Postischemic proximal tubular resistance to oxidant stress and Ca2+ ionophore-induced attack. Implications for reperfusion injury

BACKGROUND: The severity of "reperfusion injury" is dependent on the extent to which the involved pathways are activated and on the degree of tissue susceptibility to them. This study was undertaken to ascertain whether preexistent ischemic proximal tubular damage (ischemic "pre-conditioning") significantly alters the expression of two purported mediators of reperfusion damage: oxidant stress and cytosolic Ca2+ loading. EXPERIMENTAL DESIGN: Male Sprague-Dawley rats underwent 35 minutes of bilateral renal arterial occlusion. Fifteen minutes or 24 hours later, the kidneys were removed, proximal tubular segments (PTS) were isolated, and their susceptibility to oxidant stress (H2O2 or FeSO4) and to cytosolic Ca2+ loading (Ca2+ ionophore, A23187) was determined. Results were contrasted to those obtained with normal PTS. Cell injury was quantified by percentage of cellular lactate dehydrogenase released. Lipid peroxidation was gauged by PTS malondialdehyde (MDA) concentrations. As an index of endogenous antioxidant defenses, PTS catalase and superoxide dismutase activities were determined. Vulnerability to lipid peroxidation is highly dependent on phospholipid unsaturated fatty content, so PTS fatty acid concentrations also were assessed. RESULTS: Although PTS harvested at 15 minutes postischemia manifested sublethal injury (increased lactate dehydrogenase release under control conditions), no increased vulnerability to the oxidant insults or to the Ca2+ ionophore was noted. By 24 hours of reflow, cytoresistance to each of the insults had developed. Postischemic PTS demonstrated no increase in basal MDA concentrations (indicating a lack of in vivo lipid peroxidation), and when challenged with H2O2 or FeSO4, significantly less MDA generation developed (vs. the normal PTS). This resistance to lipid peroxidation was not associated with increased superoxide dismutase/catalase levels or altered PTS fatty acid content. CONCLUSIONS: Sublethal ischemic proximal tubular injury does not directly predispose to oxidant stress or cytosolic Ca2+ loading, and by 24 hours postischemia, increased resistance to these insults develops. Decreased membrane susceptibility to lipid peroxidation may contribute to this result.     

Obstruction of proximal tubules initiates cytoresistance against hypoxic damage

Following acute tubular necrosis (ATN), cytoresistance to further renal injury results. However, the initiating events and the subcellular determinants of this phenomenon have not been defined. Since tubular obstruction is a consequence of ATN, this study evaluated whether it alters tubular susceptibility to hypoxic damage. Extrarenal obstruction (ureteral ligation in rats) was used for this purpose to dissociate obstructive effects from those of ATN. Twenty-four hours following ureteral ligation or sham surgery, cortical proximal tubular segments (PTS) were isolated and subjected to hypoxic (15 or 30 min)/reoxygenation injury. Since oxidant stress, cell Ca2+ overload, and PLA2 attack are purported mediators of hypoxic/reoxygenation injury, degrees of FeS04, Ca2+ ionophore, and phospholipase A2-induced PTS damage also were assessed. The cell injury (% LDH release) which resulted from each of the above was consistently less in PTS obtained from obstructed kidneys. This cytoresistance: (a) did not require prior uremia to develop (seen with unilateral obstruction); (b) it did not appear to correlate with a tubular proliferative response (assessed by proliferating cell nuclear antigen expression); (c) it was uninfluenced by early tubular repair (unchanged by 24 hrs of obstruction release); and (d) it occurred without increased heat shock protein (HSP-70) or antioxidant enzyme (superoxide dismutase, catalase) expression. Total adenylate pools were higher in obstructed versus control PTS during injury; however, this appeared to be a correlate of the protection, rather than a mediator of it. In contrast, obstructed tubules manifested a primary increase in plasma membrane resistance to PLA2 attack (approximately 3-fold less lysophosphatidylcholine and free fatty acid generation in obstructed vs. control PTS during incubation with exogenous PLA2). In sum, these results indicate that: (1) tubular obstruction protects PTS from injury, suggesting that its development during ATN may initiate cytoresistance; and (2) this cytoresistance appears to be mediated, at least in part, by a direct increase in plasma membrane resistance to PLA2 and potentially other forms (such as, oxidant stress, cytosolic Ca2+ loading) of attack.     

