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1.
Dan Med J ; 71(2)2024 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-38314732

RESUMO

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.


Assuntos
Transplante de Rim , Infecções Urinárias , Humanos , Masculino , Idoso , Adolescente , Transplante de Rim/efeitos adversos , Sobrevivência de Enxerto , Infecções Urinárias/epidemiologia , Infecções Urinárias/etiologia , Rim , Incidência
2.
Acta Physiol (Oxf) ; 231(3): e13591, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33270356

RESUMO

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.


Assuntos
Alcalose , Regulador de Condutância Transmembrana em Fibrose Cística , Animais , Bicarbonatos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Rim/metabolismo , Camundongos , Camundongos Endogâmicos CFTR
3.
Purinergic Signal ; 15(2): 265-276, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31129780

RESUMO

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.


Assuntos
Plaquetas/efeitos dos fármacos , Infecções por Escherichia coli , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X1 , Sepse , Infecções Urinárias , Animais , Benzenossulfonatos , Proteínas Hemolisinas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pielonefrite , Suramina/análogos & derivados , Escherichia coli Uropatogênica
4.
Am J Pathol ; 189(6): 1201-1211, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30926332

RESUMO

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.


Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Rim/metabolismo , Pielonefrite , Receptores Purinérgicos P2X7/deficiência , Escherichia coli Uropatogênica , Animais , Fibrose , Rim/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Pielonefrite/genética , Pielonefrite/metabolismo , Pielonefrite/microbiologia , Pielonefrite/prevenção & controle , Receptores Purinérgicos P2X7/metabolismo , Escherichia coli Uropatogênica/metabolismo , Escherichia coli Uropatogênica/patogenicidade
5.
Cell Microbiol ; 21(6): e13017, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30761726

RESUMO

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.


Assuntos
Bacteriemia/microbiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/sangue , Proteínas Hemolisinas/sangue , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/patogenicidade , Fatores de Virulência/sangue , Animais , Bacteriemia/sangue , Bacteriemia/mortalidade , Plaquetas/metabolismo , Citocinas/sangue , Modelos Animais de Doenças , Eritrócitos/metabolismo , Eritrócitos/microbiologia , Eritrócitos/patologia , Infecções por Escherichia coli/sangue , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Hemólise , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Óperon , Infecções Urinárias/sangue , Escherichia coli Uropatogênica/metabolismo , Fatores de Virulência/genética
6.
Crit Care ; 22(1): 181, 2018 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-30071869

RESUMO

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.


Assuntos
Hematócrito/métodos , Receptores Purinérgicos P2X1/análise , Sepse/sangue , Idoso , Toxinas Bacterianas/sangue , Serviço Hospitalar de Emergência/organização & administração , Serviço Hospitalar de Emergência/estatística & dados numéricos , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/patogenicidade , Bactérias Gram-Positivas/enzimologia , Bactérias Gram-Positivas/patogenicidade , Hematócrito/estatística & dados numéricos , Humanos , Unidades de Terapia Intensiva/organização & administração , Unidades de Terapia Intensiva/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Receptores Purinérgicos P2X1/sangue , Síndrome de Resposta Inflamatória Sistêmica , Vitamina D/análise , Vitamina D/sangue
7.
8.
J Biol Chem ; 292(48): 19656-19673, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28972171

RESUMO

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.


Assuntos
Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , Tapsigargina/análogos & derivados , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Canais de Cálcio/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Humanos , Tapsigargina/farmacologia
9.
J Membr Biol ; 250(3): 301-313, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28488084

