Minimal role of xanthine oxidase and oxygen free radicals in rat renal tubular reoxygenation injury

The role of xanthine oxidase and oxygen free radicals in postischemic reperfusion injury in the rat kidney remains controversial. Proximal tubules, the focal segment affected by ischemic renal injury, were isolated in bulk, assayed for xanthine oxidase activity, and subjected to 60 min of anoxia or hypoxia and 60 min of reoxygenation to evaluate the participation of xanthine oxidase and oxygen radicals in proximal tubule reoxygenation injury. The total xanthine oxidase in isolated rat proximal tubules was 1.1 mU/mg of protein, approximately 30% to 40% of the activity found in rat intestine and liver. Lactate dehydrogenase release, an indicator of irreversible cell damage, increased substantially during anoxia (39.8 +/- 2.3 versus 9.8 +/- 1.8% in controls) with an additional 8 to 12% release during reoxygenation. Addition of 0.2 mM allopurinol, a potent xanthine oxidase inhibitor, and dimethylthiourea, a hydroxyl radical scavenger, failed to protect against the reoxygenation lactate dehydrogenase release. Analysis of xanthine oxidase substrate levels after anoxia and flux rates during reoxygenation indicates that hypoxanthine and xanthine concentrations are in a 15-fold excess over the enzyme Km and 0.3 mU/mg of protein of xanthine oxidase activity exists during reoxygenation. Hypoxic tubule suspensions had a minimal lactate dehydrogenase release during hypoxia and failed to demonstrate accelerated injury upon reoxygenation. In conclusion, although xanthine oxidase is present and active during reoxygenation in isolated rat proximal tubules, oxygen radicals did not mediate reoxygenation injury.     

Measurement of free radical generation from endothelial cells and observation of cell injury exposed to anoxia-reoxygenation]

Oxygen free radicals have been demonstrated to be important mediators in postischemic reperfusion injury. In this study, I determined the superoxide and the hydrogen peroxide generation from human umbilical endothelial cells on reoxygenation following anoxic incubation (1% O2, 5% CO2, 94% N2). The superoxide generation, detected by the reduction of cytochrome, c, was at its maximum 3 minutes after reoxygenation in any anoxic interval. The hydrogen peroxide production, detected by the fluorometric analysis, was observed later than that of superoxide. Treatment of EC with superoxide dismutase and allopurinol attenuated the superoxide production, and catalase attenuated the hydrogen peroxide. Cell injury was assessed by both fura-2 release assay and trypan blue dye exclusion methods. Although cell injury was less than 20% in anoxic condition, it was remarkably increased after reoxygenation. However this cell injury was not completely prevented in the presence of free radical scavengers. Allopurinol was more effective than superoxide dismutase or catalase. In conclusion, EC are the major source of free radicals in postischemic reperfusion which are originated mainly from xanthine-xanthine oxidase system and these radicals may also contribute, at least in part, to the EC injury.     

Prevention of free radical-induced myocardial reperfusion injury with allopurinol

Growing evidence supports the concept that oxygen free radicals are an important cause of myocardial ischemic and reperfusion injury. This study was designed to determine if toxic oxygen metabolites may exacerbate ischemic injury upon reoxygenation. Left ventricular function was studied in a group of seven dogs receiving intermittent, 4 degrees C, hyperosmolar, hyperkalemic (KCI 25 mEq/L) saline cardioplegic solution. This group was compared to a group (n = 7) receiving a hyperkalemic (KCI 25 mEq/L) cardioplegic solution designed to scavenge superoxide anion and hydroxyl radical: superoxide dismutase (3,000 U/ml) and mannitol (325 mOsm/L). A third group of five animals received allopurinol pretreatment (50 mg/kg/day) for 72 hours and hyperkalemic saline cardioplegic solution. After 60 minutes of ischemia (10 degrees to 15 degrees C) and 45 minutes of reperfusion, left ventricular mechanical function was better in the groups receiving free radical scavengers and allopurinol pretreatment than in the group receiving only hyperkalemic saline cardioplegic solution. Free radical scavengers preserved myocardial function in this model of hypothermic global ischemia and reperfusion. Our data support the concept that injury occurs primarily during reperfusion with the generation of oxygen free radicals via the hypoxanthine-xanthine oxidase reaction. Allopurinol has potential clinical application in the prevention of reperfusion injury.     

