Renal denervation abolishes the protective effects of ischaemic preconditioning on function and haemodynamics in ischaemia‐reperfused rat kidneys

Studies were conducted to investigate the role of renal sympathetic nerves in the process of acquiring ischaemic tolerance in ischaemic preconditioned ischaemia‐reperfused rat kidneys. Two periods of 3‐min occlusion of bilateral renal arteries was performed prior to 30‐min bilateral ischaemia and 90‐min reperfusion in acute renal denervated or innervated kidneys. The glomerular filtration rate (GFR), fractional excretion of sodium (FENa) and lithium (FELi), and renal blood flow (RBF) were assessed in reperfused kidneys. Ischaemic preconditioning significantly improved values for all these parameters as compared with no treated ischaemia‐reperfused kidneys. Denervation caused slight increase in GFR, diuresis and natriuresis without improving RBF after reperfusion. However, protecting effects of ischaemic preconditioning on renal function were disappeared in denervated kidneys, while in innevated kidneys the effects of ischaemic preconditioning were maintained. These results clearly showed that ischaemic preconditioning pre‐treatment protects kidneys against ischaemia–reperfusion injury, and the effects are, at least in part, mediated by sympathetic nerves, as the protective effects were abolished by denervation.     

Long-lasting genomic instability following arsenite exposure in mammalian cells: the role of reactive oxygen species

Previously, we reported that the progeny of mammalian cells, which has been exposed to sodium arsenite for two cell cycles, exhibited chromosomal instability and concurrent DNA hypomethylation, when they were subsequently investigated after two months of subculturing (about 120 cell generations) in arsenite-free medium. In this work, we continued our investigations of the long-lasting arsenite-induced genomic instability by analyzing additional endpoints at several time points during the cell expanded growth. In addition to the progressive increase of aneuploid cells, we also noted micronucleated and multinucleated cells that continued to accumulate up to the 50th cell generation, as well as dicentric chromosomes and/or telomeric associations and other complex chromosome rearrangements that began to appear much later, at the 90th cell generation following arsenite exposure. The increasing genomic instability was further characterized by an increased frequency of spontaneous mutations. Furthermore, the long-lasting genomic instability was related to elevated levels of reactive oxygen species (ROS), which at the 50th cell generation appeared higher than in stable parental cells. To gain additional insight into the continuing genomic instability, we examined several individual clones isolated at different time points from the growing cell population. Chromosomally and morphologically unstable cell clones, the number of which increased with the expanded growth, were also present at early phases of growth without arsenite. All genomically unstable clones exhibited higher ROS levels than untreated cells suggesting that oxidative stress is an important factor for the progression of genomic instability induced by arsenite.     

The role of reactive oxygen species and nitric oxide in mast cell-dependent inflammatory processes

Summary:  Reactive oxygen species (ROS) and reactive nitrogen oxide species (RNOS), including nitric oxide, are produced in cells by a variety of enzymatic and non-enzymatic mechanisms. At high levels, both types of oxidants are used to kill ingested organisms within phagocytes. At low levels, RNOS may diffuse outside cells where they impact the vasculature and nervous system. Recent evidence suggests that low levels of ROS produced within cells are involved in cell signaling. Along with these physiological roles, many pathological conditions exist where detrimental high-level ROS and RNOS are produced. Many situations in which ROS/RNOS are associated also involve mast cell activation. In innate immunity, such mast cells are involved in the immune response toward pathogens. In acquired immunity, activation of mast cells by cross-linking of receptor-bound immunoglobulin E causes the release of mediators involved in the allergic inflammatory response. In this review, we describe the principle pathways for ROS and RNOS generation by cells and discuss the existence of such pathways in mast cells. In addition, we examine the evidence for a functional role for ROS and RNOS in mast cell secretory responses and discuss evidence for a direct relationship between ROS, RNOS, and mast cells in mast cell-dependent inflammatory conditions.     