RESUMO

Uropathogenic Escherichia coli often produce the virulence factor α-hemolysin (HlyA), and the more severe the infection, the likelier it is to isolate HlyA-producing E. coli from patients. HlyA forms pores upon receptor-independent insertion of the toxin into biological membranes and it has been substantiated that HlyA-induced hemolysis is amplified by toxin-induced ATP release and activation of P2X receptors. Thus, hemolysis inflicted by HlyA is a protracted process involving signal transduction. It consists of early, marked cell shrinkage followed by swelling and eventually lysis. The initially shrinkage is a consequence of a substantial Ca2+-influx and activation of Ca2+-sensitive K+ and Cl- channels (KCa3.1/TMEM16A). The shrinkage is followed by gradual cell swelling, which ultimately lyses the cells. These findings clearly show that the HlyA pore provides a substantial volume challenge for the cells, and the fate of the given cell is co-determined by intrinsic erythrocytal volume regulation. We therefore speculated that other mechanisms involved in erythrocyte volume regulation may influence the hemolytic process inflicted by HlyA. Strikingly, HlyA-induced hemolysis is markedly reduced in erythrocytes isolated from NKCC1-deficient (NKCC1-/-) mice compared to controls. The NKCC1 inhibitors furosemide and bumetanide concentration-dependently inhibit HlyA-induced lysis of human and murine erythrocytes. However, in high concentrations bumetanide further reduced hemolysis in erythrocytes from NKCC1-/- mice and, thus, also exhibit indirect effects on hemolysis. The effect of loop diuretics on the hemolysis is not unique to HlyA but is similarly seen in LtxA- and α-toxin-induced hemolysis. Bumetanide clearly potentiates HlyA-induced volume reduction and delays the following erythrocyte swelling. This allows increased phagocytosis of damaged erythrocytes by THP-1 cell as a result of prolonged cell shrinkage. These data suggest that erythrocyte susceptibility to cytolysins is modified by NKCC1 and signifies intrinsic volume regulators as important determinants of cellular outcome of pore-forming toxins.


Assuntos
Escherichia coli/química , Proteínas Hemolisinas/farmacologia , Animais , Proteínas de Bactérias/farmacologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Furosemida/farmacologia , Hemólise/efeitos dos fármacos , Humanos , Camundongos , Camundongos Knockout , Fagocitose/efeitos dos fármacos , Membro 2 da Família 12 de Carreador de Soluto/deficiência , Membro 2 da Família 12 de Carreador de Soluto/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Células THP-1
10.
Artigo em Inglês | MEDLINE | ID: mdl-28428949

RESUMO

α-haemolysin (HlyA)-producing Escherichia coli commonly inflict severe urinary tract infections, including pyelonephritis, which comprises substantial risk for sepsis. In vitro, the cytolytic effect of HlyA is mainly mediated by ATP release through the HlyA pore and subsequent P2X1/P2X7 receptor activation. This amplification of the lytic process is not unique to HlyA but is observed by many other pore-forming proteins including complement-induced haemolysis. Since free hemoglobin in the blood is known to be associated with a worse outcome in sepsis one could speculate that inhibition of P2X receptors would ameliorate the course of sepsis. Surprisingly, this study demonstrates that [Formula: see text] and [Formula: see text] mice are exceedingly sensitive to sepsis with uropathogenic E. coli. These mice have markedly lower survival, higher cytokine levels and activated intravascular coagulation. Quite the reverse is seen in [Formula: see text] mice, which had markedly lower cytokine levels and less coagulation activation compared to controls after exposure to uropathogenic E. coli. The high cytokine levels in the [Formula: see text] mouse are unexpected, since P2X7 is implicated in caspase-1-dependent IL-1ß production. Here, we demonstrate that IL-1ß production during sepsis with uropathogenic E. coli is mediated by caspase-8, since caspase-8 and RIPK3 double knock out mice show substantially lower cytokine during sepsis and increased survival after injection of TNFα. These data support that P2X7 and P2X4 receptor activation has a protective effect during severe E. coli infection.


Assuntos
Suscetibilidade a Doenças , Infecções por Escherichia coli/patologia , Receptores Purinérgicos P2X1/deficiência , Receptores Purinérgicos P2X4/deficiência , Receptores Purinérgicos P2X7/deficiência , Sepse/patologia , Animais , Modelos Animais de Doenças , Infecções por Escherichia coli/genética , Camundongos , Camundongos Knockout , Análise de Sobrevida , Resultado do Tratamento
11.
Physiol Rep ; 5(5)2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28292889

