Erythropoietin attenuates experimental haemorrhagic shock-induced renal damage through an iNOS- dependent mechanism in male Wistar rats.

AIM: Erythropoietin (EPO) is shown to exert protective effects on different tissues in haemorrhagic shock (HS) states. Nitric oxide (NO), as a multifunctional signaling molecule, is implicated in diverse physiologic and pathologic processes. In order to understand the exact mechanism of EPO protection, in this study we evaluated the role of different NOS enzymes in the EPO signaling pathway in male rats. METHODS: Rats were randomized to five groups: 1) Sham, 2) HS 3) EPO 4) L-NAME, a non-specific NOS inhibitor 5) 1400W, a specific iNOS inhibitor. HS was induced by withdrawal of 50% of total blood volume. After 2h, resuscitation was performed with the shed blood and Ringer's lactate. In group 3, rats were treated with EPO (300IU/kg, i.v.) over 10min before HS induction. In the L-NAME and 1400W groups, L-NAME (10mg/kg, i.p.) and 1400W (2mg/kg, i.p.) were administered 30min before EPO injection. Blood and kidney tissue samples were obtained 3h after resuscitation. RESULTS: EPO increased the survival rate and significantly improved kidney function and histology compared to the HS group. There were less renal oxidative stress, apoptosis and systemic inflammatory responses in the EPO group. EPO increased eNOS and more abundantly iNOS mRNA expressions. L-NAME and 1400W significantly abolished all beneficial effects of EPO. CONCLUSION: In this in vivo animal model, we showed that EPO administration prior to HS attenuates renal injury and dysfunction in rats. The protective effects of EPO may be mediated by nitric oxide and the expression of different NOS enzymes, especially iNOS isoform.

Administration of hydrogen sulfide protects ischemia reperfusion-induced acute kidney injury by reducing the oxidative stress.

BACKGROUND: Renal ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury. Hydrogen sulfide (H2S) has been known as a novel gaseous signaling molecule. AIMS: The aim of this study was to investigate whether the efficacy of H2S in protecting against renal IRI is through its antioxidative effect. METHOD: In this study, rats were randomized into Sham, IR, or sodium hydrosulfide (NaHS, an H2S donor) groups. To establish a model of renal IRI, both renal arteries were occluded for 55 min and then declamped to allow reperfusion for 24 h. Rats in the NaHS group received intraperitoneal injections of 75 µmol/kg NaHS 10 min before the onset of ischemia and immediately after the onset of reperfusion. Sham group underwent laparotomy without cross-clamping of renal pedicles. After reperfusion, plasma and renal tissue samples were collected for functional, histological, and oxidative stress evaluation. RESULTS: The IR group exhibited significant rise in plasma creatinine, blood urea nitrogen (BUN), renal malondialdehyde (MDA) concentration, and significant reduction of renal superoxide dismutase (SOD) activity. Treatment with NaHS reduced the levels of plasma creatinine, BUN, renal MDA concentration, and increased SOD activity in the kidneys. NaHS improved renal histological changes in comparison to IR group. CONCLUSION: Our data demonstrated that H2S can protect against renal IRI and that its therapeutic effects may be mediated by reducing oxidative stress.

Hepatoprotective effects of remote perconditioning during renal ischemia.

BACKGROUND: Novel treatment strategies are required to reduce the development of hepatic injury during surgical procedure in which renal ischemia/reperfusion (IR) is inevitable. Remote perconditioning (rPeC) has been proved to reduce the extent of kidney damages induced by renal IR injury. The aim of this study was to determine the protective effect of rPeC against hepatic injury caused by renal ischemia. METHODS: Male rats were subjected to the right nephrectomy and randomized as: sham, no additional intervention; IR, 45 min of left renal pedicle occlusion; rPeC, four cycles of 5-min limb IR administered at the beginning of renal ischemia. After 24-h of reperfusion, the plasma and tissue samples were taken. RESULTS: A significant improvement in hepatic functional injury and oxidative damages were observed in the rPeC group compared to the IR group. However, histological evaluation and plasma levels of TNF-α revealed no significant difference among groups. CONCLUSIONS: It is concluded that rPeC exerted protective effects on renal IR-induced hepatic injury as a remote organ. The protection may be a consequence of the reduction in oxidative stress in the liver. This simple approach may be a promising strategy against IR-induced remote organ damages in the clinical practice (Fig. 4, Ref. 23).

