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AIM: Urinary tract infections (UTIs) rank among the most prevalent infections in humans, carrying substantial implications for public health. Women experiencing recurrent UTIs are often advised to boost their fluid intake to help eliminate bacteria. In this study, we explored the impact of elevated fluid consumption during UTIs using a mouse model of pyelonephritis. METHODS: UTI was induced in 8-10 w female BALB/cJ-mice by surgically injecting Escherichia coli (O6:K13:H1) into the bladder whereafter mice were randomized to gel food (GF) or regular chow. Immune response and infection severity were determined 24-h post-infection. In vitro bacterial growth (OD600) was determined in urine from mice or from human volunteers. RESULTS: Gel feeding increased urine output (1.40 ± 0.77 µL min-1, p < 0.01) and diluted the urine (668.7 ± 177 mOsmol kg-1, p < 0.0001) compared to controls on regular chow (urine output: 0.34 ± 0.27 µL min-1, osmolality: 1439 ± 473.5 mOsmol kg-1). Mice on GF had a higher risk of pyelonephritis (87.5%) and more severe infections (26.22 ± 9.88 CFU mg-1 tissue) compared to controls (43.75%; 3.87 ± 3.56 CFU mg-1, p < 0.01). Correspondingly, the growth of E. coli was markedly reduced at osmolalities above 1200 mOsmol kg-1 compared to 600 mOsmol kg-1 and GF mice had lower urine levels of uromodulin (13.70 ± 1.89 µg mL-1, p < 0.01) compared to controls (24.65 ± 2.70 µg mL-1). CONCLUSION: Increased water intake and urine flow in mice will markedly increase the risk of pyelonephritis. The increased risk may reflect reduced urine uromodulin combined with optimized growth conditions for E. coli. The study does not immediately support the notion that established UTIs can be eliminated by increased water intake.
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Modelos Animales de Enfermedad , Ingestión de Líquidos , Ratones Endogámicos BALB C , Pielonefritis , Uromodulina , Animales , Femenino , Humanos , Ratones , Escherichia coli , Infecciones por Escherichia coli , Pielonefritis/prevención & control , Pielonefritis/orina , Pielonefritis/microbiología , Infecciones Urinarias/prevención & control , Uromodulina/orina , MasculinoRESUMEN
Acute pyelonephritis (APN) is most frequently caused by uropathogenic Escherichia coli (UPEC), which ascends from the bladder to the kidneys during a urinary tract infection. Patients with APN have been reported to have reduced renal concentration capacity under challenged conditions, polyuria, and increased aquaporin-2 (AQP2) excretion in the urine. We have recently shown increased AQP2 accumulation in the plasma membrane in cell cultures exposed to E. coli lysates and in the apical plasma membrane of inner medullary collecting ducts in a 5-day APN mouse model. This study aimed to investigate if AQP2 expression in host cells increases UPEC infection efficiency and to identify specific bacterial components that mediate AQP2 plasma membrane insertion. As the transepithelial water permeability in the collecting duct is codetermined by AQP3 and AQP4, we also investigated whether AQP3 and AQP4 localization is altered in the APN mouse model. We show that AQP2 expression does not increase UPEC infection efficiency and that AQP2 was targeted to the plasma membrane in AQP2-expressing cells in response to the two pathogen-associated molecular patterns (PAMPs), lipopolysaccharide and peptidoglycan. In contrast to AQP2, the subcellular localizations of AQP1, AQP3, and AQP4 were unaffected both in lysate-incubated cell cultures and in the APN mouse model. Our finding demonstrated that cellular exposure to lipopolysaccharide and peptidoglycan can trigger the insertion of AQP2 in the plasma membrane revealing a new regulatory pathway for AQP2 plasma membrane translocation, which may potentially be exploited in intervention strategies.NEW & NOTEWORTHY Acute pyelonephritis (APN) is associated with reduced renal concentration capacity and increased aquaporin-2 (AQP2) excretion. Uropathogenic Escherichia coli (UPEC) mediates changes in the subcellular localization of AQP2 and we show that in vitro, these changes could be elicited by two pathogen-associated molecular patterns (PAMPs), namely, lipopolysaccharide and peptidoglycan. UPEC infection was unaltered by AQP2 expression and the other renal AQPs (AQP1, AQP3, and AQP4) were unaltered in APN.