RESUMO

Mammalian potassium homeostasis results from tightly regulated renal and colonic excretion, which balances the unregulated dietary K+ intake. Colonic K+ secretion follows the pump-leak model, in which the large conductance Ca2+-activated K+ channel (KCa1.1) is well established as the sole, but highly regulated apical K+ conductance. The physiological importance of auxiliary ß and γ subunits of the pore-forming α-subunit of the KCa1.1 channel is not yet fully established. This study investigates colonic K+ secretion in a global knockout mouse of the KCa1.1-ß2-subunit (KCNMB2-/-), which apparently is the only ß-subunit of the colonic enterocyte KCa1.1 channel. We can report that: (1) Neither KCa1.1 α- nor the remaining ß-subunits were compensatory transcriptional regulated in colonic epithelia of KCNMB2-/- mice. (2) Colonic epithelia from KCNMB2-/- mice displayed equal basal and ATP-induced KCa1.1-mediated K+ conductance as compared to KCNMB2+/+ (3) K+ secretion was increased in KCNMB2-/- epithelia compared to wild-type epithelia from animals fed an aldosterone-inducing diet. (4) Importantly, the apical K+ conductance was abolished by the specific blocker of KCa1.1 channel iberiotoxin in both KCNMB2+/+ and KCNMB2-/- mice. Recently a novel family of auxiliary γ-subunits of the KCa1.1 channel has been described. (5) We detected the γ1-subunit (LRRC26) mRNA in colonic epithelia. To investigate the physiological role of the γ1-subunit of KCa1.1 channels in colonic K+ secretion, we acquired an LRRC26 knockout mouse. (6) Unexpectedly, LRRC26 mice had a perinatal lethal phenotype, thus preventing functional measurements. On this basis we conclude that colonic K+ secretion is intact or even increased in mice lacking the ß2-subunit of KCa1.1 channel complex despite no additional compensatory induction of KCa1.1 ß-subunits.


Assuntos
Colo/metabolismo , Mucosa Intestinal/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Potássio/metabolismo , Aldosterona , Animais , Transporte Biológico , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Camundongos , Camundongos Knockout
12.
Front Physiol ; 8: 18, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28174542

RESUMO

Activation of basolateral P2X receptors markedly reduces NaCl absorption in mouse medullary thick ascending limb (mTAL). Here we tested the role of nitric oxide (NO) in the ATP-mediated (P2X) transport inhibition. We used isolated, perfused mTALs from mice to electrically measure NaCl absorption. By microelectrodes we determined the transepithelial voltage (Vte) and transepithelial resistance (Rte). Via these two parameters, we calculated the equivalent short circuit current, I'sc as a measure of the transepithelial Na+ absorption. Basolateral ATP (100 µM) acutely induced reversible inhibition of Na+ absorption (24 ± 4%, n = 10). Addition of L-arginine (100 µM) had no apparent effect on the ATP-induced transport inhibition. Acute reduction of extracellular [Ca2+] to either 100 nM or 0 nM by addition of EGTA had no effect on the ATP-induced transport inhibition. In the presence of the NO synthase (NOS) inhibitor L-NAME (100 µM) and/or ODQ to inhibit the guanylyl cyclase, the ATP effect remained unaffected. Increasing the concentration and incubation time for L-NAME (1 mM) still did not reveal any effect on the ATP-mediated transport inhibition. Acute addition of the NO donors SNAP (100 µM) and Spermine NONOate (10 µM) did not alter tubular transport. High concentrations of L-NAME (1 mM) in itself, however, reduced the transepithelial transport significantly. Thus, we find no evidence for nitric oxide (NO) as second messenger for P2X receptor-dependent transport inhibition in mTAL. Moreover, Ca2+ signaling appears not involved in the ATP-mediated effect. It remains undefined how P2X receptors trigger the marked reduction of transport in the TAL.

