Leticia Martínez-Caro

Role of peroxynitrite in sepsis-induced acute kidney injury in an experimental model of sepsis in rats

ABSTRACT The mechanisms involved in sepsis-induced acute kidney injury (AKI) are unknown. We investigated the role of nitrosative stress in sepsis-induced AKI by studying the effects of manganese (III) tetrakis-(1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP), a peroxynitrite decomposition catalyst, and aminoguanidine (AG), a selective nitric oxide synthase 2 (NOS2) inhibitor and peroxynitrite scavenger, on kidney function of rats subjected to cecal ligation and puncture (CLP). Sprague-Dawley rats (weighing 350 [SD, 50] g) were treated with MnTMPyP (6 mg/kg i.p.) or AG (50 mg/kg i.p.) at t = 12 and 24 h after CLP or sham procedure. At t = 36 h, mean arterial pressure and aortic blood flow were measured, and blood and urine samples were obtained for biochemical determinations, including creatinine clearance, fractional excretion of sodium, and neutrophil gelatinase-associated lipocalin concentration in the urine. Kidney tissue samples were obtained for (i) light microscopy, (ii) immunofluorescence and Western blot for 3-nitrotyrosine and NOS2, (iii) gene expression (quantitative real-time polymerase chain reaction) studies (NOS1, NOS2, NOS3, and superoxide dismutase 1), and (iv) matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Mean arterial pressure was unchanged and aortic blood flow decreased 25% in CLP animals. The sepsis-induced (i) decreased urine output and creatinine clearance and increased fractional excretion of sodium and urinary neutrophil gelatinase-associated lipocalin concentration, (ii) increased protein nitration and NOS2 protein, and (iii) NOS1 and NOS2 upregulation were all significantly attenuated by treatment with MnTMPyP or AG. Nitrated proteins in renal tissue from CLP animals (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) were glutamate dehydrogenase, methylmalonate-semialdehyde dehydrogenase, and aldehyde dehydrogenase, mitochondrial proteins involved in energy metabolism or antioxidant defense. Nitro-oxidative stress is involved in sepsis-induced AKI, and protein nitration seems to be one mechanism involved.

Pulmonary vascular dysfunction induced by high tidal volume mechanical ventilation.

Objectives:Acute lung injury and acute respiratory distress syndrome are characterized by increased pulmonary artery pressure and ventilation-perfusion mismatch. We analyzed the changes in the pulmonary vascular function in a model of ventilator-induced acute lung injury. Design:Controlled in vivo laboratory study. Setting:Animal research laboratory. Subjects:Anesthetized male Sprague-Dawley rats. Interventions:Rats were ventilated for 120 minutes using low tidal volume ventilation (control group, tidal volume 9 mL/kg, positive end-expiratory pressure 5 cm H2O, n = 15), high tidal volume ventilation (high tidal volume group, tidal volume 25 mL/kg, zero positive end-expiratory pressure, n = 14), or high tidal volume ventilation plus the poly-(adenosine diphosphate-ribose) polymerase inhibitor 3-aminobenzamide (10 mg/kg IP, high tidal volume group + 3-aminobenzamide group, n = 7). Vascular rings from small pulmonary arteries were mounted in a myograph for isometric tension recording. Lung messenger RNA and protein expression were analyzed by reverse transcriptase-polymerase chain reaction and Western blot, respectively. Measurements and Main Results:High tidal volume ventilation impaired phenylephrine- and acetylcholine-induced responses in pulmonary arteries in vitro, which were accompanied by induction of inducible nitric oxide synthase messenger RNA and protein. These effects, as well as hypoxemia and hypotension, were prevented by 3-aminobenzamide. Hypoxic pulmonary vasoconstriction and responses to exogenous sphingomyelinase were increased, whereas the responses to serotonin, Kv current density, and inhibition of Kv currents by hypoxia were unaffected by high tidal volume. Conclusions:High tidal volume ventilation-induced pulmonary vascular dysfunction was characterized by reduced alpha-adrenergic-induced vasoconstriction, reduced endothelium-dependent vasodilatation, and enhanced hypoxic pulmonary vasoconstriction. (Crit Care Med 2013; 41:e149–e155)

Fluidizing effects of C-reactive protein on lung surfactant membranes: protective role of surfactant protein A.