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Acuaporina 2 , Acuaporina 3 , Pielonefritis , Escherichia coli Uropatógena , Pielonefritis/metabolismo , Pielonefritis/microbiología , Pielonefritis/patología , Animales , Acuaporina 2/metabolismo , Ratones , Escherichia coli Uropatógena/metabolismo , Acuaporina 3/metabolismo , Acuaporina 3/genética , Enfermedad Aguda , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/patología , Lipopolisacáridos/toxicidad , Lipopolisacáridos/farmacología , Membrana Celular/metabolismo , Humanos , Acuaporina 4/metabolismo , Acuaporina 4/genética , Peptidoglicano/metabolismo , Riñón/metabolismo , Riñón/patología , Ratones Endogámicos C57BL , Modelos Animales de EnfermedadRESUMEN
INTRODUCTION: Renal transplant patients are prone to developing urinary tract infections (UTIs). However, the potential effect of a UTI on renal graft loss remains unclear. METHODS: We systematically surveyed the literature for a potential association between UTI and graft loss. Articles were identified in online databases using a specific search string, followed by post selection for meta-analysis following four inclusion criteria: 1) a clear definition of UTI and recurrent UTI, 2) n > 200, 3) patient age > 16 years and 4) inclusion of data on graft loss. Data on UTI and graft loss were extracted from the included studies for calculation of a combined weighted odds ratio (OR) using the Mantel-Haenszel method. This review was conducted according to the PRISMA 2020 statement. RESULTS: Unfortunately, only eight of 108 papers met the inclusion criteria. These studies reported between 276 and 2,368 patients, primarily male, aged around 50 years. The two-year incidence of overall UTI varied from 16.5% at a 27.5-month follow-up to 30.1% at a 24-month follow-up from transplantation. Seven papers were included in the OR analysis; two found an association between UTI and graft loss and five did not. However, in the meta-analysis, the weighted OR for all seven studies was 1.340 (95% confidence interval: 1.050-1.720). CONCLUSIONS: Filtering the literature for a strict definition of UTI allowed us to establish an association between UTI and graft loss in renal transplant patients. However, further investigation and stronger studies using the Goldman criteria are needed to allow stratification for UTI severity and effect on graft loss.
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Trasplante de Riñón , Infecciones Urinarias , Humanos , Masculino , Anciano , Adolescente , Trasplante de Riñón/efectos adversos , Supervivencia de Injerto , Infecciones Urinarias/epidemiología , Infecciones Urinarias/etiología , Riñón , IncidenciaRESUMEN
Renal fibrosis is tightly associated with chronic kidney disease, irrespective of the underlying pathogenesis. We previously demonstrated mild antifibrotic effects of targeting the P2X7 receptor in a pyelonephritis model. Reduced P2X7 R-activation elevated the neutrophil-to-macrophage ratio, resulting in less matrix accumulation without affecting the initial tissue healing. Here, we test if this P2X7 R-dependent modification of matrix accumulation also applies to a noninfectious fibrosis model of unilateral ureteral obstruction (7dUUO) and whether the response is gender-dependent. We found that P2X7 -/- mice show reduced fibrosis compared to wild type after 7dUUO: the effect was most pronounced in females, with a 55% decrease in collagen deposition after 7dUUO (p < 0.0068). P2X7 R deficiency did not affect early fibrosis markers (TGF-ß, α-SMA) or the renal infiltration of neutrophils. However, a UUO-induced increase in macrophages was observed in wildtypes only (p < 0.001), leaving the P2X7 -/- mice with ≈50% fewer CD68+ cells in the renal cortex (p = 0.018). In males, 7dUUO triggered an increase in diffusely interstitial scattering of the profibrotic, macrophage-attracting metalloproteinase MMP8 and showed significantly lower MMP8 tissue expression in both male and female P2X7 -/- mice (p < 0.0008). Thus, the P2X7 R is advocated as a late-stage fibrosis moderator by reducing neutrophil-dependent interstitial MMP8 release, resulting in less macrophage infiltration and reduced matrix accumulation.
