysis of P2 receptors showed that strong expression of P2Y(2) receptor and subsequent knockdown of P2Y(2) receptor mRNA for 72 and 96 hours abrogated interleukin-8 and 6 release after purinergic stimulation with adenosine triphosphate-γ-S. CONCLUSIONS: Interleukin-8 and 6 release after purinergic stimulation in uroepithelial A498 cells is mediated through P2Y(2) receptor activation.
Asunto(s)
Adenosina Trifosfato/farmacología , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Receptores Purinérgicos P2Y2/efectos de los fármacos , Urotelio/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Citocinas/efectos de los fármacos , Citocinas/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , ARN Interferente Pequeño/metabolismo , Receptores Purinérgicos P2Y2/genética , Receptores Purinérgicos P2Y2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Transducción de Señal , Infecciones Urinarias/fisiopatología , Urotelio/citología , Urotelio/metabolismoRESUMEN
BACKGROUND/AIMS: Increased nitric oxide (NO) production or inducible form of NO synthase activity have been documented in patients suffering from urinary tract infection (UTI), but the role of NO in this infection is unclear. We investigated whether NO can affect the host response in human renal epithelial cells by modulating IL-6 production and mRNA expression. METHODS: The human renal epithelial cell line A498 was infected with a uropathogenic Escherichia coli (UPEC) strain and/or the NO donor DETA/NO. The IL-6 production and mRNA expression were evaluated by ELISA and real-time RT-PCR. IL-6 mRNA stability was evaluated by analyzing mRNA degradation by real-time RT-PCR. RESULTS: DETA/NO caused a significant (p < 0.05) increase in IL-6 production. Inhibitors of p38 MAPK and ERK1/2 signaling, but not JNK, were shown to significantly suppress DETA/NO-induced IL-6 production. UPEC-induced IL-6 production was further increased (by 73 ± 23%, p < 0.05) in the presence of DETA/NO. The IL-6 mRNA expression increased 2.1 ± 0.17-fold in response to DETA/NO, while the UPEC-evoked increase was pronounced (20 ± 4.5-fold). A synergistic effect of DETA/NO on UPEC-induced IL-6 expression was found (33 ± 7.2-fold increase). The IL-6 mRNA stability studies showed that DETA/NO partially attenuated UPEC-induced degradation of IL-6 mRNA. CONCLUSIONS: NO was found to stimulate IL-6 in renal epithelial cells through p38 MAPK and ERK1/2 signaling pathways and also to increase IL-6 mRNA stability in UPEC-infected cells. This study proposes a new role for NO in the host response during UTI by modulating the transcription and production of the cytokine IL-6.
Asunto(s)
Células Epiteliales/metabolismo , Expresión Génica , Interleucina-1/biosíntesis , Interleucina-1/genética , Óxido Nítrico/farmacología , Antracenos/farmacología , Células Epiteliales/microbiología , Infecciones por Escherichia coli , Flavonoides/farmacología , Humanos , Imidazoles/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Riñón , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The inflammasome-associated proteins caspase-1, caspase-4 and NLRP3 have been emphasised to be essential in the host cell response during urinary tract infection (UTI) by regulating IL-1ß release. Our aim was to investigate how the inflammasome-associated proteins regulate the cell response of bladder epithelial cells during infection with uropathogenic Escherichia coli (UPEC). Human bladder epithelial cells (5637) and CRISPR/Cas9 generated caspase-1, caspase-4 and NLRP3 knockdown cells were stimulated with the UPEC strain CFT073. Using Olink proteomics and real time RT-PCR, we showed that caspase-1, caspase-4 and NLRP3 are vital for the expression of many inflammatory genes and proteins from bladder epithelial cells. When investigating the effect of inflammasome-associated proteins on neutrophils, we found that conditioned medium from UPEC-infected caspase-4 knockdown cells significantly increased phagocytosis of CFT073 and significantly decreased ROS production from neutrophils. In contrast, conditioned medium from UPEC-infected NLRP3 knockdown cells significantly decreased the phagocytosis of CFT073 and significantly increased the ROS production from neutrophils. In conclusion, we showed that the inflammasome-associated proteins contribute to the host cell response during UPEC infection.
