Cold storage induces time-dependent F2-isoprostane formation in renal tubular cells and rat kidneys.

BACKGROUND: Previous findings suggest a possible role for free radicals in cold-storage-associated tissue injury. Because free radical-induced lipid peroxidation catalyzes the cyclooxygenase-independent formation of vasoconstrictive F2-isoprostanes, the hypothesis that isoprostanes are produced during cold storage was tested in this study. METHODS: Total isoprostanes (free and esterified) in renal tubular epithelial (LLC-PK1) cells or whole kidneys, subjected to cold storage, were quantitated employing the gas chromatographic-mass spectroscopic method. LLC-PK1 cells were stored at 4 degrees C in a University of Wisconsin (UW) solution for 0, 24, 48, and 72 hours or 48 hours with desferrioxamine (DFO) or the lazaroid compound 2-methyl aminochroman (2-MAC). In the rat model, kidneys were perfused and stored for 48 hours in the UW solution with or without added DFO or 2-MAC. RESULTS: Isoprostanes in LLC-PK1 cells increased by fivefold following 24 hours of cold storage (36 +/- 2 pg/well to 185 +/- 6, mean +/- SE, following 24 hours of cold storage, P < 0.0001), and the levels continued to increase significantly at 48 and 72 hours. DFO and 2-MAC caused significant suppression of isoprostane formation. Cold storage of the kidneys in UW solution for 48 hours was accompanied by an eightfold increase in isoprostanes compared with control kidneys not subjected to cold storage (25.0 +/- 3.0 vs. 2.9 +/- 0.1 ng/g, P < 0.0001). The addition of 2-MAC or DFO to the UW solution was associated with a near complete suppression of 48-hour cold-induced isoprostane formation. CONCLUSION: Our findings provide evidence for the formation of large quantities of antioxidant-suppressible isoprostanes in kidney cells and whole kidney during cold-preservation. Based on this, it is hypothesized that (a) isoprostanes, which are potent renal vasoconstrictors, may contribute to immediate post-transplant vasoconstriction and dysfunction in kidneys subjected to extended cold storage, and that (b) the addition of 2-MAC or DFO to a UW solution in such circumstances may attenuate these alterations partly by suppressing isoprostane formation.

Pesticide induced changes of nitric oxide synthase in rat brain in vitro.

Organic insecticides are well known neurotoxicants. Nitric oxide (NO) is a neurotransmitter formed stoicheometrically with citrulline from L-arqinine through mediation of the enzyme nitric oxide synthase (NOS). We measured NOS activity in rat brain in vitro in the presence of selected organic insecticides such as, 10-200 microM conc carbaryl, kepone and malathion. All these three compounds inhibited NOS activity of rat brain in vitro in a concentration dependent manner. In most cases the changes observed were statistically significant. The order of potency, based on IC50 values of these insecticides, to inhibit NOS activity of rat brain, is carbaryl (105 microM) > Kepone (144 microM) > malathion (170 microM). We further demonstrated that these insecticides inhibit calmodulin (CaM)-stimulated NOS activity without affecting the basal enzyme activity. It is reported that the observed inhibition of NOS by selected insecticides may be due to interaction of these insecticides with Ca2+/CaM on which the NOS activity is well known to be dependent. This ultimately may lead to neurotoxicity of rat brain.

Chronic aminoguanidine attenuates renal dysfunction and injury in aging rats.

We have previously shown that aging is associated with increased lipid peroxidation, reductions in renal function, and increased glomerular sclerosis. The mechanism(s) responsible for these age-related changes are not clear. The purpose of the present studies was to determine if there was an increase in inducible nitric oxide synthase (iNOS) with aging, and if so, whether inhibition of iNOS would prevent aging injury by preventing free radical-mediated lipid peroxidation. iNOS protein expression in the kidney increased by approximately 90% by 24 months. Inhibition of iNOS by aminoguanidine (0.1% in drinking water) for 9 months, beginning at 13 months of age, reduced blood pressure, improved glomerular filtration rate by 70%, and renal plasma flow by 40%, whereas glomerular sclerosis was considerably reduced. Renal F2-isoprostanes and malondialdehyde levels, markers of oxidative stress and lipid peroxidation, were not reduced by aminoguanidine. Aminoguanidine also did not attenuate immunostaining for advanced glycosylation end products (AGE) in the kidneys. These findings suggest that aminoguanidine attenuates aging renal dysfunction by inhibiting a pathophysiologic function of iNOS that is independent of free radical-mediated lipid peroxidation or significant effects on AGE deposition.