13.
Infect Immun ; 84(11): 3114-3130, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27528275

RESUMO

α-Hemolysin (HlyA) from Escherichia coli and leukotoxin A (LtxA) from Aggregatibacter actinomycetemcomitans are important virulence factors in ascending urinary tract infections and aggressive periodontitis, respectively. The extracellular signaling molecule ATP is released immediately after insertion of the toxins into plasma membranes and, via P2X receptors, is essential for the erythrocyte damage inflicted by these toxins. Moreover, ATP signaling is required for the ensuing recognition and phagocytosis of damaged erythrocytes by the monocytic cell line THP-1. Here, we investigate how these toxins affect THP-1 monocyte function. We demonstrate that both toxins trigger early ATP release and a following increase in the intracellular Ca2+ concentration ([Ca2+]i) in THP-1 monocytes. The HlyA- and LtxA-induced [Ca2+]i response is diminished by the P2 receptor antagonist in a pattern that fits the functional P2 receptor expression in these cells. Both toxins are capable of lysing THP-1 cells, with LtxA being more aggressive. Either desensitization or blockage of P2X1, P2X4, or P2X7 receptors markedly reduces toxin-induced cytolysis. This pattern is paralleled in freshly isolated human monocytes from healthy volunteers. Interestingly, only a minor fraction of the toxin-damaged THP-1 monocytes eventually lyse. P2X7 receptor inhibition generally prevents cell damage, except from a distinct cell shrinkage that prevails in response to the toxins. Moreover, we find that preexposure to HlyA preserves the capacity of THP-1 monocytes to phagocytose damaged erythrocytes and may induce readiness to discriminate between damaged and healthy erythrocytes. These findings suggest a new pharmacological target for protecting monocytes during exposure to pore-forming cytolysins during infection or injury.


Assuntos
Aggregatibacter actinomycetemcomitans/metabolismo , Toxinas Bacterianas/toxicidade , Escherichia coli/metabolismo , Proteínas Hemolisinas/toxicidade , Monócitos/efeitos dos fármacos , Receptores Purinérgicos P2X/fisiologia , Toxinas Bacterianas/metabolismo , Morte Celular/efeitos dos fármacos , Citoplasma/metabolismo , Citotoxinas/metabolismo , Eritrócitos/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/toxicidade , Exotoxinas/metabolismo , Exotoxinas/toxicidade , Proteínas Hemolisinas/metabolismo , Hemólise/fisiologia , Humanos , Monócitos/metabolismo
14.
Am J Physiol Renal Physiol ; 310(10): F1035-46, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-26962098

RESUMO

The kidney is the primary organ ensuring K(+) homeostasis. K(+) is secreted into the urine in the distal tubule by two mechanisms: by the renal outer medullary K(+) channel (Kir1.1) and by the Ca(2+)-activated K(+) channel (KCa1.1). Here, we report a novel knockout mouse of the ß2-subunit of the KCa1.1 channel (KCNMB2), which displays hyperaldosteronism after decreased renal K(+) excretion. KCNMB2(-/-) mice displayed hyperaldosteronism, normal plasma K(+) concentration, and produced dilute urine with decreased K(+) concentration. The normokalemia indicated that hyperaldosteronism did not result from primary aldosteronism. Activation of the renin-angiotensin-aldosterone system was also ruled out as renal renin mRNA expression was reduced in KCNMB2(-/-) mice. Renal K(+) excretion rates were similar in the two genotypes; however, KCNMB2(-/-) mice required elevated plasma aldosterone to achieve K(+) balance. Blockade of the mineralocorticoid receptor with eplerenone triggered mild hyperkalemia and unmasked reduced renal K(+) excretion in KCNMB2(-/-) mice. Knockout mice for the α-subunit of the KCa1.1 channel (KCNMA1(-/-) mice) have hyperaldosteronism, are hypertensive, and lack flow-induced K(+) secretion. KCNMB2(-/-) mice share the phenotypic traits of normokalemia and hyperaldosteronism with KCNMA1(-/-) mice but were normotensive and displayed intact flow-induced K(+) secretion. Despite elevated plasma aldosterone, KNCMB2(-/-) mice did not display salt-sensitive hypertension and were able to decrease plasma aldosterone on a high-Na(+) diet, although plasma aldosterone remained elevated in KCNMB2(-/-) mice. In summary, KCNMB2(-/-) mice have a reduced ability to excrete K(+) into the urine but achieve K(+) balance through an aldosterone-mediated, ß2-independent mechanism. The phenotype of KCNMB2 mice was similar but milder than the phenotype of KCNMA1(-/-) mice.