Time course of growth factor expression in mercuric chloride acute renal failure

BACKGROUND: Renal EGF expression decreases in varying models of acute renal failure (ARF). We found previously that the loss of distal tubular EGF during gentamicin ARF is strongest in the cortex, where proximal tubular injury was most severe. To gain more insight into the mechanism underlying this apparent anatomical association, renal growth factor expression was investigated during mercuric chloride ARF, in which proximal tubular injury is most severe in the outer stripe of the outer medulla (OSOM). METHODS: Endogenous renal growth factor expression was investigated by RNA hybridization and by immunohistochemistry in a rat model of mercuric chloride ARF. In addition we determined temporal and spatial profiles of tubular injury, cell proliferation, and mononuclear cell infiltration during the 3-week observation period. RESULTS: Serum creatinine values were maximal 2 days after treatment and were again normalized at day 6. Tubular injury was most severe in the PST and maximal at day 2. Cell proliferation was also higher in the PST and maximal at day 4. Three weeks after treatment, normal renal morphology was restored. Increased numbers of mononuclear cells appeared transiently in the renal interstitium from day 1 on. Most of these cells were macrophages and T lymphocytes; macrophages surrounded preferentially the severely injured PST in the OSOM. In analogy to gentamicin ARF, renal EGF and IGF-I gene expression were decreased early in the setting of mercuric chloride ARF. The decrease in distal tubular EGF staining was most pronounced in the OSOM, i.e. the anatomical area where mercuric-chloride-induced proximal tubular injury was most severe. CONCLUSIONS: Renal EGF and IGF-I gene expression decreases strongly during mercuric chloride ARF. The spatial association between the initial decrease of distal tubular EGF expression and the zone of major proximal tubular injury could originate from metabolic alterations secondary to oxygen starvation. A possible role of mononuclear cells remains to be determined.     

Imipramine demethylation in vivo: impact of CYP1A2, CYP2C19, and CYP3A4

Nephron loss leads to increased production of reactive oxygen intermediates. We measured the effect of carvedilol, a beta-blocking drug with radical scavenging properties, on renal function, glomerulosclerosis, antioxidant enzyme status and in vivo hydrogen peroxide (H2O2) production in rats with chronic renal failure caused by 5/6 nephrectomy (remnant kidney) and compared results to data obtained with propranolol, a beta-blocking drug without scavenging characteristics. Carvedilol and propranolol were administered during 11 weeks following reduction of nephron number. Kidneys were examined using enzymatic and histological techniques. Both carvedilol and propranolol decreased systolic blood pressure. Compared to propranolol, carvedilol offered some additional beneficial effects on renal function, particularly with regard to glomerulosclerosis. Lipid peroxidation, evaluated by malonaldehyde and 4-hydroxynonenal concentration in cortex homogenates, was decreased in carvedilol-treated rats only. Superior beneficial effect of carvedilol treatment is not linked to a significant up-regulation of the activities of the remnant kidney antioxidant enzymes (catalase, glutathione peroxidase and superoxide dismutase) or to a decreased in vivo H2O2 production.     

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.     