Protective effects of zinc in indomethacin-induced gastric mucosal injury: evidence for a dual mechanism involving lipid peroxidation and nitric oxide.

BACKGROUND: Indomethacin causes gastric mucosal injury, although the pathogenesis is not fully understood. Zinc, is known to have gastroprotective effects in both humans and experimental animals. AIM: To determine (i) the protective effects of zinc in indomethacin-induced gastric mucosal injury in rats, and (ii) whether these cytoprotective effects are mediated by changes in gastric lipid peroxidation and/or nitric oxide synthase activity. METHODS: Gastric lesions were induced in rats by the intragastric administration of indomethacin. Morphological changes, lipid peroxidation (malondialdehyde levels) and nitric oxide synthase activity were determined in animals pre-treated with zinc sulphate and in controls. RESULTS: Indomethacin significantly increased malondialdehyde levels and decreased NOS activity. These effects were attenuated by pre-treatment with zinc (P < 0.005 and 0.0001, respectively). The protective effects of zinc were readily abolished in animals pre-treated with N-nitro-L-arginine methyl ester (L-NAME). Morphologically, indomethacin induced large areas of mucosal ulcerations, which were completely prevented by zinc pre-treatment. CONCLUSIONS: Zinc provides protection against indomethacin-induced gastric mucosal injury. These protective effects result from the inhibition of lipid peroxidation and the preservation of mucosal nitric oxide synthase.

Cisplatin induces N-acetyl cysteine suppressible F2-isoprostane production and injury in renal tubular epithelial cells.

In the low intracellular chloride milieu, chloride ions of cisplatin may exchange for cellular SH moieties resulting in glutathione depletion, H2O2 accumulation, and lipid peroxidation. Cisplatin-induced lipid peroxidation, in addition to causing direct cellular injury, may further contribute to cisplatin-induced renal dysfunction by generating vasoconstrictive E2- and F2-isoprostanes. The aim of this study was to determine whether cisplatin-induced renal epithelial (LLC-PK1 and primary human proximal tubular) cell injury is associated with increased production of isoprostanes, and whether this can be suppressed with a thiol donor, N-acetyl cysteine. It was confirmed that incubation of renal epithelial cells with cisplatin resulted in N-acetyl cysteine-inhibitable glutathione depletion, H2O2 accumulation, lipid degradation, and lactate dehydrogenase release. In additional experiments, incubation of cells with cisplatin for 48 h was accompanied by a dose-related increase in total (free plus esterified) F2-isoprostanes. An increase in F2-isoprostanes was discernible at 16.5 microM cisplatin and doubled at 66.0 microM. N-Acetyl cysteine at 50 microM concentration effectively suppressed 66.0 microM cisplatin-induced increase in isoprostanes. Similar findings were also obtained in human cells. Thus, cisplatin-induced tubular cell injury is accompanied by increased isoprostane production through a mechanism involving thiol depletion. On the basis of this new finding, it is hypothesized that these arachidonic acid peroxidation products may be partially responsible for the cisplatin-induced renal vasoconstriction demonstrable in the in vivo models.

Vitamin E ameliorates enhanced renal lipid peroxidation and accumulation of F2-isoprostanes in aging kidneys.

