The uremic toxin methylguanidine increases the oxidative metabolism and accelerates the apoptosis of canine neutrophils.

We investigated the hypothesis that the increased concentration of plasma methylguanidine (MG) increases oxidative metabolism and accelerates apoptosis of neutrophils from dogs with chronic kidney disease (CKD). To achieve this, the levels of MG were quantified in healthy (n=16) and uremic dogs with CKD stage 4 of according to the guidelines of the International Renal Interest Society (IRIS, 2015) (n=16) using high performance liquid chromatography (HPLC). To evaluate the isolated effect of MG on neutrophil oxidative metabolism and apoptosis, neutrophils isolated from 12 healthy dogs were incubated with the highest concentration of plasma MG (0.005g/L) observed in dogs with CKD. Neutrophil oxidative metabolism was assessed by flow cytometry, using the probes hydroethidine for superoxide production and 2',7'-dichlorofluorescein diacetate for hydrogen peroxide production, with or without phorbol myristate acetate (PMA) stimulus. Neutrophil apoptosis and viability were also evaluated in flow cytometer using the Annexin V-PE system, with or without the apoptosis-inducing effect of camptothecin. Uremic dogs presented higher concentrations of MG (p<0.0001), increased oxidative stress and primed neutrophils with higher apoptosis rate. The neutrophil abnormalities observed in vivo were also reproduced in vitro, using cells isolated from healthy dogs and incubated with MG. We obtained strong evidence that in dogs with CKD, increased MG levels contributed to oxidative stress and potentially compromised the non-specific immune response by altering the oxidative metabolism and viability of canine neutrophils.

Evaluation of alternatives for dysfunctional double lumen central venous catheters using a two-compartmental mathematical model for different solutes.

Double lumen (DL) central venous catheters (CVC) often suffer from thrombosis, fibrin sheet formation, and/or suction towards the vessel wall, resulting in insufficient blood flow during hemodialysis. Reversing the catheter connection often restores blood flows, but will lead to higher recirculation. Single lumen (SL) CVCs have often fewer flow problems, but they inherently have some degree of recirculation. To assist bedside clinical decision making on optimal catheter application, we investigated mathematically the differences in dialysis adequacy using different modes of access with CVCs.
A mathematical model was developed to calculate reduction ratio (RR) and total solute removal (TSR) of urea, methylguanidine (MG), beta-2-microglobulin (ß2M), and phosphate (P) during different dialysis scenarios: 4-h dialysis with a well-functioning DL CVC (DL-normal, blood flow QB 350 ml/min), dysfunctional DL CVC (DL-low flow, QB 250), reversed DL CVC (DL-reversed, QB 350, recirculation R = 10%) and 12 Fr SL CVC (effective QB 273). 
With DL-normal as reference, urea RR was decreased by 3.5% (DL-reversed), 13.0% (SL), and 15.6% (DL-low flow), while urea TSR was decreased by 3.3% (DL-reversed), 13.2% (SL), and 13.5% (DL-low flow). The same trend was found for MG and P. However, ß2M RR decreased only 1.5% with SL CVC although TSR decrease was 17.2%, while RR decreased 21.1% with DL-low flow although TSR decrease was only 4.9%.
In the case of dysfunctional DL CVCs, reversing the catheter connection and restoring the blood flow did not impair TSR, with 10% recirculation. The SL CVC showed suboptimal TSR results that were similar to those of the dysfunctional DL CVC.

The protective role of nitric oxide and nitric oxide synthases in whole-body hyperthermia-induced hepatic injury in rats.