Assuntos
Hiperaldosteronismo/etiologia , Rim/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/deficiência , Potássio/urina , Aldosterona/sangue , Animais , Pressão Sanguínea , Canais Epiteliais de Sódio/metabolismo , Eplerenona , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Poliúria/etiologia , Sódio na Dieta/administração & dosagem , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Espironolactona/análogos & derivados
15.
Am J Physiol Renal Physiol ; 309(10): F835, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26377796
16.
Am J Physiol Renal Physiol ; 309(2): F146-53, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25995110

RESUMO

The loop diuretic furosemide inhibits NaCl reabsorption in the thick ascending limb (TAL). In addition, furosemide acidifies the urine, which is traditionally explained by increased Na+ loading to the distal tubule causing an activation of H+ secretion via H+-ATPase in α-intercalated cells. The inability to acidify urine in response to furosemide serves to diagnose distal renal tubular acidosis (dysfunction of α-intercalated cells). Since the TAL is important for acid/base regulation, we speculated that it is involved in furosemide-induced urinary acidification. Luminal furosemide (100 µM) caused major, stable, and reversible intracellular alkalization (7.27 ± 0.06 to 7.6 ± 0.04) in isolated perfused murine medullary TAL and pronounced H+ secretion. This H+ secretion was fully inhibited with luminal amiloride (1 mM) and the Na+/H+ exchanger (NHE)3-specific antagonist #4167 (1 µM). Moreover, furosemide triggered a substantial drop of intracellular Na+ concentration in the medullary TAL. These results suggest that the furosemide-induced H+ secretion is a consequence of a drop in intracellular Na+ concentration, increasing the driving force for NHE3. Intriguingly, in whole animal experiments, furosemide-induced urinary acidification and net acid excretion were markedly reduced by specific NHE3 inhibition. Furthermore, the furosemide-induced urinary acidification was partially preserved during epithelial Na+ channel inhibition with benzamil. These results provide new insights in the mechanism of furosemide-induced urinary acidification and emphasize the role of the TAL in renal acid/base handling.


Assuntos
Furosemida/farmacologia , Alça do Néfron/efeitos dos fármacos , Prótons , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , Urina/química , Acidose Tubular Renal/diagnóstico , Amilorida , Animais , Canais Epiteliais de Sódio/metabolismo , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Alça do Néfron/metabolismo , Camundongos , Sódio/metabolismo , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores
17.
J Biol Chem ; 290(23): 14776-84, 2015 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-25911098

RESUMO

Urinary tract infections are commonly caused by α-hemolysin (HlyA)-producing Escherichia coli. In erythrocytes, the cytotoxic effect of HlyA is strongly amplified by P2X receptors, which are activated by extracellular ATP released from the cytosol of the erythrocytes. In renal epithelia, HlyA causes reversible [Ca(2+)]i oscillations, which trigger interleukin-6 (IL-6) and IL-8 release. We speculate that this effect is caused by HlyA-induced ATP release from the epithelial cells and successive P2 receptor activation. Here, we demonstrate that HlyA-induced [Ca(2+)]i oscillations in renal epithelia were completely prevented by scavenging extracellular ATP. In accordance, HlyA was unable to inflict any [Ca(2+)]i oscillations in 132-1N1 cells, which lack P2R completely. After transfecting these cells with the hP2Y2 receptor, HlyA readily triggered [Ca(2+)]i oscillations, which were abolished by P2 receptor antagonists. Moreover, HlyA-induced [Ca(2+)]i oscillations were markedly reduced in medullary thick ascending limbs isolated from P2Y2 receptor-deficient mice compared with wild type. Interestingly, the following HlyA-induced IL-6 release was absent in P2Y2 receptor-deficient mice. This suggests that HlyA induces ATP release from renal epithelia, which via P2Y2 receptors is the main mediator of HlyA-induced [Ca(2+)]i oscillations and IL-6 release. This supports the notion that ATP signaling occurs early during bacterial infection and is a key player in the further inflammatory response.


