Protective effect of PEG 35,000 Da on renal cells: paradoxical activation of JNK signaling pathway during cold storage.

Polyethylene glycol (PEG), a high-molecular weight colloid, is added to preservation solutions in order to decrease cold- and ischemia-induced injuries of the grafted organ. We evaluated on LLC-PK1, a porcine proximal tubular epithelial cell line (1) the efficiency of several commercial preservation solutions (University of Wisconsin, Euro-Collins, Celsior, SCOT, IGL-1), and (2) whether adding PEG (400-35,000 Da) in a simple extracellular-type buffer modified cell integrity and mitogen-activated protein kinase (MAPK) signaling pathways. SCOT was the most efficient commercial solution. Moreover, only PEG 35,000 Da totally preserved cell viability, induced a decrease on reactive oxygen species production and a decrease on p38-MAPK activation. Furthermore PEG 35,000 Da stimulated c-Jun N-terminal kinase (JNK). However, the inhibition of JNK pathway, with the specific SP600125 inhibitor, in the presence of PEG 35,000 Da did not affect cell survival. We also confirmed on whole pig kidney the protective effect of PEG 35,000 Da on cold-induced tubular injuries. This study confirms PEG antioxidative properties, but we demonstrate that its effect on JNK signaling pathway had also a paradoxical effect on cell death. This sheds a new light on PEG effects during cell preservation, independently from the classical immuno-camouflaging hypothesis.

Plasmalogen degradation by oxidative stress: production and disappearance of specific fatty aldehydes and fatty alpha-hydroxyaldehydes.

Plasmalogens are often considered as antioxidant molecules that protect cells from oxidative stress. Their vinyl ether bond could indeed be among the first targets for newly formed radicals. However, the long chain aldehydes released from plasmalogens were seldom studied and possible injurious or harmless effects were poorly examined. Thus, the sensitivity of the vinyl ether bond of plasmalogens was investigated in a cerebral cortex homogenate under UV irradiation- or Fe2+/ascorbate-induced peroxidation. Kinetics of aldehyde production was followed by gas chromatography/mass spectrometry. This confirmed that plasmalogens were highly sensitive to oxidative stress (70% cleavage after 90 min UV irradiation and 30% after 30 min of Fe2+/ascorbate). The aldehydes corresponding to sn-1 position 16:0, 18:0, or 18:1 were poorly detected. Conversely, oxidation of plasmalogens yielded preferentially 15:0, 17:0, and 17:1 aldehydes under UV and the alpha-hydroxyaldehydes 16:0-OH and 18:0-OH following a Fe2+/ascorbate oxidation. Kinetics showed that free aldehydes and above all free alpha-hydroxyaldehydes disappeared from the medium as soon as produced. Consequently, the behavior of these released aldehydes in the tissues has to be investigated in order to ascertain the protective effect of plasmalogens against oxidation.

Evidence against a major role of plasmalogens in the resistance of astrocytes in lactic acid-induced oxidative stress in vitro.

Astrocytes are known to play a key role in buffering extracellular pH variations and, in addition, they are particularly resistant to oxidative stress and subsequent lipid peroxidation. This great resistance may be ascribed to the presence of high concentrations of certain antioxidants, but another explanation may be the presence of a high quantity of plasmalogens, which are a special group of glycerophospholipids characterized by a vinyl ether bond instead of an ester bond in the sn-1 position of the glycerol backbone. Plasmalogens are sensitive to free radical attack and acidity, and numerous works have supported the hypothesis that they may be antioxidant molecules that protect cells from oxidative stress. The aim of this work was to investigate, on astrocytes in lactic acid-induced oxidative stress (pH 5.5), the behavior of phospholipids and, in particular, plasmalogens. Two main techniques, based on the susceptibility of the vinyl ether bond to hydrolysis, were employed in this study to measure plasmalogen levels. In both cases, the sn-1 vinyl ether linkage was cleaved using mercuric chloride, producing a lysophospholipid that was assessed by phosphorus measurement or using HCl treatment, producing a long-chain fatty aldehyde assayed by gas chromatography/mass spectrometry. On astrocytes in culture, only plasmenylethanolamine (PlmEtn) was evidenced, representing 40% of glycerophosphoethanolamine lipids. When astrocytes were incubated with lactic acid, no modification in the amount of PlmEtn was seen. Furthermore, free aldehydes and aldehydes corresponding to the quantity of intact plasmalogens were similar to those observed on controls. In addition, the constancy of two lipid peroxidation markers, thiobarbituric acid reactive substances and polyunsaturated fatty acids, was clear evidence of the resistance of these cells in lactic acid conditions. In conclusion, our results fail to demonstrate a major role of plasmalogens in the resistance of astrocytes in lactic acid-induced oxidative stress.