PURPOSE: The present study was designed to elucidate the role of endothelial nitric oxide (NO) synthase (eNOS), inducible NOS (iNOS)-derived NO and heat-shock protein (Hsp70) in a rat model of whole-body hyperthermia (WBH)-induced liver injury. MATERIALS AND METHODS: Real-time polymerase chain reaction, immunohistochemistry and western blot were used to observe the mRNA and protein expression of eNOS, iNOS and Hsp70. Rats were exposed to hyperthermia by immersion for 60 min at a conscious state in a water bath maintained at 41°C. Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were used to assess liver injury 15 h after the hyperthermia challenge. Nitrosative and oxidative mediators, particularly NO and hydroxyl radical were measured. RESULTS: Plasma AST, ALT, hydroxyl radical, and NO were significantly increased after WBH. There were 4.14 ± 0.42, 2.82 ± 0.34 and 2.91 ± 0.16-fold increases in the mRNA expression of eNOS, iNOS and Hsp70. Immunohistochemistry and western blot showed up-regulation of eNOS, iNOS and Hsp70 protein. An eNOS inhibitor (N(ω)-nitro-L-arginine methyl ester (L-NAME)), or an iNOS inhibitor (aminoguanidine (AG)), significantly aggravated the liver injury. On the contrary, administration of NO precursor, L-arginine (L-ARG), attenuated the liver injury. Hsp70 inhibitor quercetin reduced Hsp70, while aggravating the WBH-induced hepatic changes. CONCLUSIONS: WBH induces increases in eNOS, iNOS and Hsp70 expression with increase in NO release. The deleterious effects of L-NAME and AG and the protective effects of L-ARG and Hsp70 inhibitor on the liver function and pathology suggest that NO and heat shock protein play a beneficial role in the WBH-induced hepatic injury.

L-ascorbic acid and alpha-tocopherol attenuates liver ischemia-reperfusion induced of cardiac function impairment.

OBJECTIVES: The Pringle maneuver is a surgical procedure to minimize hemorrhage during hepatectomy, which however, can induce production of reactive oxygen species causing remote organ injury. We sought to study the impact of the Pringle maneuver on cardiac function as well as the protective effects of L-ascorbic acid and α-tocopherol pretreatments. METHODS: Rats were divided into four study groups: L-ascorbic acid (60 mg/kg/d) or α-tocopherol (200 mg/kg/d), and surgical interventions (Sham-operated or liver ischemia-reperfusion [I/R]). Liver ischemia was performed by clamping the hepatic artery and portal vein for 30 minutes, followed by reperfusion by releasing the clamps for 2 hours. Cardiac function was evaluated by a high-fidelity pressure-volume catheter positioned in the left ventricle. Myocardial injury was assessed through plasma creatine kinase-MB (CKMB) and troponin I (cTnI). Cardiac lipid peroxidation and systemic hydroxyl radical levels were assessed by cardiac tissue malondialdehyde and plasma methylguanidine, respectively. RESULTS: Cardiac function was significantly depressed in the I/R group, where plasma CKMB and cTnI were markedly increased (P < .05). L-ascorbic acid and α-tocopherol pretreatments improved cardiac function and significantly reduced cardiac injury (P < .05). L-ascorbic acid pretreatment demonstrated better heart protection than α-tocopherol, in terms of cTnI and CKMB (P < .05), but no significant difference in terms of cardiac functional improvement. CONCLUSIONS: L-ascorbic acid and α-tocopherol pretreatment 3 days prior to the Pringle maneuver attenuated myocardial injury and protected cardiac function by scavenging hydroxyl radical and reducing lipid peroxidation. L-ascorbic acid demonstrated better protection than α-tocopherol.

Preischemic treatment with melatonin attenuates liver reperfusion-induced impairment of cardiac function.

OBJECTIVES: Cardiac functional impairment is frequently observed in patients with end-stage liver disease and after reperfusion of an ischemic liver. Excessive production of reactive oxygen species (ROS) through activation of Kupffer cells and leukocytes during reperfusion may play important roles. We evaluated the cardiac protective effects of preischemic treatment with melatonin. METHODS: Studies were performed on 3 groups of male Sprague-Dawley rats; shame-operated controls, liver ischemia and reperfusion (I/R), and melatonin pretreatment prior to I/R. Liver I/R was performed by clamping the hepatic artery and portal vein for 30 minutes, followed by releasing the clamps for 2 hours. The cardiac function was assessed using a high-fidelity dual pressure-volume catheter positioned in the left ventricle (LV). We also evaluated heart injury using plasma creatine kinase-MB (CKMB), and Troponin I (cTnI). The level of hydroxyl radical production was evaluated using plasma methylguanidine (MG). RESULTS: LV function was severely impaired after 2 hours of reperfusion; stroke volume and LV contractility were significantly reduced (P < .05). Markedly increased CKMB and cTnI indicated serious myocardial injury. Preischemic treatment with melatonin protected the heart as seen by the reduced plasma CKMB and cTnI (P < .05), and decreased systemic hydroxyl radical production. CONCLUSIONS: Liver I/R severely impaired cardiac functions by production of hydroxyl radicals. Melatonin pretreatment effectively scavenged oxidants and hydroxyl radicals, protecting cardiac function against liver I/R-induced injury.

