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See detailDisruption in energy metabolism and mitochondrial function in a cellular model of inflammation-induced acute kidney injury
Quoilin, Caroline ULg; Mouithys-Mickalad, Ange ULg; Lécart, Sandrine et al

Poster (2013, September)

Sepsis is a very complex clinical condition characterized by stimulation of a systemic inflammatory response due to an infection. It has a profound deleterious effect on kidney functions leading to sepsis ... [more ▼]

Sepsis is a very complex clinical condition characterized by stimulation of a systemic inflammatory response due to an infection. It has a profound deleterious effect on kidney functions leading to sepsis-induced acute kidney injury (AKI). This failure seems to occur through complex mechanisms involving the immune system response, inflammatory pathways, cellular dysfunction and hemodynamic instability. To study the role of cellular energetic metabolism dysfunction and mitochondrial impairment in the occurrence of AKI during sepsis, we developed an inflammation-induced in vitro model using proximal tubular epithelial cells (HK-2) exposed to a bacterial endotoxin (lipopolysaccharide, LPS). This investigation has provided key features on the relationship between endotoxic stress and mitochondrial respiratory chain assembly defects. Firstly, we have shown that renal cells subjected to LPS are no longer capable to use adequately the available oxygen to maintain their metabolic functions. One hypothesis of this down-regulation suggests that impairment in mitochondria oxidative phosphorylation could prevent cells from using oxygen for adenosine triphosphate (ATP) production and potentially could cause sepsis-induced organ failure. Our study has then investigated this possible mitochondrial impairment to explain the decreased O2 consumption rate observed in LPS-treated HK-2 cells. After exposure to LPS, functionality of mitochondria was affected without any disturbance in their spatial organization. LPS seemed rather to interrupt mitochondrial oxidative phosphorylation by blocking cytochrome c oxidase activity. As a consequence, disruptions in the electron transport and the proton pumping across the system occurred, leading to a decrease of the mitochondrial membrane potential, an electron leakage as the form of superoxide anion, a release of cytochrome c in the cytosol and a decrease in ATP production. This irreversible defect in the production of cellular energy would support the concept that kidney failure in sepsis may occur on the basis of cytopathic hypoxia. [less ▲]

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See detailESR spectroscopy for the study of an inflammation-induced AKI cellular model
Quoilin, Caroline ULg; Mouithys-Mickalad, Ange ULg; Gallez, Bernard et al

Poster (2012, November)

The kidney is faced to an impairment of oxygen extraction during sepsis which is well-known to be a risk factor for the development of acute kidney injury (AKI). Recent research activities in the ... [more ▼]

The kidney is faced to an impairment of oxygen extraction during sepsis which is well-known to be a risk factor for the development of acute kidney injury (AKI). Recent research activities in the mechanisms involved in the development of AKI in sepsis emphasize the central role of hemodynamic and inflammatory events. More particularly, two mechanisms are suggested to explain the inability of the injured kidney to extract oxygen: tissue hypoxia and cellular energetic metabolism dysfunction. Our working hypothesis of the pathophysiology of AKI is based on cellular respiratory dysfunction due to the inflammatory response inherent to sepsis. To study the mechanism of oxygen regulation in inflammation-induced acute kidney injury, we investigate the effects of a bacterial endotoxin (lipopolysaccharide, LPS) on the basal respiration of proximal tubular epithelial cells (HK-2) by ESR oximetry. This method has shown that HK-2 cells exhibit a decreased oxygen consumption rate when treated with LPS. Surprisingly, this cellular respiration alteration persists even after the stress factor was removed. We suggested that this irreversible decrease in renal oxygen consumption after LPS challenge is related to a pathologic metabolic down-regulation such as a lack of oxygen utilization by cells. This decrease was accompanied by increased nitric oxide (NO) production as measured by a spin trapping technique using ESR spectroscopy. This method is based on the trapping of NO by a metal-chelator complex consisting of N-methyl-D-glucamine dithiocarbamate (MGD) and reduced iron (Fe2+) forming a water-soluble NO-FeMGD complex detected by ESR. Since inducible NO synthase (iNOS) has been shown to play an important role in sepsis-induced AKI, the iNOS inhibitor L-NMMA (L-NG-monomethyl Arginine citrate) was tested in this in vitro model. L-NMMA blocked NO generation and permitted the HK-2 cells to recover a normal cellular respiration. Overall, ESR spectroscopy and the model of HK-2 cells exposed to LPS displays some key features of inflammation-induced acute kidney injury. [less ▲]

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See detailA confocal microscopic study of mitochondrial alterations of renal HK-2 cells exposed to an endotoxic stress
Quoilin, Caroline ULg; Mouithys-Mickalad, Ange ULg; Fontaine-Aupart, Marie-Pierre et al

Poster (2012, September)

Sepsis has a profound deleterious effect on kidney functions through complex mechanisms, which involve the immune response, inflammatory pathways, intracellular dysfunction and hemodynamic instability ... [more ▼]