Age-related changes in ethanolamine glycerophospholipid fatty acid levels in rat frontal cortex and hippocampus.

Morphological and biochemical alterations are associated with a progressive age-related cognitive deficit. Plasmenylethanolamine, the major brain plasmalogen, may be modified during aging because of a possible antioxidant role and involvement in synaptic transmission. Two- and 18-month-old rats were used to study the effect of aging on the levels and acyl composition of plasmenylethanolamine (PmE), phosphatidylethanolamine (PE), and phosphatidylserine (PS) in the frontal cortex and hippocampus. Aging only reduced significantly the PE levels in the frontal cortex. In 18-month-old rats, the fatty acid composition of the three phospholipid classes studied showed an increase of monounsaturated fatty acid (18:1 n-9 and 20:1 n-9) and a decrease in polyunsaturated fatty acid (PUFAs), essentially docosahexaenoic acid (DHA). DHA was markedly decreased in hippocampus PE. DHA, but also arachidonic acid, were considerably lower in frontal cortex PmE. PS modifications were similar in both regions. Hippocampus and frontal cortex underwent specific age-induced modifications in PmE and PE acyl composition. This could produce different effects on the functional ability of these two structures involved in the processes of specific memorization.

Quantification of long-chain aldehydes by gas chromatography coupled to mass spectrometry as a tool for simultaneous measurement of plasmalogens and their aldehydic breakdown products.

The cleavage of the specific vinyl ether linkage at the sn-1 position of plasmalogens leads to the formation of two products: the 1-lyso-2-acyl glycerophospholipid and a long-chain fatty aldehyde. Plasmalogens are measured by quantifying one of these two products. In this paper, we describe a rapid and sensitive procedure for measuring plasmalogens via quantification of long-chain fatty aldehydes. After lipid extraction, the sn-1 vinyl ether bond of plasmalogens is cleaved by acidic hydrolysis. The produced aldehydes are then derivatized with (pentafluorobenzyl)hydroxylamine hydrochloride and analyzed by gas chromatography/mass spectrometry in selected-ion mode. Plasmalogens are then indirectly quantified by subtracting the free aldehydes obtained without prior HCl treatment from the total aldehydes obtained after acidic hydrolysis. This method is applied to three rat brain areas selected for this study. Two of these are affected in neurodegenerative diseases (cerebral cortex and hippocampus) and one is rich in white matter (cerebellum). In comparison to other procedures, the advantages of this method are not only its usefulness in plasmalogen quantification but also the identification of aldehydic breakdown products.

Pharmacological limitation of damage to renal medulla after cold storage and transplantation by trimetazidine.