Pulmonary reperfusion injury: evidence for oxygen-derived free radical mediated damage and effects of different free radical scavengers

Blood granulocyte-mediated reactions involving generation of oxygen-derived free radicals have recently been shown to be capable of causing injury to the lungs. These findings suggest a similar mechanism also to be involved in the development of pulmonary ischemia/reperfusion injury. In the present study, therefore, the effects of three oxygen-derived free radical scavengers, superoxide dismutase (SOD; 1 mg/kg), catalase (20000 IU/kg) and allopurinol (45 mg/kg), were evaluated during reperfusion in a rabbit model after 2 h normothermic ischemia of the lung. During reperfusion, ischemic lungs were found to have an elevated pulmonary vascular resistance, increased total and extravascular lung water content, and decreased arterial oxygen tension (PaO₂) compared to control animals. SOD and catalase, but not allopurinol, were able to reduce pulmonary injury by lowering the pulmonary vascular resistance, but could not prevent pulmonary damage as shown by total lung water (TLW) or PaO₂. It is concluded that oxygen-derived free radicals such as hydrogen peroxide and the superoxide anion may play an important role in precipitating pulmonary injury after ischemia. The failure of xanthine oxidase inhibition (allopurinol) to exert protective effects may suggest that oxygen-derived free radical generation following pulmonary ischemia occurs predominantly via leukocyte-mediated reactions.     

Inhibition of the compartment syndrome by the ablation of free radical-mediated reperfusion injury

Skeletal muscle edema secondary to an increase in capillary permeability after reflow is an important cause of the compartment syndrome after acute arterial revascularization. The purpose of this study was to investigate the possible role of oxygen free radicals, generated at reperfusion, in the pathogenesis of the compartment syndrome secondary to acute arterial ischemia/reperfusion. A reproducible model of this syndrome was produced in anesthetized rabbits by femoral artery occlusion after surgical devascularization of collateral branches from the aorta to the popliteal artery. Increasing periods of ischemia from 6 to 12 hours, followed by 2 hours of reperfusion, were associated with corresponding increases in the anterior muscle compartment hydrostatic pressure and inversely proportional decreases in tibialis anterior muscle blood flow within that compartment as assessed by xenon 133 washout (n = 46) (r = -0.62, p less than 0.001). Anterior compartment pressure increased from 5 +/- 1 to 48 +/- 5 mm Hg (n = 46) (p less than 0.001) after 7 hours of total arterial ischemia and 2 hours of reperfusion. Ablation of free radicals generated from xanthine oxidase with either allopurinol (n = 8) or oxypurinol (n = 8), by scavenging the superoxide radical at reperfusion with superoxide dismutase (n = 8), or by blocking secondary hydroxyl radical formation with deferoxamine (n = 8) significantly ameliorated the rise in compartment pressure (p less than 0.05) in each case; it also significantly improved muscle perfusion in the superoxide dismutase-, allopurinol-, and deferoxamine-treated animals (p less than 0.05). These findings indicate that development of the compartment syndrome after acute arterial revascularization may be due, at least in part, to microvascular injury mediated by oxygen-derived free radicals generated from xanthine oxidase at reperfusion.     

Changes in xanthine oxidase in ischemic rat brain

Xanthine oxidase activity in the rat brain was measured by means of high-performance liquid chromatography with electrochemical detection of uric acid. Cerebral ischemia was produced by a four-vessel occlusion method. In the control rat, the enzyme activity was 0.87 +/- 0.13 nmol/gm wet weight/min at 25 degrees C (mean +/- standard deviation), of which 92.4% was associated with the nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenase form and only 7.6% with the oxygen-dependent superoxide-producing oxidase form. However, the ratio of the latter form increased to 43.7% after 30 minutes of global ischemia, despite the total xanthine oxidase activity remaining the same. Thus, it was revealed that uric acid can be synthesized in the rat brain and that cerebral ischemia induced the conversion of xanthine oxidase from an NAD-dependent dehydrogenase to an oxygen-dependent superoxide-producing oxidase. Although the xanthine oxidase pathway has been proposed as a source of oxygen-derived free radicals in various ischemic organs other than brain, the results of the present study suggest the involvement of the oxygen free radicals generated from this pathway in the pathogenesis of the ischemic injury of the rat brain.     

Effects of superoxide dismutase and allopurinol on the survival of acute island skin flaps.