Hydrogen peroxide production by monoamine oxidase during ischemia-reperfusion in the rat brain

Monoamine oxidase (MAO) as a source of hydrogen peroxide (H2O2) was evaluated during ischemia-reperfusion in vivo in the rat brain. H2O2 production was assessed with and without inhibition of MAO during and after 15 min of ischemia. Metabolism of H2O2 by catalase during ischemia and reperfusion was measured in forebrain homogenates using aminotriazole (ATZ), an irreversible H2O2-dependent inhibitor of catalase. Catecholamine and glutathione concentrations in forebrain were measured with and without MAO inhibitors. During ischemia, forebrain blood flow was reduced to 8% of baseline and H2O2 production decreased as measured at the microperoxisome. During reperfusion, a rapid increase in H2O2 generation occurred within 5 min as measured by a threefold increase in oxidized glutathione (GSSG). The H2O2-dependent rates of ATZ inactivation of catalase between control and ischemia-reperfusion were similar, indicating that H2O2 was more available to glutathione peroxidase than to catalase in this model. MAO inhibitors eliminated the biochemical indications of increased H2O2 production and increased the catecholamine concentrations. Mortality was 67% at 48 h after ischemia-reperfusion, and there was no improvement in survival after inhibition of MAO. We conclude that MAO is an important source of H2O2 generation early in brain reperfusion, but inhibition of the enzyme does not improve survival in this model despite ablating H2O2 production.     

Myoglobin inhibits proliferation of cultured human proximal tubular (HK-2) cells

Following nephrotoxic injury, renal repair is dependent on tubular regeneration. In the case of myoglobinuric acute renal failure (ARF), persistence of myoglobin within tubular cells, or sublethal injury sustained at the height of exposure to it, might retard this process. To test this hypothesis, a human proximal tubular cell line (HK-2) was cultured for 24 hours in the absence or presence of clinically relevant myoglobin concentrations (0.5, 1, 2, 4 mg/ml). Immediately following myoglobin removal, lethal cell injury (vital dye uptake), lipid peroxidation, and DNA damage (alkaline unwinding assay) were assessed. The extent of cell proliferation was estimated over the next four days by a tetrazolium based (MTT) assay and by determining total intracellular LDH. Myoglobin's effects on protein and DNA synthesis were also assessed (35S-methionine and bromodeoxyuridine incorporation, respectively). Myoglobin induced dose-dependent lipid peroxidation (malondialdehyde generation) and cell death (up to 80% vital dye uptake with the 4 mg/ml challenge). Although 1 mg/ml myoglobin caused no cell death, it induced nearly complete growth arrest. This lasted for approximately three days following myoglobin removal from the media. Neither of two control proteins (albumin; lysozyme) nor a second nephrotoxin (gentamicin; 1 mg/ml) reproduced this effect. The 1 mg/ml myoglobin challenge caused an 80 to 90% depression in protein and DNA synthesis. It also induced significant DNA damage, as assessed by the alkaline unwinding assay (P < 0.01). Iron chelation therapy (deferoxamine) mitigated myoglobin-induced cell killing. However, its addition following myoglobin loading worsened HK-2 outgrowth by exerting a direct anti-proliferative effect. These results indicate that: (1) sublethal myoglobin toxicity can induce transient proximal tubular cell growth arrest, potentially slowing recovery from ARF; (2) this effect correlates with, and could result from, heme-induced DNA damage and a blockade in DNA/protein synthesis; and (3) deferoxamine can inhibit proximal tubular cell proliferation. This possibility needs to be considered in designing clinical trials with DFO for myohemoglobinuric ARF.     

Development of a limited sampling approach in pharmacokinetic studies: experience with the antiepilepsy drug tiagabine

To examine the mechanisms involved in the progression of mercury chloride (HgCl2)-induced acute tubular necrosis (ATN), we investigated the histopathological changes and the expression of inducible nitric oxide synthase (iNOS) mRNA and protein in renal cortices of rats at 20 hours after exposure to HgCl2. The expression of iNOS mRNA was significantly augmented in renal cortices of rats with HgCl2-induced acute renal failure (ARF). Likewise, the induction of iNOS protein was observed in damaged proximal tubule epithelial cells of rats with HgCl2-induced ARF. Pretreatment of rats with iNOS inhibitor aminoguanidine, however, suppressed the development of proximal tubule epithelial cell injury and prevented an increase in blood urea nitrogen and serum creatinine as well as resulting in a marked fall in iNOS mRNA and protein in rats with HgCl2-induced ARF. These observations indicate that the induction of iNOS may play a role in the progression of HgCl2-induced ATN through the exacerbation of proximal tubule epithelial cell damage.     