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Enfermedades Renales , Obstrucción Ureteral , Femenino , Ratones , Masculino , Animales , Obstrucción Ureteral/patología , Metaloproteinasa 8 de la Matriz/metabolismo , Modelos Animales de Enfermedad , Riñón/metabolismo , Enfermedades Renales/patología , Fibrosis , Macrófagos/metabolismo , Ratones Endogámicos C57BLRESUMEN
Background: The protease inhibitor inter-α-inhibitor heavy chain H4 (ITIH4) has been described as an acute-phase reactant and could potentially aid in sepsis monitoring and prognostication. Objectives: To investigate ITIH4 plasma levels in sepsis patients compared with healthy controls and to examine the association between ITIH4 and acute-phase response markers, blood coagulation, and organ dysfunction in sepsis. Methods: We performed a post hoc study to a prospective cohort study. Patients with septic shock (n = 39) were enrolled upon intensive care unit admission. ITIH4 was analyzed using an in-house immunoassay. Standard coagulation parameters, thrombin generation, fibrin formation and lysis, C-reactive protein, organ dysfunction markers, Sequential Organ Failure Assessment score, and disseminated intravascular coagulation (DIC) score were registered. ITIH4 levels were also investigated in a murine Escherichia coli sepsis model. Results: ITIH4 did not display acute-phase behavior as mean ITIH4 levels were not increased in patients with septic shock or in E. coli-infected mice. However, ITIH4 exhibited large interindividual variation in patients with septic shock compared with healthy controls. Low ITIH4 was associated with sepsis-related coagulopathy, including a high DIC score (mean ITIH4: DIC, 203 µg/mL vs non-DIC, 267 µg/mL, P = .01), low antithrombin (r = 0.70, P < .0001) and decreased thrombin generation (mean ITIH4: first peak thrombin tertile, 210 µg/mL vs third peak thrombin tertile, 303 µg/mL, P = .01). ITIH4 showed moderate correlation with arterial blood lactate (ρ = -0.50, P < .001) but only weak correlations with C-reactive protein, alanine transaminase, bilirubin, and Sequential Organ Failure Assessment score (all, ρ < 0.26, P > .05). Conclusion: ITIH4 is associated with sepsis-related coagulopathy but is not an acute-phase reactant during septic shock.
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Increased incidence of bone fractures in the elderly is associated with gradual sarcopenia. Similar deterioration of bone quality is seen with prolonged bed rest, spinal cord injuries or in astronauts exposed to microgravity and, preceded by loss of muscle mass. Signaling mechanisms involving uridine-5'-triphosphate (UTP) regulate bone homeostasis via P2Y2 receptors on osteoblasts and osteoclasts, whilst dictating the bone cells' response to mechanical loading. We hypothesized that muscle paralysis-induced loss of bone quality would be prevented in P2Y2 receptor knockout (KO) mice. Female mice injected with botulinum toxin (BTX) in the hind limb developed muscle paralysis and femoral DXA analysis showed reduction in bone mineral density (<10%), bone mineral content (<16%) and bone area (<6%) in wildtype (WT) compared to KO littermates (with <13%, <21%, <9% respectively). The femoral metaphyseal strength was reduced equally in both WT and KO (<37%) and <11% in diaphysis region of KO, compared to the saline injected controls. Tibial micro-CT showed reduced cortical thickness (12% in WT vs. 9% in KO), trabecular bone volume (38% in both WT and KO), trabecular thickness (22% in WT vs. 27% in KO) and increased SMI (26% in WT vs. 19% in KO) after BTX. Tibial histomorphometry showed reduced formation in KO (16%) but unchanged resorption in both WT and KO. Furthermore, analyses of DXA and bone strength after regaining the muscle function showed partial bone recovery in the KO but no difference in the bone recovery in WT mice. Primary osteoblasts from KO mice displayed increased viability and alkaline phosphatase activity but, impaired bone nodule formation. Significantly more TRAP-positive osteoclasts were generated from KO mice but displayed reduced resorptive function. Our data showed that hind limb paralysis with a single dose of BTX caused profound bone loss after 3 weeks, and an incomplete reversal of bone loss by week 19. Our findings indicate no role of the P2Y2 receptor in the bone loss after a period of skeletal unloading in mice or, in the bone recovery after restoration of muscle function.