The protective effect of curcumin on ischemia-reperfusion-induced liver injury.

OBJECTIVE: Reperfusion of the ischemic liver results in the generation of oxidative and nitrosative stresses and reaction product of peroxynitrite, which induce rapid cytotoxicity and liver injury. In this study we demonstrated that curcumin, an antioxidant, attenuated ischemia/reperfusion (I/R)-induced liver injury. MATERIALS AND METHODS: Ischemia was induced by clamping the common hepatic artery and portal vein of rats for 30 minutes. Thereafter, flow was restored and the liver was reperfused for 80 minutes. Blood samples collected prior to ischemia and after reperfusion were analyzed for methyl guanidine (MG), nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), and adenosphate triphosphate (ATP). Blood levels of serum glutamic oxaloacetic transaminase (sGOT), serum glutamate pyruvate transaminase (sGPT), and lactic dehydrogenase (LDH), which served as indexes of liver injury, were measured. RESULTS: The protocol resulted in elevation of blood NO (P < .001), TNF-α (P < .001), and MG (P < .001). sGOT, sGPT, and LDH were elevated significantly (P < .001), whereas ATP was significantly diminished (P < .001). Pretreatment with curcumin (25 mg/kg) significantly attenuated the reperfusion liver injury, while the ATP content reversed. In addition, MG, TNF-α, and NO release were attenuated. CONCLUSIONS: These results indicated that curcumin exerted potent anti-inflammatory effects in I/R-induced liver injury due to its antioxidant effects.

Curcumin attenuates airway hyperreactivity induced by ischemia-reperfusion of the pancreas in rats.

OBJECTIVES: Ischemia-reperfusion (I/R) of the rat pancreas induces acute pancreatitis with a systemic inflammatory response. Activated inflammatory cells are sequestered in the lung, and the consequent respiratory burst may increase airway reactivity. In this study, we characterized the effect of the antioxidant curcumin on airway hyperreactivity induced by pancreatic I/R. METHODS: Ischemia of the pancreas was induced by clamping the gastroduodenal and the splenic artery for 2 hours followed by reperfusion for 6 hours. The pulmonary function data of Penh, a measurement of airway resistance, were used to show the airway responses to a methacholine challenge. The blood concentration of oxygen radicals, nitric oxide, and tumor necrosis factor-alpha (TNFalpha) were measured after pancreatic I/R. mRNA expressions of inducible nitric oxide synthase (iNOS) and TNFalpha in lung tissues were measured after pancreatic I/R. Pretreatment with curcumin (20 mg/kg) was administered by intraperitoneal injection 2 hours before pancreatic I/R. RESULTS: The protocol resulted in significant elevations of the blood concentrations of amylase, hydroxyl radical, nitric oxide, TNFalpha, and white cells among the I/R group. iNOS and TNFalpha mRNA expressions also significantly increased in lung tissues. Pulmonary function data showed that pancreatic I/R induced significant increases in responses to methacholine challenge: Penh increased significantly in the I/R group when compared with the sham group. Pretreatment with curcumin significantly attenuated the inflammatory, oxidative, and nitrosative responses and lung tissue iNOS and TNFalpha expressions. Curcumin also attenuated airway reactivity to methacholine challenge. CONCLUSIONS: I/R of the pancreas induced systemic inflammatory responses with respiratory burst, nitrosative stress, and hyperresponses in the airways. Curcumin, which has antioxidant and anti-inflammatory effects, significantly attenuated the inflammatory responses and airway hyperreactivity induced by pancreatic I/R.

Enhancement effects of hypercapnia on the acute lung injury caused by acid aspiration.