Critical review of psychometric tests

Macroscopic haematuria is common in IgA nephropathy, but its significance and influence on prognosis remains uncertain. We compared the clinical and pathological features of 11 adult patients with primary IgA nephropathy who had had a renal biopsy during or shortly after a bleeding episode. Six patients developed transient acute renal failure (ARF) (group 1) and five did not (group 2). Patients of group 1 had a higher percentage of tubular red-blood-cell (RBC) casts (P < 0.05) and of glomerular crescents (P < 0.001). However, crescents were focal and involved less than 50% of glomeruli. Acute tubular necrosis was only present in patients of group 1, and ARF was attributed to the acute tubular changes rather than to the glomerular lesions. Despite a prolonged duration of ARF (mean: 38 days), further outcome did not differ in patients of both groups. We suggest that acute tubular damage and/or tubular obstruction by RBC casts should be considered in any patient who develops ARF soon after a haematuric episode.     

Neurotrophin regulation of energy homeostasis in the central nervous system

Our hypothesis is that one cause of neuronal cell death and shrinkage in the aged central nervous system is an inability of neurons to maintain oxidant homeostasis in the face of increased levels of reactive oxygen species, decreased endogenous antioxidants, and impaired energy metabolism associated with physiological senescence, Alzheimer's, and Parkinson's diseases. Since treatment with nerve growth factor (NGF) reverses behavioral impairments in aged rats and stimulates cholinergic activity in the basal forebrain, while brain-derived neurotrophic factor appears to play a similar role in the striatum, we propose that neurotrophin-mediated cell-sparing reflects effects on oxidant homeostasis. Neurotrophins may play a similar cell-sparing role in hypoxic/ischemic injury to the nervous system, which also is mediated in part by reactive oxygen species. The degradation of one such species, H2O2, is catalyzed by catalase and glutathione peroxidase (GSH Px). The activity of the latter enzyme is dependent on glutathione reductase and the availability of NADPH for regeneration of reduced GSH. The GSH redox cycle is also regulated by enzymes of the hexose monophosphate shunt. NGF protects PC12 cells from H2O2 injury by stimulating the synthesis of antioxidant enzymes including catalase, GSH Px, glucose-6-phosphate dehydrogenase, and gamma-glutamylcysteine synthetase, the rate-limiting enzyme for glutathione synthesis. NGF also enhances recovery from the NAD+ losses occurring as a consequence of H2O2 treatment.     

Potential contribution of endothelin to renal abnormalities in glycerol-induced acute renal failure in rats

We studied the possible participation of endothelin-1 (ET-1) in the pathogenesis of renal damage in glycerol-induced acute renal failure (ARF). Cortical mRNA expression of ET-1 increased, peaking at 10 hr postinjury, but this did not occur in the medulla, plasma concentration and urinary excretion of ET-1 also increased in this model. There was no change in ETA receptor mRNA, whereas the ETB receptor tended to be down-regulated in the kidney after glycerol administration. In situ hybridization study demonstrated that elevated expression of prepro ET-1 was predominantly localized in cells in the proximal tubules of the nephritic kidney. The administration (30-3 mg/kg) of S-0139, (+)-disodium 27-[(E)-3-[2-[(E)-3-carboxylatoacryloylamino]-5-hydroxyphenl ]acrylayl oxy]-3-oxoolean-12-en-28-oate, an ETA selective antagonist, after initiation of insult offered dose-dependent prevention against ARF, demonstrating preventing of renal function impairment and mortality. These findings indicate that ET-1 participates in the pathogenesis of acute tubular injury in glycerol-induced ARF and that ETA antagonist may be useful in the treatment of some types of human ARF.     