Hepatic changes during various periods of reperfusion after induction of renal ischemia in rats.

BACKGROUND: Recent studies have suggested that ischemic damage to the kidneys causes liver tissue alterations. Thus, the morbidity and mortality in patients with acute renal failure (ARF) may be related to liver complications as well as to renal injury. The aim of the present study was to assess the hepatic changes during various periods of reperfusion after induction of renal ischemia. METHODS: Forty male rats were subjected to either a sham operation or to a 45-minute ischemia followed by 1, 3, 6, or 24 hours of reperfusion. Arterial pressure was continuously monitored. Blood samples were drawn to measure serum creatinine and blood urea nitrogen (BUN). RESULTS: We evaluated hepatic concentrations of interleukin (IL)-10 and tumor necrosis factor (TNF)-alpha. Ischemia reperfusion (IR) caused significant reductions in renal function as demonstrated by increased values of serum creatinine and BUN. These rats also showed significant increases in hepatic TNF-alpha and IL-10 concentrations. The most significant changes among inflammatory factors in the liver were observed at 3 hours of reperfusion: TNF-alpha, 616 +/- 41 vs 215 +/- 16, and IL-10, 926 +/- 73 vs 125 +/- 34, pg/100 mg tissue (P

Effect of bicarbonate administration on cyclosporine-induced nephrotoxicity in rats.

BACKGROUND: Cyclosporine (CsA) is known to cause metabolic and distal tubular acidosis. There is some evidence that CsA reduces net HCO(3)(-) absorption. The aim of this study was to elucidate whether bicarbonate administration prevented CsA-induced functional or structural nephrotoxicity. METHODS: Seven days after uninephrectomy, 20 rats were divided into 4 groups. NaHCO(3) (0.28 mol/L) was added in drinking water for 7 days, whereas control rats received regular tap water. The bicarbonate pretreated rats were administered either CsA (50 mg/kg intraperitoneally) or vehicle daily for a week. At the end of the procedure, animals were placed in metabolic cages for 24 hours after which we measured creatinine clearance (Ccr), urinary total proteins, pH, and N-acetyl-beta-D-glucosaminidase (NAG) activity. The kidney was fixed in formaldehyde. RESULTS: Ccr was significantly affected by the administration of CsA. The effects of CsA on serum pH and HCO(3)(-) concentration were prevented by pretreatment with NaHCO(3). However, it did not affect the CsA-induced increased urinary NAG activity or decreased Ccr. There was no protection of CsA-induced changes in renal tissues by NaHCO(3). CONCLUSION: Overall NaHCO(3) administration did not prevent CsA-induced changes in Ccr and NAG activity. These data suggested involvement of factors other than acid-base status in CsA-induced nephrotoxicity. However, correction of acidosis should still be considered for patients receiving CsA because acidosis exacerbates tissue damage.

Proteinuria is reduced by inhibition of inducible nitric oxide synthase in rat renal ischemia-reperfusion injury.