Acid aspiration or intrapulmonary instillation of gastric particles causes lung inflammation leading to acute lung injury (ALI). Hypercapnia exerts different effects on ALI caused by various insults. The effects of hypercapnia on lung inflammation and injury due to acid aspiration are yet to be determined. The present study was designed to investigate the involvement of inducible nitric oxide synthase (iNOS) and other mediators in acid-aspiration-induced ALI. We also sought to evaluate the effects of hypercapnia on the lung and associated changes induced by acid aspiration. We used Spague-Dawley rats anesthetized with intraperitioneal pentobarbital (40 mg/kg). Gastric acid particles were prepared from the stomach contents of rats at necropsy. The rats were randomly assigned to receive intratracheal instillation of physiological saline solution (PSS) at pH 7.24 (Control group), PSS at pH 1.25 (Low pH, LPH group), gastric particles (GP group), and GP with low pH PSS (GPLPH group). There were 10 rats in each group. The animals were observed for 6 hrs. To evaluate the effects of hypercapnia, we carried out two series of experiments: one under normocapnia and the other under hypercapnia with alteration of CO2 fraction in inspired air. Arterial pressure (AP) was monitored from the femoral arterial catheter. Heart rate was obtained from AP traicing. We determined the blood gases and acid-base status. Lung weight to body weight (LW/BW) ratio, LW gain (LWG), protein concentration in bronchoalveolar lavage (PCBAL) and leakage of Evans blue dye tracer were measured. Plasma nitrate/nitrite, methyl guanidine (MG), myeloperoxidase (MPO), phospholipase A2 (PLA2), proinflammatory cytokines were assessed. Histopathological examination of the lung tissue was performed. We employed reverse-transcriptase polymerase chain reaction to detect the expression of iNOS mRNA. GP and GPLPH caused hypotension, decreases in PaO2, pH and SaO2, and an increase in PaCO2. The insults also elevated LW/BW, LWG, PCBAL and dye leakage, plasma nitrate/nitrite, MG, MPO, PLA2, tumor necrosis factor(alpha), interleukin-beta and interleukin-6. The lung pathology was characterized by alveolar edema and hemorrhage with inflammatory cells infiltration. Assessment of lung injury score revealed that GP and GPLPH caused ALI. Furthermore, hypercapnia significantly enhanced ALI and associated changes following LPH, GP and GPLPH. Intratracheal instillation of GP in normal or low pH PSS causes ALI accompanied with biochemical changes. The release of nitric oxide via iNOS isoform is detrimental to the lung. Hypercapnia tended to enhance ALI and associated changes induced by gastric acid instillation.

First indications demonstrating the preventive effects of NZ-419, a novel intrinsic antioxidant, on the initiation and/or progression of chronic renal failure in rats.

The concentration of NZ-419 (5-hydroxy-1-methylimidazolidine-2,4-dione), an intrinsic antioxidant, has been shown to increase in the sera of animals and patients with chronic renal failure (CRF). This is the first report that orally administered exogenous NZ-419 prevents the initiation and/or progression of CRF in rats using an adenine-loaded model. After 24 d of adenine loading, there was a ca. 90% decrease in creatinine clearance (C(Cr)) in the control rats. Treatment with NZ-419 from the beginning significantly inhibited the decrease in C(Cr) and also the increase in serum creatinine (sCr). Bio-markers for in vivo hydroxyl radicals, the serum methylguanidine (sMG) level, and sMG/sCr molar ratio, not only in serum but also in the urine, kidney, liver, and muscle indicated that NZ-419 inhibited the increase in oxidative stress induced by CRF in rats. An increase of guanidinosuccinic acid, an another bio-marker of oxidative stress, was also inhibited with NZ-419.

Oxidative and nitrosative mediators in hepatic injury caused by whole body hyperthermia in rats.

The involvement of oxidative and nitrosative mediators in liver injury caused by heat stress remains unclear. This study aimed to elucidate the role of endothelial nitric oxide synthase (eNOS), and inducible NOS (iNOS)-derived NO and nitrotyrosine in the whole-body hyperthermia (WBH)-induced liver injury. Rats were anesthetized with intraperitoneal pentobarbital, and were exposed to a heating lamp for 60 min to raise the core temperature to 42.5 degrees C. The rats were maintained at the hyperthermic state for an additional 50 min. Blood urea nitrogen, creatinine, aspartate aminotransferase, alanine aminotransferase, lactic dehydrogenase, creatine phosphokinase, amylase, lipase, nitrate/nitrite, methyl guanidine, and proinflammatory cytokines (tumor necrosis factoralpha, interleukin-1beta and interleukin-10) were measured before and 14 h after hyperthermia. Immunohistochemical staining was employed to detect the eNOS, iNOS and nitrotyrosine levels. Western blotting was used to examine the expression of heatshock protein 70 (HSP 70). Histopathological examination of the liver tissue was performed. WBH caused liver injury accompanied with significant increases in biochemical factors, nitrate/nitrite, methyl guanidine, and proinflammatory cytokines. In addition, WBH enhanced the eNOS, iNOS, nitrotyrosine and HSP 70 levels. WBH caused hepatic injury. The pathogenetic mechanism is likely mediated through the NOS-derived NO, free radical, proinflammatory cytokines and nitrotyrosine. The enhanced expression of HSP 70 may play a protective role.