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Enfermedades Óseas Metabólicas , Animales , Enfermedades Óseas Metabólicas/etiología , Enfermedades Óseas Metabólicas/prevención & control , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Noqueados , Músculos , ParálisisRESUMEN
AIM: Renal fibrosis is a major driver of chronic kidney disease, yet current treatment strategies are ineffective in attenuating fibrogenesis. The cyclooxygenase/prostaglandin system plays a key role in renal injury and holds great promise as a therapeutic target. Here, we used a translational approach to evaluate the role of the PGE2 -EP1 receptor in the pathogenesis of renal fibrosis in several models of kidney injury, including human (fibrotic) kidney slices. METHODS: The anti-fibrotic efficacy of a selective EP1 receptor antagonist (SC-19220) was studied in mice subjected to unilateral ureteral obstruction (UUO), healthy and fibrotic human precision-cut kidney slices (PCKS), Madin-Darby Canine Kidney (MDCK) cells and primary human renal fibroblasts (HRFs). Fibrosis was evaluated on gene and protein level using qPCR, western blot and immunostaining. RESULTS: EP1 receptor inhibition diminished fibrosis in UUO mice, illustrated by a decreased protein expression of fibronectin (FN) and α-smooth muscle actin (αSMA) and a reduction in collagen deposition. Moreover, treatment of healthy human PCKS with SC-19220 reduced TGF-ß-induced fibrosis as shown by decreased expression of collagen 1A1, FN and αSMA as well as reduced collagen deposition. Similar observations were made using fibrotic human PCKS. In addition, SC-19220 reduced TGF-ß-induced FN expression in MDCK cells and HRFs. CONCLUSION: This study highlights the EP1 receptor as a promising target for preventing both the onset and late stage of renal fibrosis. Moreover, we provide strong evidence that the effect of SC-19220 may translate to clinical care since its effects were observed in UUO mice, cells and human kidney slices.
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Enfermedades Renales , Obstrucción Ureteral , Animales , Colágeno , Ácido Dibenzo(b,f)(1,4)oxazepina-10(11H)-carboxílico, 8-cloro-, 2-acetilhidrazida , Modelos Animales de Enfermedad , Perros , Femenino , Fibrosis , Humanos , Riñón/metabolismo , Enfermedades Renales/metabolismo , Masculino , Ratones , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta/uso terapéutico , Obstrucción Ureteral/metabolismoRESUMEN
AIM: Aquaporin-2 (AQP2) shuttling between intracellular vesicles and the apical plasma membrane is pivotal in arginine vasopressin-mediated urine concentration and is dysregulated in multiple diseases associated with water balance disorders. Children and adults with acute pyelonephritis have a urinary concentration defect and studies in children revealed increased AQP2 excretion in the urine. This study aimed to analyse AQP2 trafficking in response to acute pyelonephritis. METHODS: Immunofluorescence analysis was used to evaluate subcellular localization of AQP2 and AQP2-S256A (mimicking non-phosphorylated AQP2 on serine 256) in cells stimulated with bacterial lysates and of AQP2 and pS256-AQP2 in a mouse model at day 5 of acute pyelonephritis. Western blotting was used to evaluate AQP2 levels and AQP2 phosphorylation on S256 upon incubation with bacterial lysates. Time-lapse imaging was used to measure intracellular cAMP levels in response to incubation with bacterial lysates. RESULTS: In cell cultures, lysates from both uropathogenic and nonpathogenic bacteria-mediated AQP2 plasma membrane targeting and increased AQP2 phosphorylation on serine 256 (pS256) without increasing cAMP levels. Both bacterial lysates induced plasma membrane targeting of AQP2-S256A. Immunofluorescence analysis of renal sections from mice after 5 days of acute pyelonephritis revealed apical plasma membrane targeting of AQP2 and pS256-AQP2 in inner medullary collecting ducts. CONCLUSION: Uropathogenic bacteria induce AQP2 plasma membrane targeting in vitro and in vivo. cAMP levels were not elevated by the bacterial lysates and AQP2 plasma membrane targeting could occur without S256 phosphorylation. This may explain increased AQP2 excretion in the urine during acute pyelonephritis.