BACKGROUND: Ischemia-reperfusion (IR)-induced nephrotoxicity is associated with proteinuria. There are reports on the involvement of inducible nitric oxide synthase (iNOS) in proteinuria in conjunction with renal disease. This study was designed to investigate the effect of N6-(1-iminoethyl)-L-lysine hydrochloride (L-Nil), a selective inhibitor of iNOS, to prevent proteinuria in IR injury. METHODS: Ischemia was induced by 40-minute clamping of the renal arteries followed by 6-hour reperfusion. Rats were administered either L-Nil (3 mg/kg intravenous bolus followed by infusion of 1 mg/kg/h) or saline. To monitor glomerular and tubular functional changes before and after treatment, we measured blood urea nitrogen, plasma creatinine, and urinary N-acetyl-beta-D-glucosaminidase activity. Total protein (TP), albumin, and low- (LMW) and high-molecular-weight (HMW) protein excretion rates were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis of urine samples. Kidney ultrastructure was examined through a transmission electron microscope (TEM). RESULTS: IR resulted in significant LMW and HMW proteinuria. L-Nil significantly prevented the IR-induced increases in TP, albumin, and alpha1-microglobulin excretion. TEM showed loss of microvilli of the proximal tubule cells, injured mitochondria, and foamy changes in the structure of nuclear and cytoplasm in IR group. L-Nil reduced IR-mediated renal ultrastructural changes and tubular proteinuria. DISCUSSION: This study suggested possible differences in the mechanism(s) of nephrotoxicity induced by iNOS in the glomeruli and tubular cells. The types of proteins excreted in the urine should be considered in the treatment strategy. In conclusion, this study suggested the involvement of iNOS in IR-induced tubular proteinuria.

Changes in serum and renal vitamin E levels in deoxycorticosterone acetate-salt hypertensive rats.

BACKGROUND: Hypertension is an important risk factor for the progression of chronic renal disease, which may result in the development of end-stage renal disease. Since reactive oxygen species are implicated in the induction of hypertension, antioxidants have been used to reduce blood pressure and renal impairment in animal models and in human hypertension. However, the available data are not conclusive. METHODS: To investigate oxidative stress in hypertension, we evaluated renal and serum vitamin E levels as the most effective antioxidant to reduce lipid peroxidation by high-performance liquid chromatography among rats subjected to deoxycorticosterone acetate (DOCA)-salt treatment for 4 weeks. Renal levels of malondialdehyde (MDA) as a marker of cell lipid peroxidation were also assayed in treated rats. Systolic blood pressure (SBP) was measured in conscious rats by the tail-cuff method using a PowerLab/4sp data acquisition system. RESULTS: SBP increased significantly in DOCA-salt-treated rats compared to the sham group after 4 weeks of treatment. Serum vitamin E levels were significantly lower and renal MDA concentrations significantly higher in treated compared to sham rats. However, renal vitamin E levels were also significantly higher among treated compared to sham rats. DISCUSSION: Decreased plasma vitamin E levels and increased renal MDA levels show systemic and local oxidative stress in DOCA-salt-treated rats. However, the higher renal vitamin E level suggested a local compensatory mechanism. Vitamin E administration might be appropriate, as significant decreases in vitamin E levels were observed in the serum of DOCA-salt-treated rats.

Antioxidant vitamins preserve superoxide dismutase activities in gentamicin-induced nephrotoxicity.

OBJECTIVE: The clinical use of gentamicin (G) is limited due to its known nephrotoxic actions. Generation of reactive oxygen species has been proposed as a causative factor of cell death in G-induced acute renal failure (ARF). Previous studies using superoxide dismutase (SOD) mimetics have indirectly suggested a role for the superoxide ion in G-induced ARF. In this study, we directly measured the enzyme activities using in situ isolated kidneys seeking to investigate the effects of antioxidant therapy on preservation of endogenous antioxidant levels in ARF. METHODS: Thirty-five male Sprague-Dawley rats were randomly assigned to 5 groups: control, Tyrode-perfused; G, gentamicin (200 mg/L) added to the perfusate; G + vitamin E (Vit E; 100 mg/100 g BW, IM); G + vitamin C (Vit C) added to the drinking water for 3 days (200 mg/L) and to the perfusate (100 mg/L); G + Vit E + Vit C. SOD activities were determined in renal tissues based on NAPDH oxidation at 340 nm by spectrophotometry. RESULTS: SOD activity was significantly reduced in the G group compared with the controls (P<.05). Administration of Vit E alone or in combination with Vit C significantly preserved enzyme activity levels compared with the G group (P<.05). CONCLUSION: Antioxidant vitamins have a role in preservation of renal endogenous antioxidant activities, namely SOD, in G-induced nephrotoxicity.