Asunto(s)
Caspasa 1/fisiología , Caspasas Iniciadoras/fisiología , Células Epiteliales/inmunología , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/fisiología , Infecciones Urinarias/genética , Infecciones Urinarias/inmunología , Escherichia coli Uropatógena/inmunología , Caspasas Iniciadoras/metabolismo , Línea Celular , Células Epiteliales/metabolismo , Humanos , Inflamasomas/genética , Inflamasomas/metabolismo , Inflamación , Interleucina-1beta/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Neutrófilos/metabolismo , Fagocitosis , Especies Reactivas de Oxígeno/metabolismo , Vejiga Urinaria/citologíaRESUMEN
Uropathogenic Escherichia coli (UPEC) may undergo a cyclic cascade of morphological alterations that are believed to enhance the potential of UPEC to evade host responses and re-infect host cell. However, knowledge on the pathogenic potential and host activation properties of UPEC during the morphological switch is limited. Microarray analysis was performed on mRNA isolated from human bladder epithelial cells (HBEP) after exposure to three different morphological states of UPEC (normal coliform, filamentous form and reverted form). Cells stimulated with filamentous bacteria showed the lowest number of significant gene alterations, although the number of enriched gene ontology classes was high suggesting diverse effects on many different classes of host genes. The normal coliform was in general superior in stimulating transcriptional activity in HBEP cells compared to the filamentous and reverted form. Top-scored gene entities activated by all three morphological states included IL17C, TNFAIP6, TNF, IL20, CXCL2, CXCL3, IL6 and CXCL8. The number of significantly changed canonical pathways was lower in HBEP cells stimulated with the reverted form (32 pathways), than in cells stimulated with the coliform (83 pathways) or filamentous bacteria (138 pathways). A host cell invasion assay showed that filamentous bacteria were unable to invade bladder cells, and that the number of intracellular bacteria was markedly lower in cells infected with the reverted form compared to the coliform. In conclusion, the morphological state of UPEC has major impact on the host bladder response both when evaluating the number and the identity of altered host genes and pathways.
Asunto(s)
Células Epiteliales/metabolismo , Infecciones por Escherichia coli/genética , Transcripción Genética , Vejiga Urinaria/microbiología , Escherichia coli Uropatógena/fisiología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Quimiocina CXCL2/genética , Quimiocina CXCL2/metabolismo , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Células Epiteliales/microbiología , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Vejiga Urinaria/metabolismo , Escherichia coli Uropatógena/crecimiento & desarrolloRESUMEN
Adenosine has been identified as a significant inhibitor of inflammation by acting on adenosine A(2A) receptors. In this study, we examined the role of adenosine and A(2A) receptors in the transmigration of human neutrophils across an in vitro model of the transitional bladder urothelium. Human uroepithelial cells (UROtsa) were grown on transwell inserts; uropathogenic Escherichia coli (UPEC) and neutrophils were added to the transwell system; and the number of migrating neutrophils was evaluated. Reverse transcription-PCR (RT-PCR), immunohistochemistry, and flow cytometry were used to investigate the expression of adenosine receptors, the epithelial adhesion molecule ICAM-1, and the neutrophil integrin CD11b. Levels of proinflammatory interleukin-8 (IL-8) and phosphorylated IκBα were measured by enzyme-linked immunosorbent assays (ELISA) and Luminex assays, respectively. The neutrophils expressed all four adenosine receptor subtypes (A(1), A(2A), A(2B), and A(3) receptors), but A(3) receptors were not expressed by UROtsa cells. UPEC stimulated neutrophil transuroepithelial migration, which was significantly decreased in response to the specific A(2A) receptor agonist CGS 21680. The inhibitory effect of CGS 21680 on neutrophil migration was reversed by the A(2A) receptor antagonist SCH 58261. The production of chemotactic IL-8 and the expression of the adhesion molecule ICAM-1 or CD11b were not significantly affected by CGS 21680. However, a significant decrease in the level of phosporylated IκBα was revealed in response to CGS 21680. In conclusion, UPEC infection in vitro evoked neutrophil migration through a multilayered human uroepithelium. The UPEC-evoked neutrophil transmigration decreased in response to A(2A) receptor activation, possibly through inhibition of NF-κB signaling pathways.