The detrimental role of inducible nitric oxide synthase in the pulmonary edema caused by hypercalcemia in conscious rats and isolated lungs.

We aim to test the hypothesis that hypercalcemia produces pulmonary edema (PE) and to elucidate the mechanism. Experimentations were carried out in conscious rats and isolated perfused rat lungs. We evaluated PE by lung weight changes, protein concentration in bronchoalveolar lavage, dye leakage, and microvascular permeability. Plasma nitrate/nitrite, methyl guanidine (MG), proinflammatory cytokines, procalcitonin levels, and histopathological examinations were evaluated. Immunochemical staining and reverse-transcriptase polymerase chain reaction (RT-PCR) were used to detect inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) in the lungs. Hypercalcemia was produced in the conscious rat and isolated perfused lungs. Calcitonin and L-N(6) (1-iminoethyl)-lysine (L-Nil) were administered before hypercalcemia to observe their effects. Hypercalcemia caused severe PE in rats. Pathological and immunochemical examinations revealed hemorrhagic edema with iNOS activity in the alveolar macrophages and epithelial cells. RT-PCR showed an increase in iNOS mRNA expression. Hypercalcemia increased nitrate/nitrite, MG, proinflammatory cytokines and procalcitonin levels. Pretreatment with calcitonin or L-Nil prevented these changes. In conclusion, hypercalcemia caused PE in conscious rats and isolated perfused rat lungs. The increases in nitrate/nitrite, free radicals, proinflammatory cytokines, procalcitonin and iNOS activity suggest that hypercalcemia induces a sepsis-like syndrome. The effect of hypercalcemia on the lung may involve iNOS and NO.

Ischemia/reperfusion of the liver induces heart injury in rats.

OBJECTIVE: We evaluated the cardiovascular injury induced by ischemia and reperfusion (I/R) of the liver by measuring changes in blood levels of cardiac troponin I (cTNI), an index of cardiovascular injury, as well as levels of selected indicators of an inflammatory response. MATERIALS AND METHODS: Ischemia was induced in the rat liver by clamping the common hepatic artery and portal vein for 40 minutes, after which flow was restored, and the liver reperfused for 90 minutes. Blood samples were collected prior to ischemia and after reperfusion. cTNI as well as levels of tumor necrosis factor alpha (TNFalpha), hydroxyl radical (.OH), nitric oxide (NO), and alanine transferase (ALT) were measured. RESULTS: I/R of the liver induced a significant increase in ALT (P<.001). Increased cTNI levels (P<.05) were associated with inflammatory responses, such as elevated levels of TNFalpha (P<.001), . OH (P<.001), and NO (P<.001). After administration of 3-aminobenzamide, a poly(ADP-ribose) polymerase (PARP) inhibitor, liver and heart injuries were significantly attenuated (P<.05). CONCLUSIONS: I/R-induced liver injury was associated with cardiovascular injury, perhaps resulting from inflammatory responses triggered by elevated levels of reactive radical species of nitric oxide, superoxide, and peroxynitrite, by which PARP was activated. 3-Aminobenzamide, significantly attenuated I/R-induced liver and heart injuries.

Urinary excretion of creatol, an in vivo biomarker of hydroxyl radical, in patients with chronic renal failure.

Creatol (CTL) is a hydroxyl radical adduct of creatinine (Cr). The serum methylguanidine (MG) level and the MG/Cr molar ratio are reported to be biomarkers for oxidative stress. The aim of this study was to examine whether urinary excretion of CTL, another oxidative stress-related marker, is increased in patients with chronic renal failure (CRF). One hundred twenty-four non-dialyzed patients with chronic renal failure (serum Cr level, 1.3-10.0 mg/dL) were recruited from our hospitals. Urine and serum levels of CTL and MG were determined by high-performance liquid chromatography with the use of 9, 10- phenanthrenequinone as a fluorogenic reagent. The CTL/Cr and (CTL+MG)/Cr molar ratios in spot urine samples were also compared with those in 24-h urine samples. The urinary CTL/Cr and (CTL+MG)/Cr molar ratios increased with decreases in Cr clearance in patients with CRF. Correlations between serum and spot urine (CTL+MG)/Cr and between serum and spot urine CTL/Cr were quite similar to those in 24-h urine samples. CTL/Cr and (CTL+MG)/Cr molar ratios in both 24-h urine and spot urine samples appear to be useful indices of the severity of CRF.