The regulation of blood perfusion in the renal cortex and medulla by reactive oxygen species and nitric oxide in the anaesthetised rat

Aims: The regulation of blood flow through the renal medulla is important in determining blood pressure, and its dysregulation in pathophysiological states, such as oxidative stress, may contribute to the development of hypertension. This investigation examined the hypothesis that reactive oxygen species has both direct and indirect actions, via scavenging NO, to determine the degree of blood perfusion through the renal medulla. Methods: Groups of male Wistar rats received a renal interstitial infusion of either tempol, a superoxide dismutase (SOD) mimetic, or tempol plus catalase (tem + cat), or diethyldithio‐carbamic acid (DETC) a SOD inhibitor, or L‐NAME alone or L‐NAME followed by DETC. Results: Medullary blood perfusion (MBP) increased by 16 ± 1% (P < 0.05) following the renal infusion of tempol and by 35 ± 4%% (P < 0.05) when tem + cat was infused. Cortical blood perfusion (CBP) was unchanged during the administration of tempol and tem + cat. The renal interstitial infusion of DETC reduced CBP by 13 ± 2%, (P < 0.05) and MBP by 22 ± 3% (P < 0.05). Infusion of L‐NAME to block NOS did not change CBP but decreased MBP by 12 ± 4%, which was (P < 0.05) less than the reduction obtained with DETC. Administration of DETC in the presence of L‐NAME reduced CBP and MBP by 17 and 14%, respectively, the latter response being approximately half that obtained when only DETC was infused. Conclusions: These findings demonstrated that both reactive oxygen species and NO determined the level of MBP. The findings support the hypothesis that reactive oxygen species can act both indirectly, via scavenging of NO, and directly via H₂O₂ to modulate blood perfusion in the medulla.     

Novel morphological features of developing white matter pericytes and rapid scavenging of reactive oxygen species in the neighbouring endothelia

Capillary endothelia and pericytes form a close morphological arrangement allowing pericytes to regulate capillary blood flow, in addition to contributing to vascular development and support. Vascular changes associated with oxidative stress are implicated in important pathologies in developing whiter matter, but little is known about the vascular unit in white matter of the appropriate age or how it responds to oxidative stress. We show that the ultrastructural arrangement of post-natal day 10 (P10) capillaries involves the apposition of pericyte somata to the capillary inner basement membrane and penetration of pericyte processes onto the abluminal surface where they form close connections with endothelial cells. Some pericytes have an unusual stellate morphology, extending processes radially from the vessel. Reactive oxygen species (ROS) were monitored with the ROS-sensitive dye 2',7'-dichlorofluorescin (DCF) in the endothelial cells. Exposure to exogenous ROS (100 μm H(2) O(2) or xanthine/xanthine oxidase), evoked an elevation in intracellular ROS that declined to baseline during the ongoing challenge. A second challenge failed to evoke an intracellular ROS rise unless the nerve was rested for > 4 h or exposed to very high levels of exogenous ROS. Exposure to a first ROS challenge prior to loading with DCF also prevented the intracellular ROS rise from a second challenge, proving that dye washout during exposure to ROS is not responsible for the loss of a second response. Perfusion with 30 μm extracellular Ca(2+) or the voltage-gated Ca(2+) antagonist diltiazem partially prevented this rapid scavenging of intracellular ROS, but blocking either catalase or glutathione peroxidase did not. The phenomenon was present over a range of post-natal ages and may contribute to the high ROS-tolerance of endothelial cells and act to limit the release of harmful ROS onto neighbouring pericytes.     