The purpose of this study was to investigate how surfactant membranes can be perturbed by C‐reactive protein (CRP) and whether surfactant protein A (SP‐A) might overcome CRP‐induced surfactant membrane alterations. The effect of CRP on surfactant surface adsorption was evaluated in vivo after intratracheal instillation of CRP into rat lungs. Insertion of CRP into surfactant membranes was investigated through monolayer techniques. The effect of CRP on membrane structure was studied through differential scanning calorimetry and fluorescence spectroscopy and microscopy using large and giant unilamellar vesicles. Our results indicate that CRP inserts into surfactant membranes and drastically increases membrane fluidity, resulting in surfactant inactivation. At 10% CRP/phospholipid weight ratio, CRP causes disappearance of liquid‐ordered/liquid‐disordered phase coexistence distinctive of surfactant membranes. SP‐A, the most abundant surfactant lipoprotein structurally similar to C1q, binds to CRP (Kd=56±8 nM), as determined by solid‐phase binding assays and dynamic light scattering. This novel SP‐A/CRP interaction reduces CRP insertion and blocks CRP effects on surfactant membranes. In addition, intratracheal coinstillation of SP‐A+ CRP into rat lungs prevents surfactant inhibition induced by CRP, indicating that SP‐A/CRP interactions might be an important factor in vivo in controlling harmful CRP effects in the alveolus.—Sáenz, A., López‐Sánchez, A., Mojica‐Lázaro, J., Martínez‐Caro, L., Nin, N., Bagatolli, L. A., Casals, C. Fluidizing effects of C‐reactive protein on lung surfactant membranes: protective role of surfactant protein A. FASEB J. 24, 3662–3673 (2010). www.fasebj.org

Vascular dysfunction in sepsis: effects of the peroxynitrite decomposition catalyst MnTMPyP.

The mechanisms contributing to sepsis vascular dysfunction are not well known. We tested the hypothesis that peroxynitrite scavenging ameliorates sepsis-induced macrovascular and microvascular dysfunction. Male Sprague-Dawley rats were killed 48 h after cecal ligation (n = 15) and puncture or sham procedure (n = 15). Their aortas and mesenteric vessels were mounted in organ baths for isometric tension recording. We studied contraction in resting vessels (norepinephrine 1 nM-10 &mgr;M and 10 nM-10 &mgr;M) and endothelium-dependent relaxation (acetylcholine, 10 nM-10 &mgr;M and 1 nM-10 &mgr;M) for aortas and microvessels, respectively. Vascular rings were preincubated for 30 min with the superoxide scavenger Cu-Zn-superoxide dismutase (SOD) (100 U/mL), the SOD mimetic and peroxynitrite scavenger tempol (10−4 M), the NO synthase inhibitor N-nitro-l-arginine methyl ester (10−4 M), or the peroxynitrite decomposition catalyst manganese tetrakis(4-N-methylpyridyl)porphyrin (MnTMPyP) (10−5 M). Fluorescence to 3-nitrotyrosine, oxidized dihydroethidium, and NOS2 was assessed in vascular tissue. Vascular NOS2, endothelial nitric oxide synthase (NOS1), NADPH-oxidase-1 (NOX-1), and SOD expression was analyzed by reverse transcription-polymerase chain reaction. Sepsis induced (i) in macrovessels, impairment of norepinephrine-induced contractions; (ii) in microvessels, impairment in norepinephrine-induced contractions and acetylcholine-induced relaxations; (iii) aortic and microvascular tissue increased reactivity to 3-nitrotyrosine, oxidized dihydroethidium, NOS2, and increased expression of NOS2, as well as increased expression of NOX-1 in microvascular tissue. Contractile responses in aortic and microvascular rings improved by ex vivo treatment with MnTMPyP and tempol, whereas vascular relaxation in microvessels improved only with MnTMPyP. Peroxynitrite scavenging protects from vascular dysfunction in sepsis.

A Metabolomic Approach to the Pathogenesis of Ventilator-induced Lung Injury.

Background:Global metabolic profiling using quantitative nuclear magnetic resonance spectroscopy (MRS) and mass spectrometry (MS) is useful for biomarker discovery. The objective of this study was to discover biomarkers of acute lung injury induced by mechanical ventilation (ventilator-induced lung injury [VILI]), by using MRS and MS. Methods:Male Sprague–Dawley rats were subjected to two ventilatory strategies for 2.5 h: tidal volume 9 ml/kg, positive end-expiratory pressure 5 cm H2O (control, n = 14); and tidal volume 25 ml/kg and positive end-expiratory pressure 0 cm H2O (VILI, n = 10). Lung tissue, bronchoalveolar lavage fluid, and serum spectra were obtained by high-resolution magic angle spinning and 1H-MRS. Serum spectra were acquired by high-performance liquid chromatography coupled to quadupole-time of flight MS. Principal component and partial least squares analyses were performed. Results:Metabolic profiling discriminated characteristics between control and VILI animals. As compared with the controls, animals with VILI showed by MRS higher concentrations of lactate and lower concentration of glucose and glycine in lung tissue, accompanied by increased levels of glucose, lactate, acetate, 3-hydroxybutyrate, and creatine in bronchoalveolar lavage fluid. In serum, increased levels of phosphatidylcholine, oleamide, sphinganine, hexadecenal and lysine, and decreased levels of lyso-phosphatidylcholine and sphingosine were identified by MS. Conclusions:This pilot study suggests that VILI is characterized by a particular metabolic profile that can be identified by MRS and MS. The metabolic profile, though preliminary and pending confirmation in larger data sets, suggests alterations in energy and membrane lipids.SUPPLEMENTAL DIGITAL CONTENT IS AVAILABLE IN THE TEXT