Clinical and pathological features of fat embolism with acute respiratory distress syndrome.

FES (fat embolism syndrome) is a clinical problem, and, although ARDS (acute respiratory distress syndrome) has been considered as a serious complication of FES, the pathogenesis of ARDS associated with FES remains unclear. In the present study, we investigated the clinical manifestations, and biochemical and pathophysiological changes, in subjects associated with FES and ARDS, to elucidate the possible mechanisms involved in this disorder. A total of eight patients with FES were studied, and arterial blood pH, PaO(2) (arterial partial pressure of O(2)), PaCO(2) (arterial partial pressure of CO(2)), biochemical and pathophysiological data were obtained. These subjects suffered from crash injuries and developed FES associated with ARDS, and each died within 2 h after admission. In the subjects, chest radiography revealed that the lungs were clear on admission, and pulmonary infiltration was observed within 2 h of admission. Arterial blood pH and PaO(2) declined, whereas PaCO(2) increased. Plasma PLA(2) (phospholipase A(2)), nitrate/nitrite, methylguanidine, TNF-alpha (tumour necrosis factor-alpha), IL-1beta (interleukin-1beta) and IL-10 (interleukin-10) were significantly elevated. Pathological examinations revealed alveolar oedema and haemorrhage with multiple fat droplet depositions and fibrin thrombi. Fat droplets were also found in the arterioles and/or capillaries in the lung, kidney and brain. Immunohistochemical staining identified iNOS (inducible nitric oxide synthase) in alveolar macrophages. In conclusion, our clinical analysis suggests that PLA(2), NO, free radicals and pro-inflammatory cytokines are involved in the pathogenesis of ARDS associated with FES. The major source of NO is the alveolar macrophages.

Inhibition of inducible nitric oxide synthase attenuates acute endotoxin-induced lung injury in rats.

1. In the present study, we investigated the effects of the inducible nitric oxide (iNOS) inhibitors S-methylisothiourea (SMT) and l-N(6)-(1-iminoethyl)-lysine (l-Nil) on endotoxin-induced acute lung injury (ALI), as well as the associated physiological, biomedical and pathological changes, in anaesthetized Sprague-Dawley rats and in rat isolated perfused lungs. 2. Endotoxaemia was induced by an intravenous (i.v.) infusion of lipopolysaccharide (LPS; Escherichia coli 10 mg/kg). Lipopolysaccharide produced systemic hypotension and tachycardia. It also increased the lung weight/bodyweight ratio, lung weight gain, exhaled nitric oxide (NO), the protein concentration in bronchoalveolar lavage and microvascular permeability. 3. Following infusion of LPS, plasma nitrate/nitrite, methyl guanidine, pro-inflammatory cytokines (tumour necrosis factor-alpha and interleukin-1beta) were markedly elevated. Pathological examination revealed severe pulmonary oedema and inflammatory cell infiltration. Pretreatment with SMT (3 mg/kg, i.v.) or l-Nil (3 mg/kg, i.v.) significantly attenuated the LPS-induced changes and ALI. 4. The results suggest that the inflammatory responses and ALI following infusion of LPS are due to the production of NO, free radicals and pro-inflammatory cytokines through the iNOS system. Inhibition of iNOS is effective in mitigating the endotoxaemic changes and lung pathology. Inhibitors of iNOS may be potential therapeutic agents for clinical application in patients with acute respiratory distress syndrome.

Endotoxin-induced acute lung injury is enhanced in rats with spontaneous hypertension.