Asunto(s)
Movimiento Celular , Interacciones Huésped-Patógeno , Neutrófilos/fisiología , Receptor de Adenosina A2A/metabolismo , Urotelio/inmunología , Antígeno CD11b/análisis , Ensayos de Migración de Leucocitos , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/patogenicidad , Citometría de Flujo , Humanos , Proteínas I-kappa B/análisis , Inmunohistoquímica , Molécula 1 de Adhesión Intercelular/análisis , Interleucina-8/análisis , Inhibidor NF-kappaB alfa , Neutrófilos/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Bombesin (BOM) and gastrin releasing peptide (GRP) have been located to the lower urinary tract. However, there is a paucity of data demonstrating the impact of these neuropeptides. OBJECTIVES: The present study investigates the impact of BOM and GRP in the female Sprague-Dawley rats 225 g b.w. n = 37 urethras in vitro and in vivo. Intraurethral pressure was recorded by a catheter placed at the maximum pressure zone corresponding to the intrinsic urethral spincter. MEASUREMENTS: In vitro, the intraurethral pressure was measured in response to the administration of BOM and GRP and noradrenaline from perfused intact urethral/bladder preparations. In vivo, changes in intraurethral pressure were conducted in anesthetized subjects and compared with the basal intraurethral pressure and sham controls. RESULTS: In vitro, the increase in intraurethral pressure induced by BOM was 23.6 ± 3.2 cmH(2) O, exceeding the pressure evoked with NA by 10.7 cmH(2) O whereas GRP induced 10.7 ± 1.6 cmH(2) O, an increase of 3.3 cmH(2) O but less than the NA evoked intraurethral pressure by 2.2 cmH(2) O. Incubation with scopolamine (1 µM), phentolamine (1 µM), pancuronium (1 µM), and indomethacin (1 µM) did not produce any significant difference in the contractile responses to BOM or GRP. In vivo, the mean basal pressure was 22.9 ± 1.4 cmH(2) O. The intraurethral pressure evoked by BOM was 29.7 cmH(2) O (21.3 ± 1.3 to 51.0 ± 1.6 cmH(2) O), and for GRP, the evoked intraurethral pressure was 33.8 cmH(2) O (22.3 ± 1.9 to 56.2 ± 30 cmH(2) O). CONCLUSIONS: BOM and GRP may contribute to the control of continence by their contractile action on the sphincters of the lower urinary tract outflow region.
Asunto(s)
Bombesina/administración & dosificación , Péptido Liberador de Gastrina/administración & dosificación , Contracción Muscular/efectos de los fármacos , Neurotransmisores/administración & dosificación , Uretra/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Femenino , Presión , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
AIM: To investigate risk factors and consequences of nosocomial urinary tract infection in hip fracture patients. BACKGROUND: Nosocomial urinary tract infection is a well-known problem in hip fracture patients. There are several risk factors for nosocomial urinary tract infection described in the literature. DESIGN: Prospective observational study with a descriptive and comparative design. METHODS: Hip fracture patients were included consecutively between April 2006-March 2007. Excluded were those under 50, having an indwelling urinary catheter, signs of cognitive impairment or additional severe physical problems at the time of admission. To verify nosocomial urinary tract infection, a urine specimen was taken at admission and discharge. Patients with and without nosocomial urinary tract infection were compared. RESULTS: The study included 86 hip fracture patients, of whom 45 (52·3%) contracted nosocomial urinary tract infection in hospital. Earlier reported risk factors for nosocomial urinary tract infection were not confirmed in this study, with one exception: diabetes. All diabetic patients in the study contracted urinary tract infections. Patients receiving cloxacillin as antibiotic prophylaxis for wound infection contracted UTI less often than other patients. There were no statistical differences between groups with regard to urinary tract infection frequency four months after fracture or with regard to mortality after one year. CONCLUSION: Diabetes was the only previously known risk factor for nosocomial urinary tract infection confirmed among hip fracture patients in this study. Cloxacillin as antibiotic prophylaxis for surgery seemed to offer a certain protection against nosocomial urinary tract infection. RELEVANCE TO CLINICAL PRACTICE: Nurses in clinical practice should be aware of the risk of urinary tract infections in hip fracture patients and especially in hip fracture patients with diabetes. Patients given cloxacillin as antibiotic prophylaxis seem less likely to contract nosocomial urinary tract infection.
Asunto(s)
Infección Hospitalaria/complicaciones , Fracturas de Cadera/complicaciones , Infecciones Urinarias/complicaciones , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Estudios Prospectivos , Factores de RiesgoRESUMEN
Extracellular ATP can be released by many cell types under conditions of cellular stress and signals through activation of purinergic receptors. Bladder uroepithelial cells grown in vitro have previously been shown to release ATP in response to stretch. In the present study, we investigated ATP release from uroepithelial cells infected with bacteria and the effect of ATP on the host cell proinflammatory interleukin 8 (IL-8) response. The human kidney epithelial cell line A498 and the human uroepithelial cell line UROtsa were grown in culture and stimulated by the uropathogenic Escherichia coli (UPEC) IA2 strain or the stable ATP analogue ATP-gamma-S. ATP and IL-8 levels were measured in cell culture medium with a luciferin-luciferase assay and enzyme-linked immunosorbent assay (ELISA), respectively. The results showed that UPEC infection of uroepithelial cells for 1 h significantly increased (P < 0.01) the extracellular ATP levels. ATP-gamma-S (10 and 100 microM) stimulated release of IL-8 from UROtsa and A498 cells after 6 and 24 h. Experiments with different purinoceptor agonists suggested that P2Y receptors, and not P2X receptors, were responsible for the ATP-gamma-S-induced IL-8 release. The potency profile further suggested involvement of P2Y(1), P2Y(2), and/or P2Y(11) receptors, and reverse transcription-PCR (RT-PCR) studies confirmed that the cells expressed these receptors. The amount of IL-8 released increased 12-fold in UPEC-infected cells, and apyrase, an enzyme that degrades ATP, reduced this increase by approximately 50%. The present study suggests that enhanced ATP release and P2Y receptor activation during urinary tract infection may represent a novel, non-TLR4-mediated mechanism for production of proinflammatory IL-8 in human urinary tract epithelial cells.