We have demonstrated previously that oxygen-derived free radicals are important mediators of tissue injury following ischemia (total venous occlusion) and reperfusion in small (3 cm X 6 cm) island skin flaps in rats. In this study, we evaluated extension of this concept to regional ischemia in large (8 cm X 8 cm) acute island skin flaps which were constructed to exceed their sole blood supply via unilateral inferior epigastric vessels. Under normal (control) circumstances, a significant portion of the flap would undergo necrosis at the periphery, mimicking the corresponding clinical situation. Blocking the generation of superoxide radicals from xanthine oxidase with a single dose of allopurinol prior to flap elevation significantly improved the area of flap viability from 34 +/- 12% to 57 +/- 6% (p less than 0.01) in the random portion of the flap, contralateral to the source of blood supply. Similarly, the detoxification of superoxide radicals with a single dose of superoxide dismutase improved viability from 41 +/- 6% to 58 +/- 7% (p less than 0.01). Similar results were obtained when either of these agents were administered 60 minutes after flap elevation. These findings suggest that oxygen-derived free radicals play an important role in the development of tissue necrosis in the critical transition zone between well-vascularized and devascularized skin.     

The pathogenesis of acute pancreatitis. The source and role of oxygen-derived free radicals in three different experimental models.

Recent experimental work has suggested that oxygen-derived free radicals may play an important role in initiating the early capillary injury in acute pancreatitis. Data from models of ischemic injury in other organs have suggested the enzyme xanthine oxidase is important in generating oxygen-derived free radicals. The present study was performed to determine whether xanthine oxidase is the source of free radical production in experimental pancreatitis. Utilizing the isolated, perfused, ex vivo canine pancreas preparation, three models of pancreatitis were initiated with (1) free fatty acid infusion (FFA), (2) partial duct obstruction and secretin stimulation (POSS), and (3) ischemia (ISCH). In each model, during a 4-hour perfusion, edema developed, weight gain occurred (FFA 120.6 +/- 21.1 gm; POSS 44.5 +/- 6.9 gm; ISCH 63.3 +/- 14.0 gm), and the serum amylase became elevated (FFA 1827 +/- 397 u/dl; POSS 10,171 +/- 1487 u/dl; ISCH 1860 +/- 365 u/dl). When the xanthine oxidase enzyme inhibitor allopurinol was added to the perfusate prior to the 4-hour perfusion, edema formation was absent or minimal, weight gain was significantly less (FFA 15.2 +/- 2.5 gm p less than 0.05; POSS 8.8 +/- 2.7 gm p less than 0.001; ISCH 12.3 +/- 2.8 gm p less than 0.01), and the amylase remained normal or the elevation was significantly decreased (FFA 996 +/- 189 u/dl p less than 0.05; POSS 3021 +/- 1074 u/dl p less than 0.001; ISCH 993 +/- 214 u/dl p less than 0.002). These data confirm that oxygen-derived free radicals play an important role in the pathogenesis of experimental acute pancreatitis, and suggest that the enzyme xanthine oxidase may well be the source of their production.     

Oxygen free radical induced damage in kidneys subjected to warm ischemia and reperfusion. Protective effect of superoxide dismutase.

Superoxide anion free radical (O2-.) has been implicated in the pathogenesis of tissue injury consequent to ischemia/reperfusion in several different organs, including heart and bowel. Superoxide dismutase (SOD), an enzyme free radical scavenger specific for O2-., has been used successfully to protect these organs from structural damage during reoxygenation of ischemic tissue. It has been suggested that the catalytic action of xanthine oxidase in injured tissue is an important source of O2-. during reoxygenation. In order to evaluate the potential of SOD to protect against kidney damage resulting from transient ischemia followed by reperfusion with oxygenated blood, a model of warm renal ischemia was studied. LBNF1 rats underwent right nephrectomy and occlusion of the left renal artery for 45 minutes. Survival in the group of ischemic untreated rats (N = 30) was 56% at 7 days and serum creatinine was greatly elevated (p less than 0.01) in rats remaining alive over the full 7-day period. In strong contrast to these results, all of the animals treated with SOD before reperfusion (N = 18) were alive after 7 days similar to sham operated control rats (N = 8). Serum creatinine in the SOD treated rats was significantly elevated only to postoperative day 3 and thereafter returned to normal. Rats treated with inactive SOD (N = 4) or SOD before ischemia (N = 4) had decreased survival rates compared to ischemic untreated animals and prolonged elevation of serum creatinine. When the ischemia time was extended to 60 minutes, only 19% of the untreated animals (N = 16) survived at 7 days whereas nearly 60% of the SOD-treated animals survived (N = 19). Serum creatinine was greatly elevated during the full 7-day observation period in all surviving rats in the untreated ischemic group, whereas serum creatinine returned to normal (p less than 0.05) after 4 days in the surviving rats treated with SOD. To test whether the action of xanthine oxidase contributed to the kidney damage after reoxygenation, 45 min. ischemic rat kidneys were treated with allopurinol. All of the animals treated with allopurinol (N = 12) were alive at 7 days. Serum creatinine values returned to normal after the episode of ischemia and reperfusion but more slowly than after SOD treatment. Histologic evaluation of kidney tissue taken from animals after ischemia alone showed extensive renal tubular damage, which was essentially absent in kidneys from SOD-treated animals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Free radicals and cardioplegia: allopurinol and oxypurinol reduce myocardial injury following ischemic arrest