Aging results in progressive glomerular sclerosis and reductions in glomerular filtration rate (GFR). Oxidative stress may be an important mechanism for the aging process, but to date the role of oxidative stress on renal aging has not been determined. The present study was performed to determine whether age-related alterations in renal hemodynamics and morphology were associated with oxidative stress and whether this could be attenuated by chronic administration of vitamin E. Rats, aged 13 mo, were given either control diet containing vitamin E 50 IU/kg (n = 6) or a high-vitamin E diet (5,000 IU/kg; n = 6) for 9 mo. Another group of rats (3-4 mo old; n = 7) served as young controls. Aging was accompanied by a 60% reduction in GFR, a threefold increase in renal F2 isoprostanes, newly discovered vasoconstrictive F2-like prostaglandins generated by free radical-mediated lipid peroxidation. Renal aging was also associated with an increase in oxidant-sensitive heme oxygenase, advanced glycosylation end products (AGEs), and the AGE receptor, RAGE. AGE-RAGE interaction has been shown to induce oxidative stress. With high-vitamin E diet, GFR was increased by 50%, F2 isoprostanes were suppressed, and expression of heme oxygenase and RAGE was attenuated. There was also a tendency for glomerular sclerosis to be attenuated. These data demonstrate that age-related decline in renal function is accompanied by oxidative stress and that administration of antioxidants, such as vitamin E, could attenuate the decline in renal function.

Role of oxidant stress and antioxidant protection in acephate-induced renal tubular cytotoxicity.

Acephate (AT) is an organophosphate (OP) insecticide. Due to their reputation for low environmental persistence, OP pesticides are often used indiscriminately resulting in detrimental exposure to humans and other nontarget species. Although the toxicity of OP compounds is primarily through blockade of neural transmission via inhibition of acetylcholinesterase, studies have revealed histopathological alterations in the renal proximal tubules, suggesting a role for additional mechanisms in renal toxicity. It is our hypothesis that Reactive Oxygen Species (ROS) may play a role in OP-induced renal tubular injury for the following reasoning. Renal tubular cells concentrate many nephrotoxic chemicals including OPs, and renal injury from many of these compounds has been shown to arise from excessive ROS production. Furthermore, it has been established that many phosphorothiolates, which are sulfur-containing OPs and constitute the class of OP compounds to which AT belongs, are S-oxidized to highly reactive intermediates within cells and tissues. Because of these considerations, we examined whether ROS play a role in OP-induced renal tubular epithelial cell (LLC-PK1) toxicity using AT as a prototype. AT produced a concentration- and time-dependent increase in cell damage in LLC-PK1 cells, measured by lactate dehydrogenase (LDH, % of total) leakage. The cytotoxicity (LDH) induced by 2500 ppm of AT over 72 h was significantly suppressed by antioxidants 2-methylaminochroman (2-MAC) and desferrioxamine (DFO). H2O2 levels were significantly elevated following exposure of LLC-PK1 cells to 2500 ppm of AT. Malondialdehyde (MDA) formation was also significantly increased in AT-exposed cells compared to the control cells, indicating the occurrence of enhanced lipid peroxidation. 2-MAC and DFO, in addition to providing cytoprotection, inhibited AT-induced MDA generation in a significant and concentration-dependent manner. Results from this study, which is the first to explore the toxic effects of AT on renal tubular cells, demonstrate that toxic action of AT on kidney cells is partly through an ROS-mediated mechanism. Based on these direct in vitro findings, we further hypothesize that oxidant stress may play a role in the pathogenesis of AT-induced acute tubular necrosis and renal dysfunction observed in cases of AT overdoses.

Effects of fructose-1,6-diphosphate on the activity of rat liver nitric oxide synthase in vitro.

Fructose-1,6-diphosphate (FDP) was found to cause significant stimulation of nitric oxide synthase (NOS) in rat liver homogenates in vitro. This effect was more pronounced for the inducible isoform than its constitutive counterpart. Furthermore, FDP restored rat liver inducible NOS levels following their depletion by carbon tetrachloride (CCl4). This finding may have further practical implications in hepatoprotection from various noxious chemical and biological agents.

Inhibition of rat brain nitric oxide synthase activity by cyclosporin-A.

Cyclosporin-A, a potent immunosuppressive agent is known to induce cellular toxic side effects by way of altering calcium homeostasis, including calcium/calmodulin mediated events. We studied the in vitro and in vivo effects of cyclosporin-A on rat brain nitric oxide synthase activity (the enzyme that mediates the conversion of L-arginine to citrulline and NO). CsA in concentrations of 22-4400 nM inhibited rat brain NOS activity in vitro and in 10 or 25 mg/kg wt/4 weeks of CsA treated rat brains in vivo. We report here that CsA by way of interfering with rat brain Ca2+/CaM mediated events may inhibit its NOS activity which ultimately may result in neurotoxicity.