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Acuaporina 2 , Pielonefritis , Animales , Acuaporina 2/metabolismo , Membrana Celular/metabolismo , Riñón/metabolismo , Ratones , Fosforilación , Pielonefritis/metabolismoRESUMEN
The local environment forces a selection of bacteria that might invade the urinary tract, allowing only the most virulent to access the kidney. Quite similar to the diet in setting the stage for the gut microbiome, renal function determines the conditions for bacteria-host interaction in the urinary tract. In the kidney, the term local environment or microenvironment is completely justified because the environment literally changes within a few micrometers. The precise composition of the urine is a function of the epithelium lining the microdomain, and the microenvironment in the kidney shows more variation in the content of nutrients, ion composition, osmolality, and pH than any other site of bacteria-host interaction. This review will cover some of the aspects of bacterial-host interaction in this unique setting and how uropathogenic bacteria can alter the condition for bacteria-host interaction. There will be a particular focus on the recent findings regarding how bacteria specifically trigger host paracrine signaling, via release of extracellular ATP and activation of P2 purinergic receptors. These finding will be discussed from the perspective of severe urinary tract infections, including pyelonephritis and urosepsis.
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Infecciones por Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas Hemolisinas/genética , Pielonefritis/genética , Receptores Purinérgicos P2/genética , Sepsis/genética , Infecciones Urinarias/genética , Escherichia coli Uropatógena/genética , Adenosina Trifosfato/metabolismo , Anoctamina-1/genética , Anoctamina-1/metabolismo , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/patología , Proteínas de Escherichia coli/metabolismo , Regulación de la Expresión Génica , Proteínas Hemolisinas/metabolismo , Interacciones Huésped-Patógeno/genética , Humanos , Concentración de Iones de Hidrógeno , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Comunicación Paracrina , Pielonefritis/metabolismo , Pielonefritis/microbiología , Pielonefritis/patología , Receptores Purinérgicos P2/metabolismo , Sepsis/metabolismo , Sepsis/microbiología , Sepsis/patología , Transducción de Señal , Infecciones Urinarias/metabolismo , Infecciones Urinarias/microbiología , Infecciones Urinarias/patología , Escherichia coli Uropatógena/crecimiento & desarrollo , Escherichia coli Uropatógena/metabolismo , Escherichia coli Uropatógena/patogenicidadRESUMEN
AIM: Cystic fibrosis patients have an increased risk of developing metabolic alkalosis presumably as a result of altered renal HCO3- handling. In this study, we directly assess the kidneys' ability to compensate for a chronic base-load in the absence of functional CFTR. METHODS: Comprehensive urine and blood acid-base analyses were done in anaesthetized WT mice or mice lacking either CFTR or pendrin, with or without 7 days of oral NaHCO3 loading. The in vivo experiments were complemented by a combination of immunoblotting and experiments with perfused isolated mouse cortical collecting ducts (CCD). RESULTS: Base-loaded WT mice maintained acid-base homeostasis by elevating urinary pH and HCO3- excretion and decreasing urinary net acid excretion. In contrast, pendrin KO mice and CFTR KO mice were unable to increase urinary pH and HCO3- excretion and unable to decrease urinary net acid excretion sufficiently and thus developed metabolic alkalosis in response to the same base-load. The expression of pendrin was increased in response to the base-load in WT mice with a paralleled increased pendrin function in the perfused CCD. In CFTR KO mice, 7 days of base-loading did not upregulate pendrin expression and apical Cl- /HCO3- exchange function was strongly blunted in the CCD. CONCLUSION: CFTR KO mice develop metabolic alkalosis during a chronic base-load because they are unable to sufficiently elevate renal HCO3- excretion. This can be explained by markedly reduced pendrin function in the absence of CFTR.