Oxygen-derived free radicals generated by xanthine oxidase may represent a major cause of myocardial injury during ischemia and reperfusion. We have used the isolated working rat heart model of cardiopulmonary bypass and ischemic arrest to assess whether allopurinol or oxypurinol, which should prevent free radical formation through their ability to inhibit xanthine oxidase, can improve postischemic myocardial recovery when the drugs are administered either chronically (pretreatment) or acutely (as an addition to the cardioplegic or reperfusion solution). With normothermic ischemic arrest, both drugs, when given either chronically or acutely, significantly improved postischemic recovery of function. However, under hypothermic conditions, allopurinol conferred no protection when given either as pretreatment or during reperfusion, but it was effective when added to the cardioplegic solution. When administered under the appropriate conditions, both allopurinol and oxypurinol enhanced the protective effect afforded by the St. Thomas' Hospital cardioplegic solution, possibly by inhibiting xanthine oxidase activity and preventing the formation of oxygen-derived free radicals.     

Breakdown of hepatic tight junctions during reoxygenation injury.

We investigated whether reoxygenation following anoxia increased biliary permeability and whether or not allopurinol had a protective effect. Isolated rat livers were perfused for 30 min in a one-pass system with buffer equilibrated with 100% nitrogen after stabilization, and then for 60 min with the oxygenated buffer. Hepatic tight junction permeability was assessed by quantifying the early appearance in the bile of horseradish peroxidase (HRP) injected with the perfusate. This early peak represents paracellular passage of HRP, whereas a later second peak results from transcellular passage. In the control livers, 7% of the total HRP passage (93 +/- 50 pg/g liver) was paracellular and 93% was transcellular. After 30 min of reoxygenation following anoxia, however, 516 +/- 20 pg/g liver of HRP passed paracellularly. Addition of allopurinol (5 micrograms/ml) to the perfusate from the start of perfusion reduced paracellular passage of HRP to 219 +/- 49 pg/g liver after anoxia and reperfusion (P less than 0.01). Allopurinol also reduced the cumulative lactate dehydrogenase (LDH) release during the first 30 min of reoxygenation from 2.1 +/- 0.3 x 10(4) to 1.4 +/- 0.4 x 10(4) units/g liver (P less than 0.01). Reduction of the anoxic period from 30 min to 25 min significantly reduced the change in tight junction permeability and the extent of cellular injury: Paracellular passage of HRP was 336 +/- 20 pg/g and LDH release was 0.7 +/- 0.1 x 10(4) units/g liver, both significantly lower than those at 30 min (P less than 0.01). No significant difference in hepatic ATP levels after 60 min of reoxygenation was noted among the experimental groups, but all had lower levels than the control group. The protective effect of allopurinol suggests that the mechanism of biliary reoxygenation injury involves free radical generation. Susceptibility of tight junctions suggests a pattern of injury similar to that involved in anoxic damage of the vascular endothelium.     