Cyclosporine effects on the antioxidant capacity of rat renal tissues.

OBJECTIVE: Cyclosporine (CsA) has been shown to improve long-term survival after organ transplantation. However, CsA therapy is associated with a variety of side effects, among which nephropathy is the major one. Recent studies have suggested increased oxidative stress as a cause of drug nephrotoxicity. Therefore, this study was designed to evaluate the effects of CsA administration on the antioxidant capacity of kidney tissue. METHODS: Adult male Sprague-Dawley rats were randomly assigned into 2 groups: one group received CsA (25 mg/kg/d, IP for 2 weeks) and a control group (no CsA administration). After 2 weeks, the kidneys of the rats from both groups were removed under anesthesia. A 50 mg fresh kidney tissue sample was homogenized in ice-cold phosphate buffer. Total antioxidant capacity (Ferric Reducing Ability of Plasma [FRAP]) in the homogenates was assayed based on the Benzie spectrophotometric method. RESULTS: FRAP in the kidney tissues had been significantly decreased by 2 weeks of CsA administration when compared with control rats (P<.05). CONCLUSIONS: These data suggested that CsA administration may decrease the antioxidant capacity of renal tissues. More studies on the evaluation of the protective effects of antioxidant therapy against CsA nephrotoxicity are underway.

Acid-base status determines cyclosporine-induced hypercalciuria.

OBJECTIVE: Cyclosporine (CsA) causes tubular dysfunction characterized by polyuria, calcium wasting, distal tubular acidosis, and hyperkalemia. The hypercalciuria induced by CsA administration is associated with an inhibition of calbindin D(28k) expression. It has also been shown that chronic metabolic alkalosis increased the expression of Ca(2+) transport proteins accompanied by diminished urine Ca(2+) excretion. The aim of this study, therefore, was to determine the effect of acid-base status on CsA-induced hypercalciuria. METHODS: Experiments were performed on male Sprague-Dawley rats. Metabolic alkalosis and acidosis were induced respectively by adding 0.28 mol/L NaHCO(3) and 0.28 mol/L NH(4)Cl in the drinking water for 7 days; control rats received regular tap water. Seven days after NaHCO(3) or NH(4)Cl administration, rats were treated with CsA (25 mg/kg, IP) daily for 14 days. To estimate glomerular filtration rate (GFR) over time, animals were placed in metabolic cages. Fractional urinary calcium excretion was determined by standard formula. RESULTS: The CsA group showed decreased serum calcium and increased fractional urinary calcium excretion compared with the control group. Creatinine clearance was also significantly reduced. Metabolic alkalosis alone did not affect GFR, but significantly prevented an increase in fractional urinary calcium excretion induced by CsA, whereas chronic metabolic acidosis resulted in the exact opposite effect. CONCLUSIONS: It is essential for nephrologists to fully understand the mechanisms of CsA-induced renal injury. In this study, metabolic alkalosis reduced CsA-induced hypercalciuria. Further studies are needed to elucidate whether this effect may be achieved pharmacologically by the expression of Ca(2+) transport proteins.

Effects of hypoxia/reperfusion injury on drug disposition in the rat isolated perfused liver.