Cyclosporin A-induced changes in nitric oxide synthase activity in the rat kidney: in vitro and in vivo assay.

Cyclosporin A (CsA) is a cyclic undecapeptide that has been used extensively as an immunosuppressive drug in transplantation medicine and is known to interact with L-arginine dependent pathways. We studied the in vitro and in vivo effects of CsA on nitric oxide synthase (NOS) activity in the rat kidney. CsA in concentrations of 44-2200 nM in vitro, and 12.5 or 25 mg/kg body weight per 4 weeks treated rat kidneys in vivo, significantly stimulated NOS activity. CsA may alter the Ca2+/Cam-dependent NOS activity by interacting with rat kidney Ca2+/calmodulin dependent events.

Activity of gastric mucosal nitric oxide synthase in portal hypertensive gastropathy.

OBJECTIVE: The importance of portal hypertensive gastropathy, as a potentially bleeding lesion in cirrhotics with portal hypertension, has recently been appreciated. Histologically, dilation of the mucosal and submucosal vessels of the stomach is noted in this entity. The possibility of nitric oxide acting as a mediator for this mucosal vascular dilation has not been explored. METHODS: We determined, in a group of 10 male cirrhotic patients with esophageal varices and endoscopic changes consistent with severe portal hypertensive gastropathy (Group A), the gastric mucosal nitric oxide synthase activity. This was determined by measuring the rate of conversion of [3H]-arginine to [3H]-citrulline. Serum levels of nitrates and nitrites, the end products of nitric oxide, were also measured. The results were compared with those of a group of 10 male controls with no liver disease (Group B). RESULTS: Gastric mucosal constitutive and inducible nitric oxide synthase levels were significantly higher in group A (125.4 +/- 4.3 and 259.7 +/- 5.5 pmol/mg protein/minute, respectively) than in group B (88 +/- 8.6 and 130.8 +/- 6.6 pmol/mg protein/minute, respectively) ( p < 0.002 and < 0.0001, respectively). Serum nitrate/nitrite levels were 30.1 +/- 3.2 nmol/ml in group A and 15.5 +/- 0.09 nmol/ml in group B (p < 0.001). CONCLUSIONS: We conclude that the significantly increased gastric mucosal nitric oxide synthase activity, in patients with portal hypertensive gastropathy, suggests an important role for nitric oxide in the pathogenesis of this mucosal lesion.

Comparative study of the effect of 21-aminosteroid and alpha-tocopherol on models of acute oxidative renal injury.

21-Aminosteroids have incited a great deal of interest owing to its ability to inhibit lipid peroxidation and prevent organ damage. The main mechanism by which 21-aminosteroids inhibit lipid peroxidation is similar to the naturally occurring chain-breaking antioxidant alpha-tocopherols. Therefore, to determine whether 21-aminosteroids offer any advantage over alpha-tocopherol, we compared their effects on an in vivo and in vitro models of renal injury. 21-Aminosteroid (U-74006 F) at 3 mg/kg or alpha-tocopherol succinate at 10 mg/kg was administered intravenously once before bilateral renal ischemia and again before reperfusion. Acute administration 21-aminosteroid but not alpha-tocopherol, was attended by suppression of ischemia reperfusion-induced renal lipid peroxidation and injury. However, 4 weeks of dietary enrichment of rats with alpha-tocopherol (1000 IU/kg) was effective in suppressing these ischemia reperfusion-induced changes. In cell culture system, concurrent presence of 21-aminosteroid but not alpha-tocopherol abrogated H2O2-induced renal epithelial lipid peroxidation and injury. However, alpha-tocopherol was completely effective when cells were incubated with it for 14 h. Further, only the cells incubated with vitamin E for 14 h-but not for 1 or 3 h-had a significant increase in vitamin E content, which suggests that a delay in prompt cellular up take of vitamin E may explain its lack of acute effects. Thus, unlike alpha-tocopherol, 21-aminosteroid appears readily and completely available for its chain-breaking antioxidant activity both in vitro and in vivo. 21-Aminosteroids may, therefore, offer a therapeutic advantage over alpha-tocopherols in acute injury settings.