Comparative analysis of thymic subpopulations during different modes of atrophy identifies the reactive oxygen species scavenger,N-acetyl cysteine,to increase the survival of thymocytes during infection-induced and lipopolysaccharide-induced thymic atrophy

The development of immunocompetent T cells entails a complex pathway of differentiation in the thymus. Thymic atrophy occurs with ageing and during conditions such as malnutrition, infections and cancer chemotherapy. The comparative changes in thymic subsets under different modes of thymic atrophy and the mechanisms involved are not well characterized. These aspects were investigated, using mice infected with Salmonella Typhimurium, injection with lipopolysaccharide (LPS), an inflammatory but non-infectious stimulus, etoposide (Eto), a drug used to treat some cancers, and dexamethasone (Dex), a steroid used in some inflammatory diseases. The effects on the major subpopulations of thymocytes based on multicolour flow cytometry studies were, first, the CD4⁻ CD8⁻ double-negative (DN) cells, mainly DN2–4, were reduced with infection, LPS and Eto treatment, but not with Dex. Second, the CD8⁺ CD3^lo immature single-positive cells (ISPs) were highly sensitive to infection, LPS and Eto, but not Dex. Third, treatment with LPS, Eto and Dex reduced all three subpopulations of CD4⁺ CD8⁺ double-positive (DP) thymocytes, i.e. DP1, DP2 and DP3, but the DP3 subset was relatively more resistant during infection. Fourth, both CD4⁺ and CD8⁺ single-positive (SP) thymocytes were lowered by Eto and Dex, but not during infection. Notably, LPS lowered CD4⁺ SP subsets, whereas the CD8⁺ SP subsets were relatively more resistant. Interestingly, the reactive oxygen species quencher, N-acetyl cysteine, greatly improved the survival of thymocytes, especially DNs, ISPs and DPs, during infection and LPS treatment. The implications of these observations for the development of potential thymopoietic drugs are discussed.     

Delineating the relationships among the formation of reactive oxygen species,cell membrane instability and innate autoimmunity in intestinal reperfusion injury

Acute intestinal ischemia is a medical emergency with a high mortality rate, attesting to the need for a better understanding of its pathogenesis and the development of effective therapies. The goal of this study was to delineate the relationships among intracellular and extracellular events in intestinal ischemia/reperfusion (I/R) injury, particularly the formation of reactive oxygen species (ROS), cell membrane instability associated with lipid peroxidation and the innate autoimmune response mediated by natural IgM and complement. A murine model of natural IgM-mediated intestinal I/R was used. Mice overexpressing anti-oxidant enzyme SOD1 were found to have significantly reduced intestinal tissue damage and complete blockage of IgM-mediated complement activation compared with WT controls. To determine if cell membrane instability was an event intermediate between ROS formation and natural IgM-mediated innate autoimmune response, the cell membrane stabilizer (trehalose) was administered to WT mice prior to the induction of intestinal ischemia. Treatment with trehalose significantly protected animals from I/R injury and inhibited IgM-mediated complement activation although it did not prevent membrane lipid peroxidation. These data indicate that in normal mice subjected to I/R injury, intracellular ROS formation is an event upstream of the lipid peroxidation which results in cell membrane instability. The membrane instability leads to an innate autoimmune response by natural IgM and complement. Trehalose, a nontoxic disaccharide tolerated well by animals and humans, has promise as a protective agent for patients with medical conditions related to acute intestinal ischemia.     

Comparison of the effect of a mitochondrial uncoupler, 2,4-dinitrophenol and adrenaline on oxygen radical production in the isolated perfused rat liver