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Alcalosis , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Animales , Bicarbonatos/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Riñón/metabolismo , Ratones , Ratones Endogámicos CFTRRESUMEN
Urosepsis is a potentially life-threatening, systemic reaction to uropathogenic bacteria entering the bloodstream of the host. One of the hallmarks of sepsis is early thrombocyte activation with a following fall in circulating thrombocytes as a result of intravascular aggregation and sequestering of thrombocytes in the major organs. Development of a thrombocytopenic state is associated with a poorer outcome of sepsis. Uropathogenic Escherichia coli frequently produce the pore-forming, virulence factor α-haemolysin (HlyA), of which the biological effects are mediated by ATP release and subsequent activation of P2 receptors. Thus, we speculated that inhibition of thrombocyte P2Y1 and P2Y12 receptors might ameliorate the septic response to HlyA-producing E. coli. The study combined in vitro measurements of toxin-induced thrombocyte activation assessed as increased membrane abundance of P-selectin, fibronectin and CD63 and data from in vivo murine model of sepsis-induced by HlyA-producing E. coli under infusion of P2Y1 and P2Y12 antagonists. Our data show that the P2Y1 receptor antagonist almost abolishes thrombocyte activation by pore-forming bacterial toxins. Inhibition of P2Y1, by constant infusion of MRS2500, markedly increased the survival in mice with induced sepsis. Moreover, MRS2500 partially prevented the sepsis-induced depletion of circulating thrombocytes and dampened the sepsis-associated increase in proinflammatory cytokines. In contrast, P2Y12 receptor inhibition had only a marginal effect in vivo and in vitro. Taken together, inhibition of the P2Y1 receptor gives a subtle dampening of the thrombocyte activation and the cytokine response to bacteraemia, which may explain the improved survival observed by P2Y1 receptor antagonists.
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Toxinas Bacterianas/toxicidad , Plaquetas/patología , Receptores Purinérgicos P2Y12/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Sepsis/patología , Infecciones Urinarias/patología , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Adenosina Monofosfato/uso terapéutico , Animales , Nucleótidos de Desoxiadenina/farmacología , Modelos Animales de Enfermedad , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Humanos , Masculino , Ratones Endogámicos BALB C , Sepsis/complicaciones , Sepsis/tratamiento farmacológico , Resultado del Tratamiento , Infecciones Urinarias/complicaciones , Infecciones Urinarias/tratamiento farmacológico , Escherichia coli Uropatógena/efectos de los fármacosRESUMEN
Autocrine and paracrine signaling in the kidney adds an extra level of diversity and complexity to renal physiology. The extensive scientific production on the topic precludes easy understanding of the fundamental purpose of the vast number of molecules and systems that influence the renal function. This systematic review provides the broader pen strokes for a collected image of renal paracrine signaling. First, we recapitulate the essence of each paracrine system one by one. Thereafter the single components are merged into an overarching physiological concept. The presented survey shows that despite the diversity in the web of paracrine factors, the collected effect on renal function may not be complicated after all. In essence, paracrine activation provides an intelligent system that perceives minor perturbations and reacts with a coordinated and integrated tissue response that relieves the work load from the renal epithelia and favors diuresis and natriuresis. We suggest that the overall function of paracrine signaling is reno-protection and argue that renal paracrine signaling and self-regulation are two sides of the same coin. Thus local paracrine signaling is an intrinsic function of the kidney, and the overall renal effect of changes in blood pressure, volume load, and systemic hormones will always be tinted by its paracrine status.
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Comunicación Autocrina/fisiología , Riñón/fisiología , Comunicación Paracrina/fisiología , Animales , Humanos , Transducción de Señal/fisiologíaRESUMEN
Urosepsis is a severe condition often caused by Escherichia coli that spontaneously have ascended the urinary tract to the kidneys causing pyelonephritis and potentially bacteraemia. The number of sepsis cases has been steadily increasing over the last decades, and there are still no specific, molecular supportive therapies for sepsis to supplement antibiotic treatment. P2X1 receptors are expressed by a number of immune cells including thrombocytes, which presently have been established as an important player in the acute immune response to bacterial infections. P2X1 receptor-deficient mice have been shown to be relatively protected against urosepsis, with markedly reduced levels of circulating proinflammatory cytokines and intravascular coagulation. However, here we show that continuous intravenous infusion with P2X1 receptor antagonist markedly accelerates development of a septic response to induced bacteraemia with uropathogenic E. coli. Mice exposed to the P2X1 receptor antagonists die very early with haematuria, substantially elevated plasma levels of proinflammatory cytokines, massive intravascular coagulation and a concomitant reduction in circulating thrombocytes. Interestingly, infusion of P2X1 receptor antagonists causes a marked acute reduction in circulating thrombocytes and a higher number of bacteria in the blood. These data support the notion that the number of functional thrombocytes is important for the acute defence against bacteria in the circulation and that the P2X1 receptor potentially could be essential for this response.