1. Acute lung injury (ALI), or acute respiratory distress syndrome, is a major cause of mortality in endotoxaemia. The present study tested whether the endotoxaemia-induced changes and associated ALI were enhanced in rats with established hypertension and to examine the possible mechanisms involved. 2. Fifty spontaneously hypertensive rats (SHR) and the same number of normotensive Wistar Kyoto (WKY) rats, aged 12-15 weeks, were used. The experiments were performed in conscious, unanaesthetized rats. Endotoxaemia was produced by intravenous lipopolysaccharide (LPS; 10 mg/kg). N(G)-Nitro-L-arginine methyl ester (L-NAME; 10 mg/kg, i.v.), L-N(6)-(1-iminoethyl)-lysine (L-Nil; 5 mg/kg, i.v.) and 3-morpholinosydnonimine (SIN-1; 5 mg/kg, i.v.) were given 5 min before LPS to observe the effects of nitric oxide synthase (NOS) inhibition and nitric oxide (NO) donation. 3. We monitored arterial pressure and heart rate and evaluated ALI by determining the lung weight/bodyweight ratio, lung weight gain, leakage of Evans blue dye, the protein concentration in bronchoalveolar lavage and histopathological examination. Plasma nitrate/nitrite, methyl guanidine, pro-inflammatory cytokines, including tumour necrosis factor-alpha and interleukin-1beta, and lung tissue cGMP were determined. Expression of mRNA for inducible and endothelial NOS was examined using reverse transcription-polymerase chain reaction. 4. Lipopolysaccharide caused systemic hypotension, ALI and increases in plasma nitrate/nitrite, methyl guanidine, pro-inflammatory cytokines and lung cGMP content. The LPS-induced changes were greater in SHR than in WKY rats. Pretreatment with L-NAME or L-Nil attenuated, whereas the NO donor SIN-1 aggravated, the endotoxin-induced changes. 5. In conclusion, rats with genetic hypertension are more susceptible to endotoxaemia and this results in a greater extent of ALI compared with normotensive WKY rats.

Propofol exerts protective effects on the acute lung injury induced by endotoxin in rats.

Acute lung injury (ALI) is a major culprit of mortality in endotoxemia. Propofol has been commonly used in critical ill patients for sedation. This experiment attempted to elucidate the effects and possible mechanisms of propofol on the ALI induced by endotoxin. Experimentations were carried out using anesthetized, ventilated rats and isolated perfused rat lungs. Endotoxemia was induced by intravenous lipopolysaccharide (LPS, 10 mg kg(-1)). Various groups of rats received infusion of physiological saline solution (PSS) and LPS. Five min after LPS, propofol at low dose (5 mg kg(-1)h(-1)) or high dose (10 mg kg(-1)h(-1)) was infused for 6h. In isolated perfused rat lungs, PSS, LPS, and propofol (30 or 60 mg kg(-1)) were added into the perfusion circuit. During or after 6h observation, we determined the lung weight (LW)/body weight ratio, LW gain, exhaled nitric oxide (NO) and protein concentration in broncheoalveolar lavage. Lung pathology was evaluated to quantify the lung injury score. Plasma nitrate/nitrite, methyl guanidine (MG), tumor necrosis factor(alpha), and interleukin-1(beta) were examined. Blood leukocytes were counted. Capillary filtration coefficient (K(fc)) was obtained in isolated perfused lungs. Posttreatment of propofol at low or high dose attenuated or prevented the extent of ALI. It also reduced the plasma nitrate/nitrite, MG, and pro-inflammatory cytokines including tumor necrosis factor(alpha) (TNF(alpha)) and interleukin-1(beta) (IL-1(beta)). In the isolated perfused rat lungs, propofol significantly reduced the LPS-induced increase in K(fc). This agent did not affect the leukocytopenia caused by LPS. Accordingly, the effects of propofol on the ALI were not related to leukocyte activation or sequestration. Our results suggest that propofol exerts protective effect on the endotoxin-induced ALI. The mechanisms of actions may be mediated through inhibition on the release of pro-inflammatory cytokines, free radicals and NO. In addition, propofol abrogates the microvascular leakage of water and protein in the lungs. The results imply that the use of propofol in critically ill is not only for sedation, but also useful for the prevention of inflammatory progression and lung damage.

N-acetylcysteine abrogates acute lung injury induced by endotoxin.