Sepsis has a profound deleterious effect on kidney functions through complex mechanisms, which involve the immune response, inflammatory pathways, intracellular dysfunction and hemodynamic instability. Those factors are difficult to discriminate in vivo. To get a better understanding of renal respiratory dysfunction, we developed an in vitro model of sepsis-induced acute kidney injury using proximal tubular epithelial cell lines (HK-2) exposed to a bacterial endotoxin (lipopolysaccharide, LPS). Using this model, our first work has demonstrated that the basal respiration of renal HK-2 cells subjected to endotoxins was altered and presented a strong decrease in the oxygen consumption rates. Our working hypothesis of the pathophysiology of sepsis-induced AKI is based on a change in mitochondrial function that has been termed cytopathic hypoxia. A consequence of mitochondrial function alterations is an inability of the cell to use molecular oxygen for ATP production. The oxidative phosphorylation within mitochondria is interrupted because of the inhibition of cytochrome oxidase. The present investigation was carried out to establish whether mitochondrial alterations might be a mechanism of renal tubular epithelial injury during sepsis. To reach this goal the mitochondrial alterations of renal HK-2 cells exposed to an endotoxic stress was studied by confocal laser-scanning microscope. Confocal microscope allowed observation of the evoked phenomena at the single cell level and in real time. More particulary, mitochondrial morphology, mitochondrial membrane potential (ΔΨm) and generation of reactive oxygen species were recorded using specific vital fluorescent probes and quantified by image processing and analysis. Mitochondrial membrane potential is generated by the mitochondrial electron transport chain. This gradient is critical for the formation of ATP, and a fall in membrane potential is an indicator of mitochondrial dysfunction. ΔΨm was measured using the lipophilic cationic probe TMRE and it was shown that LPS produced a decrease in ΔΨm. In parallel, superoxide generation was measured by using MitoSOX which is selectively targeted to the mitochondria. There was a significant increase in mitochondrial superoxide-specific oxidation of MitoSOX when HK-2 cells were submitted to LPS. Overall, the model of HK-2 cells exposed to LPS displays some key features of sepsis-induced acute kidney injury. The confocal microscopy study has suggested a mechanism of toxicity dependent on mitochondrial oxidant generation and mitochondrial dysfunction. Indeed, the exposure to LPS has resulted in an increased generation of superoxide and a loss of mitochondrial function probably initiated by a fall in mitochondrial potential. [less ▲]

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Peer Reviewed
See detailEndotoxin-induced basal respiration alterations of renal HK-2 cells: A sign of pathologic metabolism down-regulation
Quoilin, Caroline ULg; Mouithys-Mickalad, Ange ULg; Duranteau, Jacques et al

in Biochemical and Biophysical Research Communications (2012), 423(2), 350-354

To study the mechanism of oxygen regulation in inflammation-induced acute kidney injury, we investigate the effects of a bacterial endotoxin (lipopolysaccharide, LPS) on the basal respiration of proximal ... [more ▼]

To study the mechanism of oxygen regulation in inflammation-induced acute kidney injury, we investigate the effects of a bacterial endotoxin (lipopolysaccharide, LPS) on the basal respiration of proximal tubular epithelial cells (HK-2) both by high-resolution respirometry and electron spin resonance spectroscopy. These two complementary methods have shown that HK-2 cells exhibit a decreased oxygen consumption rate when treated with LPS. Surprisingly, this cellular respiration alteration persists even after the stress factor was removed. We suggested that this irreversible decrease in renal oxygen consumption after LPS challenge is related to a pathologic metabolic down-regulation such as a lack of oxygen utilization by cells. [less ▲]

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See detailIn vitro ESR measurements: powerful tool to study toxic effects on cells
Grammenos, Angeliki ULg; Quoilin, Caroline ULg; Guelluy, Pierre-Henri ULg et al

Poster (2011, October)

Electron spin resonance spectroscopy (ESR) is a highly efficient technique able to access a wide range of information about the unfavourable effects caused by a chemical or a drug. ESR in spin labelling ... [more ▼]

Electron spin resonance spectroscopy (ESR) is a highly efficient technique able to access a wide range of information about the unfavourable effects caused by a chemical or a drug. ESR in spin labelling fits well in with the study of membranes and particularly with the changes in lipid bilayer organisation induced by drug. Our team previously developed a way to quantify the effective lipid bilayer microviscosity of cell membranes and consequently put in evidence the fluidity effect of the propofol. Recently, the importance of lipid raft domains has been shown due to their important role as a platform for signal transduction and protein sorting. We propose to highlight the effect of the Randomly methylated beta cyclodextrin (Rameb) on these domains on membrane model (liposomes) as well as on colon carcinoma cell line (HCT-116). Futhermore, ESR in spin trapping is used in order to identify and quantify the generation of Reactive Oxygen Species (ROS) in cells. An ESR study on human colon carcinoma cell line has highlighted the cytotoxicity of the photosensitizer pyrophephorbide-a methyl ester. Using an intracellular located spin trap (4-pyridyl 1-oxide-N-tert-butylnitrone, POBN), it has been shown that the photoexcitation of the dye is able to generate superoxide anions, hydroxyl radicals and singlet oxygen. Moreover, ESR is one of the most sensitive method for measuring cellular oxygen consumption. Our team has studied the alterations of oxygen respiratory in human tubular renal cells treated with an endotoxin (lipopolysaccharide, LPS). The incubation of HK-2 cells with LPS elicited a decreased in oxygen consumption suggesting a down-regulation of the cells metabolism. [less ▲]