Delayed graft function remains an important complication after renal transplantation. In this study, we investigated the influence of trimetazidine (TMZ), a cytoprotective agent, on renal medullary damage after prolonged preservation and autotransplantation. Pig kidneys were cold-flushed and preserved (48 h at 4 degrees C) with two standard renal preservation solutions Euro-Collins and University of Wisconsin supplemented or not with TMZ (10(-6) M). Analysis of plasma and urine from 48-h-cold-stored and autotransplanted kidneys was performed with biochemical methods and proton NMR spectroscopy. Histological study by light and electron microscopy was performed after reperfusion (30-40 min) and on day 14. The results showed that the preservation in either Euro-Collins or University of Wisconsin solution containing TMZ improved significantly glomerular filtration rate compared with kidneys preserved without TMZ. TMZ significantly reduced renal medullary damage, evidenced by decreased excretion of trimethylamine-N-oxide, dimethylamine, dimethylglycine, and acetate in urine. Proximal tubular injury in TMZ-free groups was assessed by significantly greater Na(+) excretion, amino aciduria, and lactic aciduria than in TMZ-supplemented groups. Urinary concentrating ability was significantly improved in TMZ-preserved groups compared with TMZ-free groups. In TMZ-supplemented groups, there was also a greater excretion of citrate, which is a citric acid cycle metabolite. An extensive reduction in apical brush border of tubular cells, notably those of the proximal tubules, was noted in TMZ-free groups. This study clearly shows that TMZ has a beneficial action on in vivo renal preservation and its major impact is the vulnerable renal medulla.

Early evaluation of renal reperfusion injury after prolonged cold storage using proton nuclear magnetic resonance spectroscopy.

BACKGROUND: Proton nuclear magnetic resonance (NMR) spectroscopy can be used as a non-invasive tool to measure renal damage. In the present investigation, proton NMR spectroscopy of urine was assessed in order to detect cellular damage after different periods of cold ischaemia in two standard preservation solutions. METHODS: The isolated perfused pig kidney was used to assess initial renal function after in situ cold flush and cold storage (CS) for 24 or 48 h in two standard preservation solutions: EuroCollins (EC) and University of Wisconsin (UW) solutions. Kidneys flushed with cold heparinized saline and immediately perfused were used as a control group. Kidneys were perfused for 2 h at 37.5 degrees C for functional evaluation. During reperfusion, renal perfusion flow rate was measured. Glomerular filtration rate (GFR), tubular reabsorption of sodium ions, and lactate dehydrogenase (LDH) and N-acetyl-beta-D-glucosaminidase (NAG) excretion were determined. Impairment caused by ischaemia and reperfusion was also determined by histological techniques and proton NMR spectroscopy. RESULTS: The perfusion flow rate, GFR and tubular reabsorption of sodium were significantly decreased in experimental groups compared with the control group. There was no significant difference between experimental groups after 24 h of CS. The perfusion flow rate was significantly decreased in the EC group after 48 h of cold ischaemia compared with that in the UW group. After 48 h of CS, GFR and tubular reabsorption of sodium were significantly reduced in the EC group compared with those in the UW group. The release of LDH into the effluent and the urinary excretion of NAG were not significantly different after 24 h of CS. After more than 45 and 60 min of reperfusion respectively, LDH and NAG excretion was no different in the 48-h CS groups. The most relevant resonances determined by proton NMR spectroscopy were of citrate, trimethylamine-N-oxide, lactate, acetate and amino acids. Excretion of these markers was significantly more accurate and efficient to assess renal ischaemia-reperfusion injury than that of biochemical markers. A resonance (P) detected particularly in the EC group after 48 h of CS was identified and correlated well with renal dysfunction. After CS for 48 h and 2 h of reperfusion, renal injury was histologically more pronounced in EC groups than in UW groups. However, the difference was not significant after CS for 24 h. CONCLUSION: NMR spectroscopy, which is a non-invasive and non-destructive technique, is more accurate and efficient when assessing kidney damage after cold ischaemia and reperfusion when compared to conventional histological and biochemical analysis.

Lactic acid-induced increase of extracellular dopamine measured by microdialysis in rat striatum: evidence for glutamatergic and oxidative mechanisms.