Free radicals and cardioplegia. Free radical scavengers improve postischemic function of rat myocardium

Oxygen-derived free radicals, such as the superoxide (O2-) anion, hydrogen peroxide (H2O2) and the hydroxyl (OH.) radical, may be involved in exacerbating myocardial injury during reoxygenation of ischemic tissue. The naturally occurring antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), prevent the formation of the cytotoxic OH. radical during physiological conditions but may not be able to cope with the free radical generation that follows ischemia and reperfusion. We have used the isolated perfused working rat heart model of cardiopulmonary bypass and ischemic arrest to assess whether exogenous addition of SOD (20 IU/ml) and CAT (100 IU/ml) during ischemia and/or reperfusion can improve postischemic recovery of function following normothermic or hypothermic global ischemic arrest induced by St. Thomas' Hospital cardioplegic solution. Under conditions of normothermia, the addition of SOD alone or CAT alone to both the cardioplegic solution (CS) and the reperfusion solution (RS) had no effect on postischemic recovery (after 20-min working reperfusion) of aortic flow (27.9 +/- 2.7% and 16.1 +/- 6.3%, respectively) when compared with the nontreated control value of 28.1 +/- 3.7%. However, recovery was improved when SOD plus CAT were added to the CS alone (39.3 +/- 8.7%) and was significantly improved when they were added either to both the CS and the RS (48.4 +/- 6.0%; P = less than 0.02) or to the RS alone (51.3 +/- 3.7%; P = less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence against increased hydroxyl radical production during oxygen deprivation-reoxygenation proximal tubular injury.

The purpose of this study was to assess whether proximal renal tubules generate excess hydroxyl radical (.OH) during hypoxia/reoxygenation or ischemia/reperfusion injury, thereby supporting the hypothesis that reactive oxygen species contribute to the pathogenesis of postischemic acute renal failure. In the first phase of the study, rat isolated proximal tubular segments (PTS) were subjected to hypoxia (95% N2- 5% CO2) for 15, 30, or 45 min, followed by 15 to 30 min of reoxygenation in the presence of sodium salicylate, a stable .OH trap. Cellular injury after hypoxia and reoxygenation was assessed by lactate dehydrogenase release; .OH production was gauged by hydroxylated salicylate by-product generation (2,3-, 2,5-dihydroxybenzoic acids (DHBA); quantified by HPLC/electrochemical detection). Continuously oxygenated PTS served as controls. Despite substantial lactate dehydrogenase release during hypoxia (8 to 46%) and reoxygenation (8 to 11%), DHBA production did not exceed that of the coincubated, continuously oxygenated control PTS. In the second phase of the study, salicylate-treated rats were subjected to 25 or 40 min of renal arterial occlusion +/- 15 min of reperfusion. No increase in renal DHBA concentrations occurred during ischemia or reperfusion, compared with that in sham-operated controls. To validate the salicylate trap method, PTS were incubated with a known .OH-generating system (Fe2+/Fe3+); in addition, rats were treated with antioxidant interventions (oxypurinol plus dimethylthiourea). Fe caused marked DHBA production, and the antioxidants halved in vivo DHBA generation. In conclusion, these results suggest that exaggerated .OH production is not a consequence of O2 deprivation/reoxygenation tubular injury.     

Modification of oxidative stress in response to intestinal preconditioning

Previous studies have demonstrated that intestinal preconditioning protects the organ from ischemia reperfusion damage. Xanthine oxidase mediating free radical generation contributes to the development of injury associated to ischemia reperfusion. Thus, any process able to modulate the oxygen free radical generation system could attenuate the injury. Also, it is known that nitric oxide is implicated in the preconditioning response. The aim of this work is to determine: (1) the effect of intestinal preconditioning on the xanthine oxidase system, (2) the relevance of this system in the development of injury, and (3) its relationship with nitric oxide. For this purpose, we have determined the activity of the xanthine dehydrogenase/xanthine oxidase system, the levels of its substrate (xanthine), and end-product (uric acid) and oxidant stress status in rat small intestine subjected to ischemic pre-conditioning. The effects of nitric oxide inhibition have also been evaluated. Results show that the percentage of xanthine dehydrogenase to xanthine oxidase conversion, xanthine, uric acid concentration, lipoperoxides, and reduced glutathione were significantly reduced in preconditioned rats irrespectively of nitric oxide inhibition. In summary, this work shows that oxidative stress in intestinal preconditioning is reduced as consequence of the diminished conversion of xanthine dehydrogenase to xanthine oxidase, and also as a consequence of the reduced availability of xanthine.     

Histamine: a promoter of xanthine oxidase activity in intestinal ischemia/reperfusion.