Redox Balance and Cellular Inflammation in the Diaphragm, Limb Muscles, and Lungs of Mechanically Ventilated Rats

Background:High tidal volume (VT) mechanical ventilation was shown to induce organ injury other than lung injury and systemic inflammation in animal models of ventilator-induced lung injury. The authors aimed to explore whether high VT mechanical ventilation per se induces early oxidative stress and inflammation in the diaphragm, limb muscles, and lungs of healthy rats exposed to ventilator-induced lung injury. Methods:Protein carbonylation and nitration, antioxidants (immunoblotting), and inflammation (immunohistochemistry) were evaluated in the diaphragm, gastrocnemius, soleus, tibialis anterior, and lungs of mechanically ventilated healthy rats and in nonventilated control animals (n = 8/group) for 1 h, using two different strategies (moderate VT [VT = 9 ml/kg] and high VT [VT = 35 ml/kg]). Results:The main findings are summarized as follows: compared with controls, (1) the diaphragms and gastrocnemius of high-VT rats exhibited a decrease in reactive carbonyls, (2) the soleus and tibialis of high- and moderate-VT rodents showed a reduction in reactive carbonyls and malondialdehyde-protein adducts, (3) the lungs of high-VT rats exhibited a significant rise in malondialdehyde-protein adducts, (4) the soleus and tibialis of both high- and moderate-VT rats showed a reduction in protein nitration, (5) the lungs of high- and moderate-VT rats showed a reduction in antioxidant enzyme levels, but not in the muscles, and (6) the diaphragms and gastrocnemius of all groups exhibited very low inflammatory cell counts, whereas the lungs of high-VT rats exhibited a significant increase in inflammatory infiltrates. Conclusions:Although oxidative stress and inflammation increased in the lungs of rats exposed to high VT, the diaphragm and limb muscles exhibited a decline in oxidative stress markers and very low levels of cellular inflammation.

Inhibition of Nitro-Oxidative Stress Attenuates Pulmonary and Systemic Injury Induced by High-Tidal Volume Mechanical Ventilation.

ABSTRACT Mechanisms contributing to pulmonary and systemic injury induced by high tidal volume (VT) mechanical ventilation are not well known. We tested the hypothesis that increased peroxynitrite formation is involved in organ injury and dysfunction induced by mechanical ventilation. Male Sprague-Dawley rats were subject to low- (VT, 9 mL/kg; positive end-expiratory pressure, 5 cmH2O) or high- (VT, 25 mL/kg; positive end-expiratory pressure, 0 cmH2O) VT mechanical ventilation for 120 min, and received 1 of 3 treatments: 3-aminobenzamide (3-AB, 10 mg/kg, intravenous, a poly adenosine diphosphate ribose polymerase [PARP] inhibitor), or the metalloporphyrin manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP, 5 mg/kg intravenous, a peroxynitrite scavenger), or no treatment (control group), 30 min before starting the mechanical ventilation protocol (n = 8 per group, 6 treatment groups). We measured mean arterial pressure, peak inspiratory airway pressure, blood chemistry, and gas exchange. Oxidation (fluorescence for oxidized dihydroethidium), protein nitration (immunofluorescence and Western blot for 3-nitrotyrosine), PARP protein (Western blot) and gene expression of the nitric oxide (NO) synthase (NOS) isoforms (quantitative real-time reverse transcription polymerase chain reaction) were measured in lung and vascular tissue. Lung injury was quantified by light microscopy. High-VT mechanical ventilation was associated with hypotension, increased peak inspiratory airway pressure, worsened oxygenation; oxidation and protein nitration in lung and aortic tissue; increased PARP protein in lung; up-regulation of NOS isoforms in lung tissue; signs of diffuse alveolar damage at histological examination. Treatment with 3AB or MnTMPyP attenuated the high-VT mechanical ventilation-induced changes in pulmonary and cardiovascular function; down-regulated the expression of NOS1, NOS2, and NOS3; decreased oxidation and nitration in lung and aortic tissue; and attenuated histological changes. Increased peroxynitrite formation is involved in mechanical ventilation-induced pulmonary and vascular dysfunction.