EGF-Receptor phosphorylation and signaling are targeted by H2O2 redox stress

Inflammation of the respiratory tract is associated with the production of reactive oxygen species, such as hydrogen peroxide (H2O2) and superoxide (O2-), which contribute extensively to lung injury in diseases of the respiratory tract. The mechanisms and target molecules of these oxidants are mainly unknown but may involve modifications of growth-factor receptors. We have shown that H2O2 induces epidermal growth factor (EGF)-receptor tyrosine phosphorylation in intact cells as well as in membranes of A549 lung epithelial cells. On the whole, total phosphorylation of the EGF receptor induced by H2O2 was lower than that induced by the ligand EGF. Phosphorylation was confined to tyrosine residues and was inhibited by addition of genistein, indicating that it was due to the activation of protein tyrosine kinase (PTK). Phosphoamino acid analysis revealed that although the ligand, EGF, enhanced the phosphorylation of serine, threonine, and tyrosine residues, H2O2 preferentially enhanced tyrosine phosphorylation of the EGF receptor. Serine and threonine phosphorylation did not occur, and the turnover rate of the EGF receptor was slower after H2O2 exposure. Selective H2O2-mediated phosphorylation of tyrosine residues on the EGF receptor was sufficient to activate phosphorylation of an SH2-group-bearing substrate, phospholipase C-gamma (PLC-gamma), but did not increase mitogen-activated protein (MAP) kinase activity. Moreover, H2O2 exposure decreased protein kinase C (PKC)-alpha activity by causing translocation of PKC-alpha from the membrane to the cytoplasm. These studies provide novel insights into the capacity of a reactive oxidant, such as H2O2, to modulate EGF-receptor function and its downstream signaling. The H2O2-induced increase in tyrosine phosphorylation of the EGF receptor, and the receptor's slower rate of turnover and altered downstream phosphorylation signals may represent a mechanism by which EGF-receptor signaling can be modulated during inflammatory processes, thereby affecting cell proliferation and thus having implications in wound repair or tumor formation.     

Synergistic renal protection by combining alkaline-diuresis with lipid peroxidation inhibitors in rhabdomyolysis: possible interaction between oxidant and non-oxidant mechanisms

BACKGROUND AND PURPOSE: Heme-proteins, besides causing renal tubular obstruction, may contribute to rhabdomyolysis-induced renal injury through a heme-iron-mediated lipid peroxidation process. In the present study, we compared the combined therapy of a lipid peroxidation inhibitor, 21-aminosteroid (21-AS) and fluid-alkaline-mannitol (FAM) diuresis with either of them alone to determine the efficacy of the combination therapy and to delineate the roles of lipid peroxidation and cast formation. METHODS AND RESULTS: Employing Raman spectroscopy, we confirmed in vitro the ability of 21-AS to inhibit iron-induced fatty acid peroxidation. 21-AS was then administered to rats developing renal failure from glycerol-induced rhabdomyolysis. Although 21-AS inhibited rhabdomyolysis-induced plasma and renal lipid peroxidation, renal protection was incomplete. Administration of FAM to inhibit cast formation afforded a better renal protection. However, when these therapies were combined to inhibit both lipid peroxidation and cast formation, there was a synergistic renal functional protection. This was accompanied by a maximum inhibition of renal and plasma lipid peroxidation, as well as, renal tubular necrosis and cast formation. Compared to combination therapy, FAM therapy alone, despite identical volume, was accompanied by a higher tubular necrosis and cast formation. CONCLUSIONS: That combining a lipid peroxidation inhibitor with fluid-alkaline diuresis in rhabdomyolysis further lowers renal lipid peroxidation, tubular necrosis and cast formation and synergistically limits renal dysfunction (i) supports a role for lipid peroxidation in the pathophysiology of rhabdomyolysis ARF, (ii) underscores the role of the intratubular heme retention, a cause for tubular obstruction as well as a source for prodigious amount of iron, likely involved in the lipid peroxidation, and (iii) raises the possibility of interactions between non-oxidant and oxidant mechanisms.     