1. Ischaemia-reperfusion injury is known to be associated with a range of functional and structural alterations in the liver. However, the effect of this injury on drug disposition is not well understood. The present study was designed to examine the effects of hypoxia/reperfusion on the disposition of glutamate and propranolol in the rat isolated perfused liver. Both glutamate and propranolol are mainly metabolised in the pericentral region of the liver. 2. Hypoxia/reperfusion was established using the slow flow-reflow method of perfusion in both anterograde and retrograde perfusion. Glutamate metabolism was measured by the recovery of [(14)C]-glutamic acid and [(14)C]-labelled metabolites in a single pass in both anterograde and retrograde perfusion in the presence of a steady state concentration of unlabelled glutamic acid. Propranolol disposition, mean transit time and normalized variance were assessed from the outflow concentration-time profile of unchanged [(3)H]-propranolol determined after a bolus injection of [(3)H]-propranolol using HPLC and liquid scintillation counting. 3. Hypoxia/reperfusion of livers did not affect oxygen consumption, but caused significant changes in enzyme release, lignocaine hepatic availability and bile flow. 4. Hypoxia/reperfusion did not affect the hepatic metabolism of glutamate to carbon dioxide or the hepatic extraction of propranolol. Small but significant changes were evident in the distribution parameters of mean transit time and vascular disposition for the hypoxic-ischaemic liver. 5. It is concluded that reperfusion injury induced by slow flow-reflow perfusion did not influence the extraction of glutamate or propranolol, but may have affected pericentral morphology and solute distribution.

Comparative effects of selective and non-selective nitric oxide synthase inhibition in gentamicin-induced rat nephrotoxicity.

Different nitric oxide synthase (NOS) isoforms are found in the kidney. Some studies provided evidences that increased endothelial NOS (eNOS) activity leads to restoration of renal function after injury, but activation of inducible NOS (iNOS) aggravates renal failure. In the present study, the beneficial effects of selective iNOS blockade in gentamicin (GM) induced nephrotoxicity have been investigated. Four groups of rats were studied. Untreated control rats received saline. In GM group, GM was injected (IV, 4 mg kg(-1)). In GM + L-NAME group rats received L-NAME (N-omega-L-arginine methyl ester, a non-selective NOS inhibitor) simultaneously with GM (IV, 30 mg kg(-1)). Additional doses of L-NAME were administered 2 and 4 h after GM (IP, 30 mg kg(-1)). In GM + L-NIL group rats were treated by N-imino-ethyl lysine (L-NIL, a selective iNOS inhibitor). First dose (IV, 3 mg kg(-1)) administrated simultaneously with GM. Next doses (IP, 3 mg kg(-1)) were administered 2 and 4 h after GM. In all groups, serum and urine creatinine levels were measured. Creatinine clearance was calculated and considered as an estimation of glomerular filtration rate (GFR). Urine N-acetyl-b-D-glucose aminidase (NAG) activities were also determined. After experiments, kidney sections were histologically studied. Selective iNOS inhibition by L-NIL prevented the GM-induced decrease in GFR and increase in creatinine levels, while complete non-selective NOS inhibition by L-NAME aggravated the GFR reduction, elevation of creatinine levels and enzyme release (P < 0.05). Histological studies showed that GM-treated kidneys had evidences of tubular damages and these damages were less evident by the administration of L-NIL. In conclusion, selective inhibition of iNOS may prevent GM-induced nephrotoxicity, whereas non-selective inhibition of NOS aggravates it.

Effects of co-administration of an iNOS inhibitor with a broad-spectrum reactive species scavenger in rat renal ischemia/reperfusion injury.