Xanthine oxidase (XO)-derived oxygen radicals are thought to play an important role in the intestinal injury resulting from ischemia and reperfusion. In vitro data shows enhanced XO activity in the presence of histamine. Histamine is known to be released during intestinal ischemia and reperfusion. The purpose of this study was to evaluate the relationship between histamine and XO in vivo in intestinal ischemia/reperfusion injury. Using an established model of gut ischemia and reperfusion, portal venous plasma was obtained and assayed for histamine levels, XO activity, and xanthine dehydrogenase (XD) activity following injury. Intestinal ischemia for 120 minutes resulted in a 200% increase in plasma histamine levels (263.4 +/- 36.9 nmol/mL control, v 548.7 +/- 35.1 nmol/mL experimental, P less than .05). Reperfusion for 15 minutes resulted in a further increase in plasma histamine (to 658.3 +/- 33.9 nmol/mL), compared with 120 minutes of ischemia alone. No significant change in plasma XO activity resulted after simple ischemia for 120 minutes. However, XO activity doubled within 15 minutes of reperfusion of the ischemic intestine (6.37 +/- 0.53 nmol O2- per milliliter per minute v 3.12 +/- 0.25 nmol O2- per milliliter per minute, P less than .05). Reperfusion for 60 minutes resulted in the maximal observed increase in plasma XO activity (9.49 +/- 0.67 nmol O2- per milliliter per minute). Analysis of XD activity demonstrated no significant decrease compared with controls until 120 minutes of ischemia and 60 minutes of reperfusion (1.62 +/- 0.49 nmol uric acid per milliliter per minute at 60 minutes of reperfusion, versus 5.02 +/- 0.52 nmol uric acid per milliliter per minute control, P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of oxygen free radicals in mediating the reperfusion injury of cold-preserved ischemic kidneys

We evaluated the hypothesis that postischemic renal failure is caused primarily at reperfusion by oxygen-derived free radicals in a swine model designed to realistically mimick human cadaveric renal transplantation. Both kidneys were removed, flushed with Euro-Collins solution, stored 24 hr at 4 degrees C, and then transplanted to a second pig. Experiments were paired, each pig receiving one treated and one control kidney. All pigs received the optimal conventional regimen of hydration, phenoxybenzamine, furosemide, and mannitol to allow assessment of free radical treatment superimposed thereupon. Two days later creatinine clearance (CCR) was measured from each kidney via separate ureterostomies. Untreated kidneys developed severe functional impairment, CCR falling from a normal level of 25.5 +/- 6.3 ml/min (n = 8) to 7.7 +/- 0.9 ml/min (n = 14, P less than .05 vs. control). The infusion of 20 mg of the free radical scavenger superoxide dismutase (SOD) into the renal artery at reperfusion substantially ameliorated this injury (CCR = 15.9 +/- 1.7 ml/min, n = 18, P less than 0.05 vs. control). A dose-response curve to SOD showed no effect of doses of 0.2 mg (CCR = 8.0 +/- 1.1 ml/min, n = 4) or 2 mg (CCR = 7.7 +/- 0.9, n = 5), and no greater benefit from 100 mg (CCR = 16.1 +/- 2.1 ml/min, n = 3, P less than 0.05 vs. control). Blocking the generation of superoxide radicals from xanthine oxidase with allopurinol (50 mg/kg) afforded similar protection (CCR = 18.2 +/- 1.8; n = 11, P less than 0.01 vs. control). On the other hand, following an 18-hr period of cold ischemia, little damage was sustained by the untreated (control) kidneys (CCR = 22.1 +/- 0.6 ml/min). Consequently, under these conditions the ablation of free radical generation with allopurinol provided no significant benefit. These findings suggest that after a critical period of cold ischemic preservation, metabolic changes take place within the kidney that lead to free radical generation and consequent tissue injury upon reperfusion, despite optimal preservation by conventional methods. This damage can be prevented by simple nontoxic measures--which, therefore, show great promise for use in the prevention of early renal failure following cadaveric renal transplantation.     