Assuntos
Infecções por Escherichia coli/imunologia , Proteínas de Escherichia coli/imunologia , Escherichia coli/fisiologia , Proteínas Hemolisinas/imunologia , Interleucina-6/imunologia , Rim/microbiologia , Receptores Purinérgicos P2Y2/imunologia , Urotélio/microbiologia , Trifosfato de Adenosina/imunologia , Animais , Sinalização do Cálcio , Linhagem Celular , Cães , Escherichia coli/imunologia , Humanos , Rim/imunologia , Camundongos , Urotélio/imunologia
19.
Am J Physiol Cell Physiol ; 308(3): C198-208, 2015 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-25428884

RESUMO

The primary cilium is an extraordinary organelle. For many years, it had the full attention of only a few dedicated scientists fascinated by its uniqueness. Unexpectedly, after decades of obscurity, it has moved very quickly into the limelight with the increasing evidence of its central role in the many genetic variations that lead to what are now known as ciliopathies. These studies implicated unique biological functions of the primary cilium, which are not completely straightforward. In parallel, and initially completely unrelated to the ciliopathies, the primary cilium was characterized functionally as an organelle that makes cells more susceptible to changes in fluid flow. Thus the primary cilium was suggested to function as a flow-sensing device. This characterization has been substantiated for many epithelial cell types over the years. Nevertheless, part of the central mechanism of signal transduction has not been explained, largely because of the substantial technical challenges of working with this delicate organelle. The current review considers the recent advances that allow us to fill some of the holes in the model of signal transduction in cilium-mediated responses to fluid flow and to pursue the physiological implications of this peculiar organelle.


Assuntos
Comunicação Celular/fisiologia , Cílios/fisiologia , Fluidez de Membrana/fisiologia , Proteínas de Membrana/metabolismo , Transdução de Sinais/fisiologia , Animais , Sinalização do Cálcio/fisiologia , Células Epiteliais/fisiologia , Humanos
20.
J Biol Chem ; 289(27): 19098-109, 2014 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-24860098

RESUMO

ATP is as an extracellular signaling molecule able to amplify the cell lysis inflicted by certain bacterial toxins including the two RTX toxins α-hemolysin (HlyA) from Escherichia coli and leukotoxin A (LtxA) from Aggregatibacter actinomycetemcomitans. Inhibition of P2X receptors completely blocks the RTX toxin-induced hemolysis over a larger concentration range. It is, however, at present not known how the ATP that provides the amplification is released from the attacked cells. Here we show that both HlyA and LtxA trigger acute release of ATP from human erythrocytes that preceded and were not caused by cell lysis. This early ATP release did not occur via previously described ATP-release pathways in the erythrocyte. Both HlyA and LtxA were capable of triggering ATP release in the presence of the pannexin 1 blockers carbenoxolone and probenecid, and the HlyA-induced ATP release was found to be similar in erythrocytes from pannexin 1 wild type and knock-out mice. Moreover, the voltage-dependent anion channel antagonist TRO19622 had no effect on ATP release by either of the toxins. Finally, we showed that both HlyA and LtxA were able to release ATP from ATP-loaded lipid (1-palmitoyl-2-oleoyl-phosphatidylcholine) vesicles devoid of any erythrocyte channels or transporters. Again we were able to show that this happened in a non-lytic fashion, using calcein-containing vesicles as controls. These data show that both toxins incorporate into lipid vesicles and allow ATP to be released. We suggest that both toxins cause acute ATP release by letting ATP pass the toxin pores in both human erythrocytes and artificial membranes.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/farmacologia , Proteínas Hemolisinas/química , Proteínas Hemolisinas/farmacologia , Aggregatibacter actinomycetemcomitans , Animais , Conexinas/deficiência , Conexinas/genética , Eritrócitos/citologia , Técnicas de Inativação de Genes , Hemoglobinas/metabolismo , Hemólise/efeitos dos fármacos , Humanos , Membranas Artificiais , Camundongos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Fosfatidilcolinas/metabolismo , Porosidade
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