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Plaquetas/efectos de los fármacos , Infecciones por Escherichia coli , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X1 , Sepsis , Infecciones Urinarias , Animales , Bencenosulfonatos , Proteínas Hemolisinas , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Pielonefritis , Suramina/análogos & derivados , Escherichia coli UropatógenaRESUMEN
Severe urinary tract infections are commonly caused by sub-strains of Escherichia coli secreting the pore-forming virulence factor α-hemolysin (HlyA). Repeated or severe cases of pyelonephritis can cause renal scarring that subsequently can lead to progressive failure. We have previously demonstrated that HlyA releases cellular ATP directly through its membrane pore and that acute HlyA-induced cell damage is completely prevented by blocking ATP signaling. Local ATP signaling and P2X7 receptor activation play a key role in the development of tissue fibrosis. This study investigated the effect of P2X7 receptors on infection-induced renal scarring in a murine model of pyelonephritis. Pyelonephritis was induced by injecting 100 million HlyA-producing, uropathogenic E. coli into the urinary bladder of BALB/cJ mice. A similar degree of pyelonephritis and mortality was confirmed at day 5 after infection in P2X7+/+ and P2X7-/- mice. Fibrosis was first observed 2 weeks after infection, and the data clearly demonstrated that P2X7-/- mice and mice exposed to the P2X7 antagonist, brillian blue G, show markedly less renal fibrosis 14 days after infection compared with controls (P < 0.001). Immunohistochemistry revealed comparable early neutrophil infiltration in the renal cortex from P2X7+/+ and P2X7-/- mice. Interestingly, lack of P2X7 receptors resulted in diminished macrophage infiltration and reduced neutrophil clearance in the cortex of P2X7-/- mice. Hence, this study suggests the P2X7 receptor to be an appealing antifibrotic target after renal infections.
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Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Riñón/metabolismo , Pielonefritis , Receptores Purinérgicos P2X7/deficiencia , Escherichia coli Uropatógena , Animales , Fibrosis , Riñón/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Pielonefritis/genética , Pielonefritis/metabolismo , Pielonefritis/microbiología , Pielonefritis/prevención & control , Receptores Purinérgicos P2X7/metabolismo , Escherichia coli Uropatógena/metabolismo , Escherichia coli Uropatógena/patogenicidadRESUMEN
α-Haemolysin (HlyA) from uropathogenic Escherichia coli has been demonstrated to be a significant virulence factor for ascending urinary tract infections. Once the E. coli reach the well-vascularised kidneys, there is a high risk of bacteraemia and a subsequent septic host response. Despite this, HlyA has the potential to accelerate the host response both directly and via its ability to facilitate adenosine triphosphate release from cells. It has not been settled whether HlyA aggravates bacteraemia into a septic state. To address this, we used an E. coli strain in a model of acute urosepsis that was either transfected with a plasmid containing the full HlyA operon or one with deletion in the HlyA gene. Here, we show that HlyA accelerates the host response to E. coli in the circulation. Mice exposed to HlyA-producing E. coli showed massively increased proinflammatory cytokines, a substantial fall in circulating thrombocytes, extensive haematuria, and intravascular haemolysis. This was not seen in mice exposed to either E. coli that do not secrete HlyA or vehicle controls. Consistent with the massive host response to the bacteria, the mice exposed to HlyA-producing E. coli died exceedingly early, whereas mice exposed to E. coli without HlyA production and vehicle controls survived the entire observation period. These data allow us to conclude that HlyA is a virulence factor that accelerates a state of bacteraemia into fulminant sepsis in a mouse model.
