Lethal Oxidative Injury Research Articles

Endothelial PINK1 mediates the protective effects of NLRP3 deficiency during lethal oxidant injury.

High levels of inspired oxygen, hyperoxia, are frequently used in patients with acute respiratory failure. Hyperoxia can exacerbate acute respiratory failure, which has high mortality and no specific therapies. We identified novel roles for PTEN-induced putative kinase 1 (PINK1), a mitochondrial protein, and the cytosolic innate immune protein NLRP3 in the lung and endothelium. We generated double knockouts (PINK1(-/-)/NLRP3(-/-)), as well as cell-targeted PINK1 silencing and lung-targeted overexpression constructs, to specifically show that PINK1 mediates cytoprotection in wild-type and NLRP3(-/-) mice. The ability to resist hyperoxia is proportional to PINK1 expression. PINK1(-/-) mice were the most susceptible; wild-type mice, which induced PINK1 after hyperoxia, had intermediate susceptibility; and NLRP3(-/-) mice, which had high basal and hyperoxia-induced PINK1, were the least susceptible. Genetic deletion of PINK1 or PINK1 silencing in the lung endothelium increased susceptibility to hyperoxia via alterations in autophagy/mitophagy, proteasome activation, apoptosis, and oxidant generation.

Role of [Ca2+]i in lethal oxidative injury in rat cultured inner medullary collecting duct cells.

Reactive oxygen metabolites have been implicated in the pathogenesis of toxic, ischaemic and immunologically mediated renal injury. An increase in the cytosolic free Ca2+ concentration ([Ca2+]i) has been proposed as a mechanism of oxidative stress-induced cell injury. We used a fluorescence spectrometer and a fluorescence probe to measure the [Ca2+]i and viability of rat primary cultured inner medullary collecting duct (IMCD) cells during oxidative stress induced by 5 mM tert-butyl hydroperoxide (TBHP). Initially, this oxidative stress evoked a small increase in [Ca2+]i which was followed by a slower sustained increase from the resting level of 170.8 +/- 38.8 nM to 1490.5 +/- 301.7 nM after 60 min, and this preceded the loss of plasma membrane integrity, measured by the propidium iodide fluorescence method. The elimination of extracellular Ca2+ from the culture medium prevented the TBHP-induced [Ca2+]i increase and improved cell viability. Restoration of extracellular Ca2+ resulted in an immediate and large increase in [Ca2+]i and extensive cell death. Verapamil, a Ca2+ channel blocker, inhibited the [Ca2+]i increase and afforded significant protection against cellular injury following exposure to TBHP-induced oxidative stress. Extracellular acidosis also prevented the increase in [Ca2+]i and cell death caused by this oxidative stress. These results are consistent with the hypothesis that oxidative stress-induced IMCD cellular injury may be the result of increased [Ca2+]i caused, in part, by activation of voltage-dependent Ca2+ channels.

Ruthenium red delays the onset of cell death during oxidative stress of rat hepatocytes

Our objective was to determine if ruthenium red protects against lethal oxidative injury of rat hepatocytes. tert-Butyl hydroperoxide, 100 μmol/L, was used to produce oxidative stress. After 2 hours of oxidative stress, cell viability was greater with than without 25 μmol/L ruthenium red (37% vs. 4.6%; P < 0.01). Despite this cytoprotection, ruthenium red did not alter the rate or extent of glutathi one depletion, malondialdehyde generation, or adenosine triphosphate depletion. In contrast, ruthenium red did retard loss of the mitochondrial membrane potential (78% vs. 42% within 30 minutes; P < 0.01). However, the protective effect of ruthenium red could not solely be explained by preserving the mitochondrial membrane potential. Indeed, ruthenium red still improved cell survival after 2 hours of exposure to 10 μmol/L carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler (39% vs. 13%; P < 0.01). Cytosolic free calcium values did not change during the uncoupling of mitochondria, suggesting that the cytoprotective properties of ruthenium red cannot be explained by blocking mitochondrial calcium transport. Ruthenium red did inhibit proteolysis after 2 hours of exposure to tert-butyl hydroperoxide (434 ± 62 vs. 242 ± 20 nmol/106 cells; P = 0.016) or CCCP (236 ± 50 vs. 99 ± 38 nmol/106 cells; P = 0.04). The results indicate that ruthenium red appears to protect against hepatocellular injury by inhibiting degradative proteolytic activity. It is concluded that proteolysis may be an important mechanism contributing to lethal oxidative injury of hepatocytes.

Cytosolic free Ca2+ and proteolysis in lethal oxidative injury in endothelial cells

Oxygen free radicals (OFR) are thought to mediate ischemia-reperfusion injury to endothelium of heart, lung, brain, liver, and kidney and contribute to development of atherosclerosis, pulmonary O2 toxicity, and adult respiratory distress syndrome. Increased cytosolic free Ca2+ (Cai2+) has been proposed as a mechanism of injury from oxidative stress, yet the pathways by which an increase in Cai2+ may cause OFR-mediated endothelial cell injury remain unknown. Using multiparameter digitized video microscopy and the fluorescent probes, fura-2 acetoxymethyl ester and propidium iodide, we measured Cai2+ and cell viability in human umbilical endothelial cells during oxidative stress with xanthine (50 microM) plus xanthine oxidase (40 mU/ml). Oxidative stress caused a sustained increase in Cai2+ from a resting level of 90-100 nM to near 500 nM, which was preceded by formation of plasma membrane blebs. The increase in Cai2+ was prevented by removal of extracellular Ca2+ (Cao2+). Prevention of the increase in Cai2+ was associated with prolonged cell viability. Readdition of Cao2+ resulted in an immediate large increase in Cai2+ and rapid onset of cell death. The protease inhibitors, leupeptin and pepstatin, delayed the increase in Cai2+ and prolonged cell viability. The results are consistent with the hypothesis that endothelial cell injury due to oxidative stress may be the result of Cai2+ influx and resultant activation of Ca(2+)-dependent proteases.

Acidosis protects against lethal oxidative injury of liver sinusoidal endothelial cells

The purposes of this study were to determine the pH dependence of lethal endothelial cell injury during oxidative stress and the pH dependence of those cellular mechanisms proposed to result in endothelial cell killing. Oxidative stress was produced in rat liver sinusoidal endothelial cells with H2O2 (5 mmol/L). Cell survival was dependent on the extracellular pH. Indeed, after 180 min of incubation with H2O2, cell survival was only 27% at pH 7.4, 45% at pH 6.8 (p less than 0.05) and 62% at pH 6.4 (p less than 0.05). Despite improved cell survival at pH 6.4 compared with pH 7.4, the magnitude of ATP hydrolysis and glutathione depletion was similar. In contrast to cell survival, lipid peroxidation as measured by malondialdehyde generation was increased twofold at pH 6.4 compared with pH 7.4. A rapid and profound loss of the mitochondrial membrane potential occurred during oxidative stress at pH 7.4 that was delayed at pH 6.4 (0.3% vs. 20% of the initial value at 30 min, p less than 0.0001). After 60 min of incubation with H2O2, NAD(P)H oxidation was greater at pH 7.4 than at pH 6.4 (100% vs. 64%, p less than 0.05). The results indicate that the protective effect of acidosis against cell death during oxidative stress is associated with the inhibition of NAD(P)H oxidation and delayed loss of the mitochondrial membrane potential. Acidosis appears to maintain organelle and cell integrity during oxidative stress by influencing the redox status of NAD(P)H.