BACKGROUND: It is generally believed that reactive oxygen species (ROS) formation and nitric oxide (NO) generation by the inducible isoform of nitric oxide synthase (iNOS) are the key mediators of ischemia-reperfusion (IR)-induced damage to the kidney. The present study was designed to investigate the effects of ROS and NOS inhibition in prevention of renal IR injury. MnTBAP (Manganese (III) meso-tetrakis (4-benzoic acid) porphyrin), a broad-spectrum reactive species scavenger was administered to inhibit ROS formation and L-Nil (N6-(1-iminoethyl)-L-lysine hydrochloride) was used for iNOS inhibition. METHODS: Ischemic acute renal failure (ARF) was induced by 40-min clamping of the renal arteries followed by a 6-hour reperfusion. Rats were administered saline, MnTBAP (10 mg/kg i.v.), L-Nil (3 mg/kg i.v. bolus followed by infusion of 1 mg/kg/h) or co-administration of MnTBAP and L-Nil. Plasma creatinine (Cr) and BUN levels as well as fractional excretion of Na+ (FE(Na+)) and urinary N-acetyl-beta-D-glucosaminidase (NAG) activities were measured. Renal damages were evaluated by light microscopy. RESULTS: MnTBAP, L-Nil and their co-administration significantly improved renal functional and histological indices. Co-administration of the mentioned drugs did not demonstrate significant difference with the administration of either drug alone. CONCLUSION: These results suggest that the significant portion of ROS and iNOS nephrotoxicities in this model of ARF may be mediated by peroxynitrite (ONOO-). These results emphasize the multifactorial nature of ARF and the need for a multidrug therapy in the future.

Oxidative stress in operating room personnel: occupational exposure to anesthetic gases.

Health professionals exposed to anesthetic gases are at higher risk of reproductive, neurological, hematological, immunological, hepatic and renal system diseases. We investigated if oxidative stress induced by chronic exposure to anesthetic gases has any association with this matter. Plasma lipid peroxidation, total antioxidant capacity and total thiol molecule levels were measured in 66 operating room staff in comparison with 66 controls. The exposed group had a significantly higher level of lipid peroxidation with decreased thiol groups compared to control subjects. Total antioxidant capacity of the body was no different among exposed and not exposed subjects. Increased lipid peroxidation in the blood of exposed subjects warns that oxygen free radicals have increased in the body and thus might attack cells, which, in the long-term, results in multi-organ damage. The remaining blood total antioxidant capacity at normal values is promising and means that other non-thiol antioxidants, such as uric acid, transferrin, ceruloplasmin, albumin, and vitamin antioxidants, such as alpha-tocopherol and ascorbic acid, have been stimulated to maintain the total anti-oxidant power of the body at normal state.

Inhibition of inducible nitric oxide synthase reduces lipopolysaccharide-induced renal injury in the rat.

1. Gram-negative bacterial lipopolysaccharide (LPS) release and subsequent septic shock is a major cause of death in intensive care units. Lipopolysaccharide has been reported to increase the production of nitric oxide (NO) and the formation of oxygen-derived free radicals (OFR) in different organs. The aim of the present study was to evaluate the role of an inducible form of NO synthase (iNOS) and OFR production in LPS-induced renal impairment. 2. Measurement of vitamin E as the most important fat-soluble anti-oxidant was used as a marker of tissue oxidative stress. Lipopolysaccharide (10 mg/kg), L-iminoethyl lysine (L-Nil; 3 mg/kg, i.p.; a specific inhibitor of iNOS activity) and dimethyl thiourea (DMTU; 500 mg/kg i.p.; a well-known OFR scavenger) were used. Four groups of eight rats were studied. One group received LPS, whereas a second group received LPS + L-Nil. A third group received LPS + DMTU and the fourth group, receiving saline, acted as a control group. To evaluate renal function, plasma creatinine and blood urea nitrogen (BUN) were measured. High-pressure liquid chromatography and ultraviolet detection were used to measure plasma and tissue vitamin E levels. Light microscopy was used to examine histopathological changes in the four groups. 3. Lipopolysaccharide markedly decreased the vitamin E content of renal plasma and tissue (P < 0.05). Administration of L-Nil attenuated renal dysfunction and preserved vitamin E levels. However, DMTU failed to prevent renal injury, as indicated by plasma BUN levels and renal histology, despite the fact that it maintained renal vitamin E levels and increased plasma vitamin E levels. Thus, the overproduction of NO by iNOS may have a role in this model of LPS-induced renal impairment.