Use of oxygen radical scavengers on autografted pig kidneys after warm ischemia and 48-hour perfusion preservation

Oxygen free radicals generated during the reperfusion of an ischemic organ may cause further cellular injury; removal of these oxygen radicals by scavengers protects tissue from reperfusion injury. Thus, oxygen radical scavengers could protect kidneys after warm ischemia and long hypothermic perfusion. Porcine kidneys were incubated at 37 degrees C for 45 minutes, placed on a pulsatile perfusion apparatus at 7 degrees C for 48 hours, and then autografted to iliac vessels. Superoxide dismutase (10 mg) and catalase (10 mg) in 10 mL of phosphate-buffered saline solution were infused into the renal artery during a three-minute interval before reperfusion. The kidneys treated with the superoxide dismutase-catalase solution had significantly improved function compared with controls receiving only phosphate-buffered saline solution. The mean (+/- SEM) serum creatinine level on postoperative day 5 was 510 +/- 100 mumol/L (5.75 +/- 1.12 mg/dL) (n = 12) vs the control value of 840 +/- 90 mumol/L (9.54 +/- 1.01 mg/dL) (n = 11). There was more extensive cellular damage in the control kidneys. This demonstrates the efficacy of oxygen radical scavengers in protecting pig kidneys after warm ischemia and prolonged preservation.     

Inhibition of surgically induced ischemia/reperfusion injury by oxygen free radical scavengers

Recent experimental work implicates oxygen free radicals as mediators of ischemia/reperfusion injury. A simple cardioplegic solution was designed to scavenge superoxide anion and hydroxyl free radical with superoxide dismutase (10 micrograms/ml), mannitol (325 mOsm/L), and KCl 25 mEq/L (FRS). Hemodynamic and subcellular functions were studied in seven in situ canine models of hypothermic global ischemia receiving FRS, compared to a group (n = 7) receiving hyperosmolar, hyperkalemic saline (HSK) and to a standard model of topical hypothermia (TH, n = 5). Following 60 minutes of ischemia (10 degrees to 15 degrees C), hearts were reperfused and rewarmed. After 45 minutes of reperfusion, left ventricular peak systolic pressure (LVPSP), developed pressure (LVDP), dP/dt max, -dP/dt max, compliance, and elastic stiffness constant (K) were improved in the FRS group and not significantly different from control. Sarcoplasmic reticulum (SR) calcium transport in the FRS group was significantly improved (control = 1.077 +/- 0.022, TH = 0.754 +/- 0.018, HSK = 0.725 +/- 0.05, and FRS = 0.966 +/- 0.05 mumol/mg-min). Calcium adenosine triphosphatase (ATPase) activity did not differ significantly from control at pH 7.0. In this model of hypothermic global ischemia and reperfusion, free radical scavengers provide significant protection of mechanical and subcellular function. These findings support the hypothesis that oxygen free radicals are important mediators of myocardial ischemia and reperfusion injury.     

Allopurinol prevents intestinal permeability changes after ischemia-reperfusion injury.

Under normal conditions the intestinal mucosa is impermeable to potentially harmful materials from the intestinal lumen. Mucosal disruption promotes bacterial translocation, which is postulated to be a fuel source for sepsis and multiorgan failure. We have previously demonstrated that mesenteric ischemia-reperfusion (I/R) injury increases intestinal permeability (IP); however, the mechanism remains unclear. This study was designed to examine the hypothesis that changes in IP, after I/R injury, are mediated by xanthine oxidase-generated, oxygen-derived free radicals. Thirty-three Sprague-Dawley rats (weighing 300 to 400 g) were included in this study. Group 1 (n = 10) received enteral allopurinol, a xanthine oxidase inhibitor, 10 mg/kg daily for 1 week prior to mesenteric ischemia. Group 2 consisted of 11 untreated, ischemic animals. Groups 1 and 2 were subjected to superior mesenteric artery occlusion with interruption of collateral flow for 20 minutes to produce ischemic injury to the intestine. An additional 12 rats (group 3), served as nonischemic controls (sham). A loop of distal ileum was isolated and cannulated proximally and distally to allow luminal perfusion with warmed Ringer's lactate at 1 mL/min. IP was determined in all groups by quantitatively measuring the plasma-to-luminal clearance of chromium (51Cr)-labeled ethylenediaminetetraacetate (EDTA) at baseline, during ischemia and 20, 40, and 60 minutes after reperfusion. Complete ischemia produced significant increases in IP over baseline values in the untreated rats (group 2, baseline: 0.49 +/- 0.006, ischemia: 0.149 +/- 0.039) compared with sham rats (baseline: 0.41 +/- 0.006; ischemia: 0.047 +/- 0.009) or allopurinol-treated rats (baseline: 0.098 +/- 0.020, ischemia: 0.073 +/- 0.012, P less than .001).(ABSTRACT TRUNCATED AT 250 WORDS)