Asunto(s)
Adenosina Trifosfato/metabolismo , Interleucina-8/metabolismo , Receptores Purinérgicos P2/biosíntesis , Receptores Purinérgicos P2/inmunología , Sistema Urinario/inmunología , Sistema Urinario/patología , Escherichia coli Uropatógena/inmunología , Línea Celular , Medios de Cultivo/química , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Perfilación de la Expresión Génica , HumanosRESUMEN
During the course of urinary tract infection (UTI) nitric oxide (NO) is generated as part of the host response. This study investigates the significance of the NO-detoxifying enzyme flavohemoglobin (Hmp) in protection of uropathogenic Escherichia coli (UPEC) against nitrosative stress. An hmp (J96Deltahmp) knockout mutant of UPEC strain J96 was constructed using single-gene deletion. The viability of J96Deltahmp was significantly reduced (P<0.001) compared to the wild-type strain after exposure to the NO-donor DETA/NO. The NO consumption in J96Deltahmp was significantly (P<0.001) impaired compared to J96wt. Screening UPEC isolates from patients with UTI revealed increased hmp expression in all patients. In a competition-based mouse model of UTI, the hmp mutant strain was significantly (P<0.05) out-competed by the wild-type strain. This study demonstrates, for the first time, that Hmp contributes to the protection of UPEC against NO-mediated toxicity in vitro. In addition, hmp gene expression occurs in UPEC isolates from the infected human urinary tract and UPEC that were hmp-deficient had a reduced ability to colonize the mouse urinary tract. Taken together the results suggest that NO detoxification by Hmp may be a fitness advantage factor in UPEC, and a potentially interesting target for development of novel treatment concepts for UTI.
Asunto(s)
Dihidropteridina Reductasa/metabolismo , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/metabolismo , Hemoproteínas/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico/toxicidad , Infecciones Urinarias/microbiología , Escherichia coli Uropatógena/fisiología , Adulto , Animales , Recuento de Colonia Microbiana , Dihidropteridina Reductasa/genética , Proteínas de Escherichia coli/genética , Femenino , Eliminación de Gen , Perfilación de la Expresión Génica , Hemoproteínas/genética , Humanos , Ratones , Ratones Endogámicos C3H , Viabilidad Microbiana/efectos de los fármacos , Persona de Mediana Edad , NADH NADPH Oxidorreductasas/genética , Estrés FisiológicoRESUMEN
OBJECTIVES: To examine the effect of nitric oxide (NO), an endogenous component of the host defence in urinary tract infection, on the adherence of P-fimbriated uropathogenic Escherichia coli (UPEC) to human renal epithelial cells. MATERIALS AND METHODS: Two wild-type UPEC strains (AD110 and IA2) and the P-fimbriated recombinant strain HB101pPIL-75 were used. Bacteria were allowed to adhere to the human renal epithelial cell line A498 and attachment was evaluated in the absence or presence of the NO donor DETA/NONOate (1 mm). Total RNA was extracted from NO-exposed bacteria in static urine cultures, followed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of the papG gene that encodes the P-fimbriae adhesin PapG. RESULTS: Bacterial adherence to A498 cells was fimbriae-dependent and the ability to agglutinate human P(1) positive erythrocytes confirmed that the used strains were P-fimbriated. UPEC strains AD110 and IA2 attached by a mean of 8 bacteria/cell and 20 bacteria/cell, respectively. In the presence of DETA/NONOate, the attachment of AD110 and IA2 to A498 cells was significantly reduced by a mean (sem) of 34 (3.9)% and 45 (14)%, respectively. The expression of papG was decreased after DETA/NONOate exposure as shown by semiquantitative RT-PCR. CONCLUSION: NO disrupted functional adhesion of P-fimbriated UPEC to kidney epithelial cells, suggesting that NO-production from epithelial cells in the urinary tract may limit bacterial colonization at the mucosal surface. The reduced adherence may involve transcriptional effects of NO on papG expression, but further studies are needed to establish the underlying mechanisms.