Oxidant stress causes alteration in the attachment of mononuclear cells to collagen

The effect of oxidant stress on the attachment of blood mononuclear cells to collagen was investigated. Study of the kinetics of attachment of mononuclear cells pretreated with 0.2% H2O2 for 10' showed significantly lower attachment to collagen I substrata when compared to untreated controls. Reduced glutathione at 2.5mM concentration partially reversed the H2O2 induced alteration in attachment. Pretreatment with H2O2 caused a reduction in the number of free thiol groups both at the cell surface and intracellular sites. Changes in cell surface free thiol groups could be reversed by reduced glutathione. These results point to the importance of a stable redox status in the cellular environment for normal interaction of mononuclear cells with extracellular matrix components.     

Activation of mitogen-activated protein kinase by H2O2. Role in cell survival following oxidant injury

The mitogen-activated protein kinase (MAPK) family is comprised of key regulatory proteins that control the cellular response to both proliferation and stress signals. In this study we investigated the factors controlling MAPK activation by H2O2 and explored the impact of altering the pathways to kinase activation on cell survival following H2O2 exposure. Potent activation (10-20-fold) of extracellular signal-regulated protein kinase (ERK2) occurred within 10 min of H2O2 treatment, whereupon rapid inactivation ensued. H2O2 activated ERK2 in several cell types and also moderately activated (3-5-fold) both c-Jun N-terminal kinase and p38/RK/CSBP. Additionally, H2O2 increased the mRNA expression of MAPK-dependent genes c-jun, c-fos, and MAPK phosphatase-1. Suramin pretreatment completely inhibited H2O2 stimulation of ERK2, highlighting a role for growth factor receptors in this activation. Further, ERK2 activation by H2O2 was blocked by pretreatment with either N-acetyl-cysteine, o-phenanthroline, or mannitol, indicating that metal-catalyzed free radical formation mediates the initiation of signal transduction by H2O2. H2O2-stimulated activation of ERK2 was abolished in PC12 cells by inducible or constitutive expression of the dominant negative Ras-N-17 allele. Interestingly, PC12/Ras-N-17 cells were more sensitive than wild-type PC12 cells to H2O2 toxicity. Moreover, NIH 3T3 cells expressing constitutively active MAPK kinase (MEK, the immediate upstream regulator of ERK) were more resistant to H2O2 toxicity, while those expressing kinase-defective MEK were more sensitive, than cells expressing wild-type MEK. Taken together, these studies provide insight into mechanisms of MAPK regulation by H2O2 and suggest that ERK plays a critical role in cell survival following oxidant injury.     

H2O2-driven reduction of the Fe3+-quin2 chelate and the subsequent formation of oxidizing species

The objective of this study was to compare effects of quin2 and EDTA in iron-driven Fenton-type reactions. Seven different assays for detection of strong oxidants were used: the DMSO, deoxyribose, benzoate hydroxylation, and plasmid DNA strand breakage assays, detection of 8-oxo-deoxyguanosine in deoxyguanosine mononucleosides and calf thymus DNA, and electron spin resonance with the spin-trap (4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in the presence of ethanol or DMSO. With H2O2 and Fe3+, quin2 generally strongly increased the formation of reactive species in all assays, whereas with EDTA the results varied between the assays from barely detectable to highly significant increases compared to H2O2 and unchelated Fe3+. We found that the species produced in the reaction between Fe3+-quin2 and H2O2 behaved like the hydroxyl radical in all assays, whereas with Fe3+-EDTA no clear conclusion could be drawn about the nature of the oxidant. The effect of quin2 on the formation of oxidants on Fe2+ autoxidation, varied from generally inhibiting to slightly promoting, depending on the assay used. EDTA had a promoting effect on the amount of oxidant detected by all but one assay. None of the autoxidation systems produced DMSO or ethanol radical adducts with 4-POBN. In the presence of either chelator, H2O2, and Fe2+ DMSO and ethanol radical adducts of 4-POBN were produced. Using the Fe2+ indicator ferrozine, evidence for direct reduction of Fe3+-quin2 by H2O2 was found. Superoxide anion radical appeared to be less efficient than H2O2 as reductant of Fe3+-quin2 as addition of superoxide dismutase in the ferrozine experiments only decreased the amount of Fe2+ available for Fenton reaction by 10-20%. The main conclusions from our study are that the reduction of Fe3+-quin2 can be driven by H2O2 and that Fe2+ in the following oxidation step produces a species indistinguishable from free hydroxyl radical.     