Striatal lactacidosis was induced by direct lactic acid perfusion to obtain a local pH as close as possible to that observed in ischemia. In a previous study we showed that such lactacidosis produces a diphasic increase in extracellular dopamine (DA). The present work investigated whether DA accumulation is related to a glutamatergic mechanism and/or production of reactive oxygen species (ROS) in the striatum. Concentrations of extracellular DA, glutamate and hydroxyl radicals ((.)OH) were measured in the presence or absence of an N-methyl-D-aspartate (NMDA) receptor blocker (dizocilpine, MK-801) or an antioxidant (Trolox). Measurements were performed using high-performance liquid chromatography (HPLC) with electrochemical and fluorimetric detection on samples obtained by an in vivo microdialysis perfusion technique and stored at -80 degrees C. The increase in lactic acid-induced DA was entirely suppressed by MK-801 and Trolox. Lactacidosis also induced an increase in extracellular glutamate and (.)OH concentrations at the same time as the first DA accumulation, as well as another (.)OH accumulation which preceded and accompanied the second DA concentration peak. Glutamate release was totally inhibited by MK-801 or Trolox. The first peak of (.)OH production was completely suppressed by MK-801 and Trolox, but the second one was only suppressed by Trolox. These data showed that the increase in DA induced by lactic acid was related to glutamatergic excitotoxicity and ROS production, suggested that the kinetic of events was different for the two DA accumulations.

Membrane carbohydrate conjugates desialylation does not alter [3H]-dopamine uptake in rat striatal slices.

Incubation of rat striatal slices induced a large decrease (about 50%) of DA uptake and a slight desialylation of polysialogangliosides (GT1b, GD1b, GD1a) with an increase of monosialogangliosides (GM1). Moreover, a pretreatment of slices by exogenous added neuraminidase of Vibrio cholerae did not modify DA uptake, although the pattern of gangliosides was modified and there was considerable loss (about 45%) of sialic acid in gangliosides and glycoproteins. It was verified that neuraminidase activity occured in synaptic membrane. Thus, DA uptake was apparently not altered by desialylation of plasma membrane carbohydrate conjugates.

Efficiency of trimetazidine in renal dysfunction secondary to cold ischemia-reperfusion injury: a proposed addition to University of Wisconsin solution.

Nonspecific injury in cadaveric renal transplants adversely affects early graft function and influences long-term graft survival after organ transplantation. Trimetazidine (TMZ) has been reported to exert a protective action against normothermic ischemia and reperfusion injury in several experimental and clinical studies. In an isolated perfused pig kidney model, we investigated the effects of TMZ added to University of Wisconsin solution (UW) during 48 or 72 h of cold storage (CS) and the consequence during reperfusion. Under all conditions tested renal perfusate flow rate (PFR), renal functions, and tubular injury markers were determined during a 120-min perfusion period. Lipid peroxidation and histological examination (optical and electron microscopy) were also determined after CS and reperfusion. The addition of TMZ (10(-6) M) to the UW solution improved dramatically the quality of preserved kidneys and consequently the functional recovery during reperfusion. TMZ + UW also significantly had a protecting role against reperfusion injury and lipid peroxidation when compared to UW alone. These results were correlated with both a better preservation of the proximal brush border membrane and reduced cellular and mitochondrial swelling. These results also suggested that the TMZ-induced renoprotection correlated well with the observed decrease membrane lipid peroxidation. Therefore, trimetazidine may be useful for clinical kidney graft preservation.

Autoxidation of rat brain homogenate: evidence for spontaneous lipid peroxidation. Comparison with the characteristics of Fe2+- and ascorbic acid-stimulated lipid peroxidation.