Asunto(s)
Bacteriemia/microbiología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/sangre , Proteínas Hemolisinas/sangre , Infecciones Urinarias/microbiología , Escherichia coli Uropatógena/patogenicidad , Factores de Virulencia/sangre , Animales , Bacteriemia/sangre , Bacteriemia/mortalidad , Plaquetas/metabolismo , Citocinas/sangre , Modelos Animales de Enfermedad , Eritrocitos/metabolismo , Eritrocitos/microbiología , Eritrocitos/patología , Infecciones por Escherichia coli/sangre , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Hemólisis , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Operón , Infecciones Urinarias/sangre , Escherichia coli Uropatógena/metabolismo , Factores de Virulencia/genéticaRESUMEN
BACKGROUND: Pore-forming proteins released from bacteria or formed as result of complement activation are known to produce severe cell damage. Inhibition of purinergic P2X receptors markedly reduces damage inflicted by cytolytic bacterial toxin and after complement activation in both erythrocytes and monocytes. P2X expression generally shows variation throughout the population. Here, we investigate correlation between P2X receptor abundance in blood cell plasma membranes and haematocrit during sepsis, in patients admitted to the emergency department (ED) or intensive care unit (ICU). METHOD: Patients admitted to the ED and successively transferred to ICU with the diagnosis sepsis (< 2 systemic inflammatory response syndrome (SIRS) criteria and suspected infection), were grouped as either blood pathogen-positive (14 patients) or blood pathogen-negative (20 patients). Blood samples drawn at ICU admission were analysed for P2X1 and P2X7 receptor abundance using indirect flow cytometry. RESULTS: Here, we find inverse correlation between P2X1 receptor expression and change in haematocrit (rs - 0.80) and haemoglobin (rs - 0.78) levels from admission to ED to arrival at ICU in patients with pathogen-positive sepsis. This correlation was not found in patients without confirmed bacteraemia. Patients with high P2X1 expression had a significantly greater change in both haematocrit (- 0.59 ± 0.36) and haemoglobin levels (- 0.182 ± 0.038 mg/dl) per hour, during the first hours after hospital admission compared to patients with low P2X1 expression (0.007 ± 0.182 and - 0.020 ± 0.058 mg/dl, respectively). CONCLUSION: High levels of P2X1 are correlated with more pronounced reduction in haematocrit and haemoglobin in patients with confirmed bacteraemia. This supports previous in vitro findings of P2X activation as a significant component in cell damage caused by pore-forming bacterial toxins and complement-dependent major attack complex. These data suggest a new potential target for future therapeutics in initial stages of sepsis.
Asunto(s)
Hematócrito/métodos , Receptores Purinérgicos P2X1/análisis , Sepsis/sangre , Anciano , Toxinas Bacterianas/sangre , Servicio de Urgencia en Hospital/organización & administración , Servicio de Urgencia en Hospital/estadística & datos numéricos , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Bacterias Gramnegativas/enzimología , Bacterias Gramnegativas/patogenicidad , Bacterias Grampositivas/enzimología , Bacterias Grampositivas/patogenicidad , Hematócrito/estadística & datos numéricos , Humanos , Unidades de Cuidados Intensivos/organización & administración , Unidades de Cuidados Intensivos/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Receptores Purinérgicos P2X1/sangre , Síndrome de Respuesta Inflamatoria Sistémica , Vitamina D/análisis , Vitamina D/sangreRESUMEN
The link between rheumatoid arthritis and exposure to a bacterial toxin was not found in a population of rheumatoid arthritis patients from Netherlands.
Asunto(s)
Artritis Reumatoide , Periodontitis , Aggregatibacter actinomycetemcomitans , Autoinmunidad , Humanos , Países BajosRESUMEN
Calcium (Ca2+) is a fundamental regulator of cell signaling and function. Thapsigargin (Tg) blocks the sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA), disrupts Ca2+ homeostasis, and causes cell death. However, the exact mechanisms whereby SERCA inhibition induces cell death are incompletely understood. Here, we report that low (0.1 µm) concentrations of Tg and Tg analogs with various long-chain substitutions at the O-8 position extensively inhibit SERCA1a-mediated Ca2+ transport. We also found that, in both prostate and breast cancer cells, exposure to Tg or Tg analogs for 1 day caused extensive drainage of the ER Ca2+ stores. This Ca2+ depletion was followed by markedly reduced cell proliferation rates and morphological changes that developed over 2-4 days and culminated in cell death. Interestingly, these changes were not accompanied by bulk increases in cytosolic Ca2+ levels. Moreover, knockdown of two key store-operated Ca2+ entry (SOCE) components, Orai1 and STIM1, did not reduce Tg cytotoxicity, indicating that SOCE and Ca2+ entry are not critical for Tg-induced cell death. However, we observed a correlation between the abilities of Tg and Tg analogs to deplete ER Ca2+ stores and their detrimental effects on cell viability. Furthermore, caspase activation and cell death were associated with a sustained unfolded protein response. We conclude that ER Ca2+ drainage and sustained unfolded protein response activation are key for initiation of apoptosis at low concentrations of Tg and Tg analogs, whereas high cytosolic Ca2+ levels and SOCE are not required.