Erythropoietin production in rats with post-ischemic acute renal failure

To study the role of erythropoietin (Epo) in the pathogenesis of anemia in acute renal failure (ARF), organ Epo mRNA was measured by RNase protection assay in rats with ARF induced by a one hour-occlusion of the left renal artery. Hematocrit was significantly decreased two hours, 24 hours and one week after renal artery occlusion. A significant reduction in serum haptoglobin at two hours and an increase in serum LDH at 24 hours indicated that hemolysis was the likely cause of the initial fall in hematocrit. However, despite the reduced hematocrit, serum Epo concentrations were not significantly different from controls, suggesting that the anemia is maintained because of lack of an appropriate Epo response. Right renal Epo mRNA levels were not significantly different in all groups, but Epo mRNA levels in post-ischemic kidneys were 50 to 67% lower than in contralateral kidneys. However, Epo mRNA in the post-ischemic kidney was increased sixfold by acute hemorrhage, a rise comparable to the ninefold increase observed in contralateral kidneys. In ARF rats exposed to 7.5% O2 for four hours, right kidney Epo mRNA increased 200-fold over normoxic levels, to a value similar to sham-operated hypoxic controls. Epo mRNA in the post-ischemic kidney also increased 200-fold, to 50% of the level in the contralateral kidney. Hepatic Epo mRNA levels were elevated to comparable levels in both groups. In this ARF model, mild anemia is associated with relative Epo deficiency. In the post-ischemic kidney, a substantial capacity for Epo production is retained but the sensitivity of the Epo response to blood oxygen availability is significantly reduced.     

Glutathione is a cofactor for H2O2-mediated stimulation of Ca2+-induced Ca2+ release in cardiac myocytes

Reactive oxygen species are known to cause attenuation of cardiac muscle contraction. This attenuation is usually preceded by transient augmentation of twitch amplitude as well as cytosolic Ca2+. The present study examines the role of an endogenous antioxidant, glutathione in the mechanism of H2O2-mediated augmentation of Ca2+ release from the sarcoplasmic reticulum. Whole-cell patch-clamped single rat ventricular myocytes were dialyzed with the Cs+-rich internal solution containing 200 microM fura-2 and 2 mM glutathione (reduced form). After equilibration of the myocyte with intracellular dialyzing solution, Ca2+ current-induced Ca2+ release from the sarcoplasmic reticulum was monitored. Rapid perfusion with H2O2 (100 microM or 1 mM) for 20 s inhibited Ca2+ current, but enhanced the intracellular Ca2+ transients for 3-4 min. Thus, the efficacy of Ca2+-induced Ca2+ release mechanism was augmented in 71% of myocytes (n = 7). This enhancement ranged between 1.5- to threefold as the concentrations of H2O2 were raised from 100 microM to 1 mM. If glutathione were excluded from the patch pipette or replaced with glutathione disulfide, the enhancement of Ca2+-induced Ca2+ release was seen in only a minority (20%) of the myocytes. H2O2 exposure did not increase the basal intracellular Ca2+ levels, suggesting that the mechanism of H2O2 action was not mediated by inhibition of the sarcoplasmic reticulum Ca2+ uptake or activation of passive Ca2+ leak pathway. H2O2-mediated stimulation of Ca2+-induced Ca2+ release was also observed in myocytes dialyzed with dithiothreitol (0.5 mM). Therefore, reduced thiols support the action of H2O2 to enhance the efficacy of Ca2+-induced Ca2+ release, suggesting that redox reactions might regulate Ca2+ channel-gated Ca2+ release by the ryanodine receptor.