Aerobically-incubated brain homogenates are known to undergo autoxidation characterized by spontaneous TBARS production, presumably as a result of lipid peroxidation. However, TBARS measurement alone, because of its lack of specificity, is not sufficient to demonstrate the occurrence of lipid peroxidation in complex biological systems. This study, undertaken to determine whether or not spontaneous oxidation of rat brain homogenate is due to lipid peroxidation, measured different specific markers of this process (fatty acids, lipid aldehydes and the formation of fluorescence products) and studied changes in alpha-tocopherol. Incubation of rat brain homogenates at 37 degrees C under air led to spontaneous TBARS formation, which was accompanied by lipid aldehydes and lipid fluorescence products as well as polyunsaturated fatty acid (PUFA) degradation. Alpha-tocopherol was also consumed. On the whole, these results demonstrate that autoxidation of brain homogenate is a spontaneous lipid peroxidation process. When homogenates were exposed to Fe2+ and ascorbic acid-induced oxidative stress, lipid peroxidation was enhanced. However, spontaneous and stimulated peroxidation showed similar patterns not characteristic of classical lipid peroxidation, i.e. without the lag and accelerating phases typical of a propagating chain reaction. PUFA degradation was limited despite stimulation of peroxidation.

Chronic dietary n-3 polyunsaturated fatty acids deficiency affects the fatty acid composition of plasmenylethanolamine and phosphatidylethanolamine differently in rat frontal cortex, striatum, and cerebellum.

As chronic consumption of a diet devoid of n-3 fatty acid induced modification of neurotransmission pathways in the frontal cortex of rats, plasmalogen alteration could occur in this area. Because of the propensity to facilitate membrane fusion, plasmenylethanolamine (PmE), a major plasmalogen of brain, may be involved in synaptic transmission. Female rats were fed diet containing peanut oil [(n-3)-deficient diet] through two generations. Two weeks before mating, half of the female rats of the second generation received a diet containing peanut oil and rapeseed oil (control group). The distribution and acyl composition of major phospholipids, phosphatidylethanolamine and PmE, were measured in the frontal cortex, striatum, and cerebellum of the male progeny of the two groups at 60 d of age. The n-3 polyunsaturated fatty acid (PUFA) deficiency had no effect on the distribution of phospholipids in all brain regions but affected their acyl composition differently. The level of 22:6n-3 was significantly lower and compensated for by higher levels of n-6 fatty acids in all regions and phospholipids studied. However, docosahexaenoic acid, being more concentrated in the PmE of frontal cortex, is also more decreased in the n-3-deficient rats compared to the striatum. By contrast, striatum PmE has retained more 22:6n-3 than PmE of the other regions. In addition, the increase of n-6 PUFA was significantly lower in frontal cortex PmE compared to the striatum and cerebellum PmE. In association with altered neurotransmission observed in frontal cortex of n-3-deficient rats, our results suggest that frontal cortex PmE might be more affected in chronically alpha-linolenic-deficient rats. However, by retaining 22:6n-3, striatum PmE could be most resilient.

Changes in excitatory amino acid levels and tissue energy metabolites of neonate rat brain after hypoxia and hypoxia-ischemia.

Lactate accumulation, amino acid aspartate and glutamate levels, and hypoxanthine, xanthine and malondialdehyde (MDA) concentrations were compared in neonate rat brain after transient global hypoxia induced alone or in association with unilateral ligation of a carotid artery. Lactate production in both hemispheres was higher in cerebral hypoxia-ischemia (CHI) than in cerebral hypoxia (CH), and was lower in CHI after 2 h than at 15 min of recovery. Aspartate and glutamate levels were reduced 15 min after CHI in both hemispheres, but aspartate alone was decreased 2 h after CHI in the ipsilateral (left) hemisphere and 15 min after CH in both hemispheres. Hypoxanthine was increased 15 min after CHI in the ipsilateral hemisphere but decreased at 2 h, whereas xanthine was increased. MDA production was not modified after CH or CHI. These data, compared to those obtained in adult animals suggest that glutamate release and the capacity to generate oxygen-derived radicals are lower in neonates after ischemia. These differences might explain why the brain of the mammalian neonate is much more resistant to CH and CHI than that of the adult.