1. Acute lung injury (ALI) or acute respiratory distress syndrome is a serious clinical problem with high mortality. N-Acetylcysteine (NAC) is an anti-oxidant and a free radical scavenger. It has been reported recently that NAC ameliorates organ damage induced by endotoxin (lipopolysaccharide; LPS) in conscious rats. The present study was designed to evaluate the effects of NAC on LPS-induced ALI and other changes in anaesthetized rats. 2. Sprague-Dawley rats were anaesthetized with pentobarbital (40 mg/kg, i.p.). Endotracheal intubation was performed to provide artificial ventilation. Arterial pressure and heart rate were monitored. The extent of ALI was evaluated with the lung weight (LW)/bodyweight ratio, LW gain, exhaled nitric oxide (NO) and protein concentration in bronchoalveolar lavage (PCBAL). Haematocrit, white blood cells, plasma nitrate/nitrite, methyl guanidine (MG), tumour necrosis factor (TNF)-alpha and interleukin (IL)-1b were measured. Pathological changes in the lung were examined and evaluated. 3. Endotoxaemia was produced by injection of 10 mg/kg, i.v., LPS (Escherichia coli). Animals were randomly divided into three groups. In the vehicle group, rats received an i.v. drip of physiological saline solution (PSS) at a rate of 0.3 mL/h. The LPS group received an i.v. drip of PSS for 1 h, followed by LPS (10 mg/kg by slow blous injection, i.v., over 1-2 min). Rats in the LPS + NAC group received NAC by i.v. drip at a rate of 150 mg/kg per h (0.3 mL/h) for 60 min starting 10 min before LPS administration (10 mg/kg by slow blous injection, i.v., over 1-2 min). Each group was observed for a period of 6 h. 4. N-Acetylcysteine treatment improved the LPS-induced hypotension and leukocytopenia. It also reduced the extent of ALI, as evidenced by reductions in LW changes, exhaled NO, PCBAL and lung pathology. In addition, NAC diminished the LPS-induced increases in nitrate/nitrite, MG, TNF-a and IL-1b. 5. In another series of experiments, LPS increased the mortality rate compared with the vehicle group (i.v. drip of PSS at a rate of 0.3 mL/h) during a 6 h observation period. N-Acetylcysteine, given 10 min prior to LPS, significantly increased the survival rate. 6. The results of the present study suggest that NAC exerts a protective effect on the LPS-induced ALI. The mechanisms of action may be mediated through the reduction of the production of NO, free radicals and pro-inflammatory cytokines.

Ascorbate supplement reduces oxidative stress in dyslipidemic patients undergoing apheresis.

OBJECTIVE: The effect of ascorbate treatment on apheresis-induced oxidative stress in uremic and dyslipidemic patients was evaluated. METHODS AND RESULTS: We developed a chemiluminescence-emission spectrum and high-performance liquid chromatography analysis to assess the effect of ascorbate supplement on plasma reactive oxygen species (ROS) scavenging activity and oxidized lipid/protein production in hyperlipidemic and uremic patients undergoing apheresis. Apheresis was efficient in reduction of atherogenic lipoproteins, complement, fibrinogen, soluble intercellular adhesion molecule-1, and oxidative parameters including phosphatidylcholine hydroperoxide (PCOOH), malonaldehyde, methylguanidine, and diotyrosine. Apheresis itself, however, activated leukocytes to increase ROS activity and reduced the plasma ROS scavenging activity. Ascorbate administration selectively diminished apheresis-enhanced H2O2 and inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant protein-1. Chronically dyslipidemic and uremic patients undergoing biweekly apheresis plus ascorbate treatment had lower levels of C-reactive protein and PCOOH than did those without ascorbate treatment during a 6-month follow-up study period. CONCLUSIONS: We demonstrate that apheresis with ascorbate treatment provides a therapeutic potential in reducing atherosclerotic risk via inhibition of H2O2-induced oxidative stress in patients with uremia or dyslipidemia.

N-acetylcysteine ameliorates lipopolysaccharide-induced organ damage in conscious rats.

Lipopolysaccharide is strongly associated with septic shock, leading to multiple organ failure. It can activate monocytes and macrophages to release proinflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and nitric oxide (NO). The present experiments were designed to induce endotoxin shock by an intravenous injection of Klebsiella pneumoniae lipopolysaccharide (LPS, 10 mg/kg) in conscious rats. Arterial pressure and heart rate (HR) were continuously monitored for 48 h after LPS administration. N-Acetylcysteine was used to study its effects on organ damage. Biochemical substances were measured to reflect organ functions. Biochemical factors included blood urea nitrogen (BUN), creatinine (Cre), lactic dehydrogenase (LDH), creatine phosphokinase (CPK), aspartate transferase (GOT), alanine transferase (GPT), TNF-alpha, IL-1 beta, methyl guanidine (MG), and nitrites/nitrates. LPS caused significant increases in blood BUN, Cre, LDH, CPK, GOT, GPT, TNF-alpha, IL-1 beta, MG levels, and HR, as well as a decrease in mean arterial pressure and an elevation of nitrites/nitrates. N-Acetylcysteine suppressed the release of TNF-alpha, IL-1 beta, and MG, but enhanced NO production. These actions ameliorate LPS-induced organ damage in conscious rats. The beneficial effects may suggest a potential chemopreventive effect of this compound in sepsis prevention and treatment.