Anti-infarct effect of magnesium is not mediated by adenosine A1 receptors in rat globally ischaemic isolated hearts.

1. The aim of present study was to investigate the effects of magnesium (Mg) on cardiac function and infarct size and to compare it effects with those of adenosine. The mechanism of Mg-mediated cardioprotection was explored by combined use of Mg and a selective adenosine A(1) receptor antagonist. 2. Rat isolated hearts were used for Langendorff perfusion. Hearts were either non-preconditioned or preconditioned with Mg (6 mmol/L) or adenosine (1 mmol/L) before 30 min sustained ischaemia followed by 120 min reperfusion. Within each of these protocols, hearts were divided into two groups; one group was exposed to the A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 200 nmol/L). Infarct size was measured by the triphenyltetrazolium chloride method. Left ventricular function was assessed by left ventricular developed pressure (LVDP), the product of heart rate x LVDP and coronary flow (CF). 3. The administration of Mg had an anti-infarct effect independent of its effect on postischaemic functional recovery in rats. Both Mg and adenosine equipotently reduced infarct size, but this effect of Mg was not blocked by the simultaneous administration of DPCPX. Cardiac function was improved by both adenosine and Mg and blockade of adenosine A(1) receptors attenuated these effects for both agents. 4. In conclusion, the results of the present study indicate that stimulation of adenosine A(1) receptors is not responsible for the anti-infarct effect of Mg in ischaemic myocardium in rats, but that the Mg-mediated protection of postischaemic functional recovery in rats is mediated by these receptors.

The role of nitric oxide in iron-induced rat renal injury.

Iron overload and enhanced hydroxyl radical (*OH) formation have been implicated as the causative factors of oxidative stress in different organs. Both pro-oxidant and anti-oxidant properties have been reported for nitric oxide (NO) in iron-mediated tissue injury. To determine the contribution of NO to iron-induced renal injury, eight groups of rats (eight in each group) were studied as follows: control (normal saline), L-Arg (L-arginine as a substrate of NO synthase, 400 mg/kg), L-NAME (an inhibitor of NO synthase, 8 mg/kg), Fe (iron dextran, 600 mg/kg), DFO (deferroxamine as a chelator of iron, 150 mg/kg), Fe+L-Arg, Fe+L-NAME, DFO+L-Arg. Twenty-four hours after the injections, blood samples were taken and kidneys removed for biochemical analysis. Plasma creatinine and urea were used to stimulate renal function. Renal tissue and plasma vitamin E levels, the most important endogenous fat soluble antioxidant, were measured by HPLC and UV detection. In this study, renal function was markedly reduced in the Fe group compared to controls (creatinine, 1.02+/-0.05 mg/dL versus 0.78+/-0.04 P <0.05; urea, 49.59+/-1.69 mg/dL versus 40.75+/-0.86, P <0.01). Vitamin E levels were significantly lower in the Fe group compared to controls (plasma P <0.01; renal tissue P <0.05). Administration of L-Arg to Fe-treated groups prevented these reductions. L-NAME increased iron-induced toxicity significantly, demonstrated by further reduction in the vitamin E levels and renal function compared to the Fe group alone. We concluded that NO plays an important role in protecting the kidney from iron-induced nephrotoxicity. NO synthase blockade enhances iron-mediated renal toxicity in this model.

Enhanced susceptibility to oxidation and diminished vitamin E content of LDL from patients with stable coronary artery disease.