Effects of 4-hydroxynonenal, a lipid peroxidation product, on dopamine transport and Na+/K+ ATPase in rat striatal synaptosomes.

Incubation of rat striatal synaptosomes in ascorbic acid induced the production of thiobarbituric acid reactive substances, a marker of lipid peroxidation, and 4-hydroxynonenal (4-HNE), a lipid peroxidation aldehydic product. Incubations with 4-HNE, used at a range of concentrations comparable to those obtained during peroxidation, induced a simultaneous, dose-dependent decrease of dopamine (DA) uptake and Na+/K+ ATPase activity and a loss of sulfhydryl (SH) groups. Similar results were observed in a previous study when lipid peroxidation was induced after incubation of synaptosomes in ascorbic acid. Taken together, these data suggest that 4-HNE is an important mediator of oxidative stress and may alter DA uptake after binding to SH groups of the DA transporter and to Na+/K+ ATPase. These toxic events may contribute to the onset and progression of Parkinson's disease.

MPTP toxicity in rat striatal slices: dopamine uptake alteration does not appear to be related to lipid peroxidation.

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which is used to create experimental models of parkinsonism, induces both dopaminergic neurotoxicity and peroxidation reactions. The present work investigated the interaction between the dopamine (DA) uptake system, lipid peroxidation and MPTP in a rat striatum slice model. [3H]DA uptake was decreased and the concentration of thiobarbituric acid reactive substances (TBARS) increased after a plain preincubation in Krebs-Ringer bicarbonate buffer for 150 min. The decrease in [3H]DA uptake and the increase in TBARS were suppressed by the iron-chelating agent desferrioxamine. Inhibition of [3H]DA uptake was intensified, [3H]GBR 12 935 binding to DA uptake sites was decreased and TBARS production was inhibited in slices after preincubation with MPTP. MPTP effects were inhibited by L-deprenyl, a MAO-B inhibitor. These results suggest that the spontaneous decrease in DA uptake during simple preincubation in pure Krebs-Ringer solution was related to spontaneous TBARS generation. During MPTP preincubation, alteration of the DA uptake mechanism was not due to additional lipid peroxidation since TBARS production was decreased. MPTP effects could have resulted from other events which are discussed.

Alterations in the ganglioside composition of rat cortical brain slices during experimental lactic acidosis: implications of an enzymatic process independent of the oxidative stress.

Several in vitro studies have shown that lactic acidosis plays a role in brain damage by enhancing free radical formation and lipid peroxidation. The purpose of this study was to determine whether gangliosides are affected by lactic acid-induced oxidation in rat brain tissues. Cortical brain slices were incubated at 37 degrees C for 5 or 17 h in Krebs-Ringer buffer containing 20 mM lactic acid (final pH 5.5) previously equilibrated with 100% O2. Damage from lipid peroxidation was estimated by measurement of thiobarbituric acid-reactive substances (TBARS) and analysis of polyunsaturated fatty acids (PUFAs). Gangliosides were studied by high-performance thin-layer chromatography. Incubation with lactic acid induced overproduction of TBARS, whereas PUFAs were only slightly degraded, even after 17 h of incubation. However, the major modifications in the ganglioside profile occurred after 17 h of incubation. Gangliosides GD1a and GT1b decreased in conjunction with a substantial increase in the GM1 percentage. The addition of butylated-hydroxytoluene and desferrioxamine in the incubation medium, or incubation under 100% nitrogen, abolished TBARS production but not the ganglioside modifications, indicating that the change in ganglioside distribution was not related to oxidative stress induced by lactic acid. To investigate the possibility of an enzymatic process activated by the pH shift, slices were incubated with lactic acid in presence of 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, a specific inhibitor of sialidase. In these conditions, no change in gangliosides profile occurred. These results demonstrate that sialidase is responsible for the alterations in the gangliosides composition of rat cortical brain slices during lactic acidosis.