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See detailEndotoxin-induced alterations in renal oxygen consumption: an ESR oximetry study
Quoilin, Caroline ULg; Grammenos, Angeliki ULg; Gallez, Bernard et al

Poster (2011, August)

The kidney, one of the most injured organs in critically ill patients, is faced with unique challenges for molecular oxygen regulation. Recent research activities in the pathophysiological mechanism of ... [more ▼]

The kidney, one of the most injured organs in critically ill patients, is faced with unique challenges for molecular oxygen regulation. Recent research activities in the pathophysiological mechanism of acute renal injury (ARI) emphasize the central role of hemodynamic and inflammatory events in septic shock. More particularly, two mechanisms have been postulated to explain the inability of the injured kidney to extract oxygen: tissue hypoxia and cellular energetic metabolism dysfunction. The present investigation was carried out to characterize the effects of bacterial endotoxin on the oxygen consumption of human tubular proximal cell line (PTC) by using the very sensitive electron spin resonance oximetry method. Oxygen consumption was shown to decrease quite markedly in cells treated with lipopolysaccharide (LPS) from 16.52 ± 2.51 (n=6) in the control group to: 12.94 ± 2.62 (n=3) in the short incubation time group (6h) and 10.86 ± 2.20 (n=3) in the long incubation time group (18h). This decrease in oxygen consumption in renal cells after LPS challenge may be in relation with a metabolic down-regulation. Renal energetic are deranged in sepsis not just because O2 delivery is impaired but perhaps also because the ability of cells to utilize available O2 is compromised. [less ▲]

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See detailESR evidence of superoxide anion, hydroxyl radical and singlet oxygen generation during photosensitization of PPME in HCT-116 cells
Quoilin, Caroline ULg; Guelluy, Pierre-Henri ULg; Grammenos, Angeliki ULg et al

Poster (2010, March)

Pyropheophorbide-a methyl ester (PPME), a derivative of chlorophyll a, is a second-generation photosensitizer and is studying largely in vitro for nearly a decade on cancerous cells. It has been ... [more ▼]

Pyropheophorbide-a methyl ester (PPME), a derivative of chlorophyll a, is a second-generation photosensitizer and is studying largely in vitro for nearly a decade on cancerous cells. It has been previously established on HCT-116 (human colon carcinoma cell line) that PPME is a molecule able to create apoptotic and necrotic death (Matroule et al). The cytotoxicity of PPME is presumed to be induced by reactive oxygen species (ROS) generated by the photoexcited molecule. Actually, to the best of our knowledge, no experimental evidence enables to confirm this supposition in an indubitable manner. Electron spin resonance (ESR) associated with spin trapping technique is a powerful method to detect, quantify and identify the ROS produced after photoactivation of PPME. Previous results indicate that PPME penetrates inside cells and localizes inside specific organelles (endothelial reticulum, Golgi apparatus and lysosome) (Matroule et al). The recent researches of Guelluy et al have also clearly demonstrated the presence of PPME inside mitochondrion. Consequently, ESR experiments were performed using an intracellular located spin trap, POBN (4-pyridyl 1-oxide-N-tert-butylnitrone), in order to detect in situ the ROS production. It has been shown that PPME is able to generate superoxide anions and hydroxyl radicals. Irradiation of the dye in HCT-116 cells in the presence of POBN spin trap and ethanol scavenger (2%, a non-toxic concentration) leads to the apparition of the ESR spectrum characteristics of POBN/ethoxy adduct. To assess the extent of contribution of ROS and to determine a possible reaction mechanism, competition experiments with specific quencher agents were carried out. Addition of catalase (CAT), a hydrogen peroxide quencher, or superoxide dismutase (SOD), a superoxide anion quencher, inhibits 30% of the signal. The parallel effect of SOD and CAT suggest that superoxide anion and hydrogen peroxide are involved in the generation of hydroxyl radicals via a Fenton reaction. This assertion is reinforced by the 20% reduction of signal intensity when adding desferroxamine, a Fe3+ chelator also implicated in Fenton reaction. Addition of DABCO, a quencher of singlet oxygen, to cells reduces 70% of the POBN/ethoxy adduct signal intensity. [less ▲]

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