BACKGROUND: Convincing evidence points to oxidative modification of LDL as an important trigger in a complex chain of events leading to atherosclerosis. We investigated the occurrence of enhanced susceptibility of LDL to oxidation and decreased vitamin E concentration in LDL as additional risk factors promoting atherosclerosis among patients with established coronary artery disease (CAD). METHODS: We examined 132 patients with angiographically confirmed CAD and compared them with 111 healthy control individuals. We measured conjugated diene production to assess susceptibility of LDL to copper-mediated oxidation. Vitamin E content of LDL was measured by HPLC. RESULTS: The mean lag time of LDL oxidation and LDL alpha-tocopherol/LDL-cholesterol ratio were lower in the patients with CAD (55 +/- 14 min and 2.4 +/- 1.0 mmol/mmol) than in the controls (63 +/- 13 min and 2.9 +/- 1.1 mmol/mmol; P <0.0001 and <0.001, respectively). Multiple stepwise regression analysis demonstrated the lag time (odds ratio, 1.96; 95% confidence interval, 1.34-2.87; P <0.0001) and concentration of vitamin E in LDL (odds ratio, 1.65; 95% confidence interval, 1.16-2.33; P <0.005) as independent determinants of CAD. Significant inverse Spearman rank correlations were found between lag time (r = -0.285; P <0.001) or concentration of vitamin E in LDL (r = -0.197; P <0.002) and severity of CAD. Lag times were not significantly correlated with serum C-reactive protein or ferritin. CONCLUSIONS: Our data suggest that a short LDL oxidation lag time and a low concentration of vitamin E in LDL might be independent coronary risk factors for stable CAD in Iranian people.

Detection of hydroxyl and carbon-centred radicals by EPR spectroscopy after ischaemia and reperfusion of the rat kidney.

Recent studies suggest that oxygen-derived free radicals are involved in mediating renal reperfusion injury. EPR spectroscopy and spin trapping with the spin traps DMPO and PBN, were used to detect and quantitate the formation of hydroxyl radicals in rat kidney after ischaemia-reperfusion in vivo and in vitro in the isolated rat kidney perfused in the absence of leucocytes. EPR analysis of homogenised kidneys and of venous samples did not detect radical adducts with either spin trap. With PBN, radical adducts were not detected in vitro. When DMPO was used as the spin trap in kidneys perfused without albumin in the perfusate, EPR signals characteristic of hydroxyl and carbon-centred radical adducts were detected during early reperfusion following ischaemia. These studies confirm the generation of hydroxyl radicals during ischaemia-reperfusion in kidney. During reperfusion the total DMPO adduct concentration reached 4.35 +/- 1.05 nmol/g kidney/3 min, p < 0.05. In control kidneys total adduct were present at lower concentration (2.55 +/- 1.1 nmol/g kidney/3 min). Addition of 15 mM dimethylthiourea abolished formation of these adducts following ischaemia-reperfusion but did not prevent a reduction in glomerular filtration rate. These results indicate that significant levels of hydroxyl and carbon-centred radicals are formed in the absence of circulating neutrophils during early renal reperfusion following ischaemia.

Hydroxyl radical generation following ischaemia-reperfusion in cell-free perfused rat kidney.

The difficulty in direct detection of oxygen-derived free radicals (OFR) in the intact kidney has left uncertain the role of OFR in renal hypoperfusion injury. Salicylate hydroxylation was used as a sensitive method of estimating the extent of production of highly reactive hydroxyl radicals in renal ischaemia-reperfusion injury in the intact rat kidney perfused with recirculating cell-free medium. The reaction products were detected and quantified by HPLC with electrochemical detection. Hydroxyl radicals were detected as 2,5-dihydroxybenzoic acid (2,5-DHBA). Ischaemia for 15 min followed by reperfusion for 15 min caused more than a twofold increase in 2,5-DHBA concentration (to 2279 +/- 225 pg/g tissue weight) compared to controls (933 +/- 103, P < 0.001). Addition of 15 mM dimethylthiourea (DMTU) before induction of ischaemia prevented this increase. Induction of hypoxia for 15 min with continued perfusion (as a model of low-flow ischaemia) had no significant effect on hydroxyl radical formation. We conclude that significant quantities of hydroxyl radicals form in the absence of circulating leucocytes during reperfusion following ischaemia, but not during hypoxia in the perfused rat kidney.