Using the isolated perfused rat liver, we examined the effect of stimulation of mitochondrial respiration by 2,4-dinitrophenol (2,4-DNP) and adrenaline on reactive oxygen species (ROS) production, liver damage and lipid peroxidation. ROS production was monitored by luminol- and lucigenin-enhanced chemiluminescence and oxygen uptake was measured simultaneously. Liver damage and lipid peroxidation were evaluated by measuring hepatic lactate dehydrogenase (LDH) and thiobarbituric acid reacting substances (TBARS) release. Tissue ROS level decreased and oxygen uptake increased soon after 2,4-DNP infusion. On termination of 2,4-DNP infusion, there was a sharp increase in lucigenin-enhanced chemiluminescence, which declined slowly, but luminol-enhanced chemiluminescence did not change prominently. Hepatic LDH and TBARS release increased gradually during 2,4-DNP infusion and were manifested by termination of the infusion. Allopurinol did not affect ROS production and TBARS release, but delayed increases in LDH release after termination of 2,4-DNP infusion. Adrenaline, which stimulates mitochondrial respiration without uncoupling caused similar but smaller ROS changes observed in 2,4-DNP. LDH and TBARS release were not affected significantly by adrenaline infusion. These results indicate that uncoupling of oxidative phosphorylation decreases ROS production and restoration of oxidative phosphorylation enhances ROS production and liver damage. Xanthine oxidase is unlikely to contribute to enhanced ROS production after termination of 2,4-DNP but has some protective effect during uncoupling.     

Characterization of CoCl2-induced reactive oxygen species (ROS): Inductions of neurite outgrowth and endothelin-2/vasoactive intestinal contractor in PC12 cells by CoCl2 are ROS dependent, but those by MnCl2 are not

CoCl(2) and MnCl(2) are hypoxic mimetic agents. We previously found that expression of ET-2/VIC, one of hypoxia-related factors, and the induction of neurite outgrowth in PC12 cells through ROS induced by CoCl(2). MnCl(2) also are known to induce neurite outgrowth in PC12 cells. However, it is unclear whether the mechanism of the effect induced by these metals is same. In the present study, we evaluated biological effects induced by MnCl(2) and compared with those induced by CoCl(2). Furthermore, we analyzed sources of CoCl(2)-induced ROS generation. MnCl(2) up-regulated ET-2/VIC gene expression and ET-2/VIC peptide production as CoCl(2) did, but not affect ET-1 gene expression, in the neurite outgrowth of PC12 cells. NAC did not at all inhibit the effects induced by MnCl(2). Furthermore, addition of MnCl(2) to the culture medium did not generate ROS as CoCl(2) did. These results indicate that ET-2/VIC expression is a common pathway in neurite outgrowth induced by CoCl(2) and MnCl(2), but the effects induced by CoCl(2) are ROS dependent, whereas the effects induced by MnCl(2) are ROS independent. Taken together, the mechanism for the effects by CoCl(2) was different from that by MnCl(2). The ROS, were not decomposed by catalase or SOD, were rapidly generated by reaction of CoCl(2) mainly with components of HS rather than with FBS or DMEM. Some ROS generated by reaction of CoCl(2) with components of HS may participate in the observed neurite outgrowth of PC12 cells.     

Reactive oxygen species produced in mitochondria are involved in age-dependent changes of hematopoietic and mesenchymal progenitor cells in mice. A study with the novel mitochondria-targeted antioxidant SkQ1

Lifelong treatment of mice with the effective mitochondria-targeted antioxidant SkQ1 [10-(6′-plastoquinonyl) decyltriphenylphosphonium] does not affect hematopoietic stem cells (HSC) and more differentiated hematopoietic progenitors but significantly decelerates age-dependent changes in peripheral blood. During the first 13 months, SkQ1 (0.9 or 28.8 nmol/kg day) prevents age-dependent myeloid shift (increase in the proportion of granulocytes and decrease in the proportion of lymphocytes). During the next year of treatment the effect disappears, and the hemogram of 2-year-old treated mice does not differ from the control. The number of mesenchymal stem cells (MSC) in the bone marrow does not change during 2 years of treatment with SkQ1, but the concentration of MSC progeny fibroblast colony-forming units (CFU-F) increases with dose of SkQ1. The concentration of CFU-F after 1 and 2 years treatment with SkQ1 is twice higher than in young mice. Our data indicate that the stromal environment of hematopoietic cells could be the primary target of age-dependent changes mediated by reactive oxygen species produced in mitochondria. The anti-aging effects of SkQ1 described here are in perfect agreement with the inhibitory effects of this antioxidant on aging observed in the other models.