G. Healing

REDUCED SUSCEPTIBILITY TO LIPID-PEROXIDATION IN COLD ISCHEMIC RABBIT KIDNEYS AFTER ADDITION OF DESFERRIOXAMINE, MANNITOL, OR URIC-ACID TO THE FLUSH SOLUTION

Rabbit kidneys were stored for 24 hr at 0 degree C after single passage arterial flush with 30 ml of cold isotonic 0.9% sodium chloride (saline) solution alone or saline to which was added 12, 30, or 60 mM desferrioxamine, 1 or 3 mM uric acid, or 100 mM mannitol. They were then subjected to in vitro biochemical assay for evidence of free radical damage immediately after storage. Results were compared to those obtained with fresh, unstored kidneys. Levels of Schiff base fluorescence, diene conjugates, and thiobarbituric acid-reactive material were each significantly elevated in kidneys stored for 24 hr after flush with saline alone. These levels were in turn each significantly reduced by the addition of 60 mM desferrioxamine, 3 mM uric acid, and 100 mM mannitol to the flush solution. Likewise, glutathione redox activity fell in those flushed with saline alone, presumably in line with increased lipid peroxidation, but was restored to control levels by inclusion of the three scavenging agents.

Intracellular iron redistribution: An important determinant of reperfusion damage to rabbit kidneys

These studies were designed to examine the possible role of low molecular weight intracellular iron chelates (desferrioxamine-available (DFX-A) iron) in the damage which occurs during cold storage and subsequent reperfusion of kidneys. The level of DFX-A iron increased significantly (P less than 0.005) in the cortex of rabbit kidneys rendered cold ischaemic (CI) for 24 hr and the amount of iron available for DFX chelation increased significantly (P less than 0.05) in both the cortex and medulla of kidneys stored for 48 or 72 hr compared with fresh non-ischaemic controls. During ex vivo reperfusion of the organs with an oxygenated asanguinous perfusate, DFX-A iron returned rapidly to pre-ischaemic levels in 24 hr CI kidneys, but remained elevated following 48 and 72 hr CI (P less than 0.05 compared with 24 hr CI kidneys after 5 min reperfusion), returning to control levels only after 30 min reperfusion. There was no concurrent increase in total iron levels, indicating that a redistribution of iron to more accessible pools had occurred within the tissue. We suggest that decompartmentalization of intracellular iron during ischaemia and raised DFX-A iron levels over an extended period during subsequent reperfusion are responsible for increased catalysis of oxygen-derived free radical-mediated lipid peroxidation, and are an important factor in the deterioration of physiological function observed in rabbit kidneys following extended periods of cold storage.

Determination of desferrioxamine-available iron in biological tissues by high-pressure liquid chromatography

Intracellular iron loosely bound to proteins such as ferritin or in the form of low molecular weight chelates is available to catalyze adverse reactions such as the formation of reactive free radicals. A method to measure this small but important iron pool by utilizing the highly specific iron-chelator desferrioxamine is described. Following incubation of tissue fractions with desferrioxamine, the parent compound and its iron-bound form, ferrioxamine, are extracted using solid-phase cartridges and quantitated by reversed-phase HPLC using uv detection. Calculation of the ferrioxamine:desferrioxamine ratio and comparison with a standard curve constructed using a series of known iron concentrations allow the determination of micromolar amounts of desferrioxamine-available iron in biological samples.

Desferrioxamine reduces susceptibility to lipid peroxidation in rabbit kidneys subjected to ward ischaemia and reperfusion

Rabbit kidneys were clamped and subjected to warm ischaemia for 60 or 120 min then reperfused with blood for 60 min or for 24 hr. Treated rabbits received desferrioxamine at 15 or 50 mg/kg i.v. 15 min before reperfusion. Their kidneys were then removed and assayed for phospholipid Schiff base fluorescence (ex. 360 nm, em. 435 nm), diene and triene conjugates by UV spectrophotometry (240 nm and 268 nm respectively), for superoxide dismutase and for reduced and oxidised glutathione to provide an index of glutathione redox activity. All indices of lipid peroxidation were significantly elevated in untreated rabbits and glutathione redox activity was reduced. Treatment with desferrioxamine however effectively prevented these deviations and in many cases maintained them at the levels in fresh rabbit kidneys. These data provide further evidence that lipid peroxidation occurring during the reperfusion period is superimposed on the damage set up during warm ischaemia and may be preventable by administration of suitable therapeutic agents.

The Importance of Iron, Calcium and Free Radicals in Reperfusion Injury: An Overview of Studies in Ischaemic Rabbit Kidneys

An overview of a series of experiments attempting to link iron and calcium redistribution and release of free fatty acids with falls in pH and adenine nucleotide levels during cold storage of rabbit kidneys is presented. The data reviewed strongly suggest that these events are inextricably linked to subsequent reperfusion injury. Circumstantial evidence incriminating iron was provided by experiments showing that iron chelation decreased reperfusion injury after warm (WI) and cold ischaemia (CI) in rat skin flap and rabbit kidney models. Evidence for a role for calcium was provided when it was found that a calcium channel blocking agent added to the saline flush solution before storage inhibited lipid peroxidation, whereas chemicals which caused release or influx of calcium into the cell exacerbated oxidative damage. Additional involvement of breakdown products of adenine nucleotides was suggested by the protection from lipid peroxidation afforded by allopurinol. Involvement of calcium-activated phospholipase A2 was strongly suggested by increases in free fatty acids during cold storage and both this increase and lipid peroxidation were inhibited by addition of dibucaine to the storage solution.

Protection against oxidative damage in cold-stored rabbit kidneys by desferrioxamine and indomethacin

The storage of rabbit kidneys in hypertonic citrate solution at 0 degree C for 48-72 hr of cold ischemia resulted in oxidative damage to membranes as measured by the in vitro formation of two markers of lipid peroxidation (Schiffs base and thiobarbituric acid (TBA)-reactive material). This damage was further increased when the organs were autografted and reperfused for 60 min. The intravenous (iv) administration of desferrioxamine (a powerful iron-chelating agent) prior to the removal of the kidneys reduced the production of Schiffs bases and TBA-reactive material to low levels in the cortex of stored kidneys and decreased these measures of lipid peroxidation in the medulla by approximately 50%. Intravenous administration of indomethacin (a cyclooxygenase inhibitor) had no effect on the rate of lipid peroxidation in the renal cortex, but significantly reduced the formation of TBA-reactive material and Schiffs bases in the medulla of kidneys following storage for 72 hr. The existence of two separate pathways of lipid peroxidation (one iron-catalyzed and the other cyclooxygenase-catalyzed) in the medulla of stored kidneys was further confirmed when administration of desferrioxamine and indomethacin together resulted in significantly greater protection against lipid peroxidation than when these compounds were administered singly. The value of this combination of agents for protecting kidneys against the damage due to cold ischemia followed by reperfusion was further suggested by a trend toward improved long-term survival of the animals following replantation of the stored kidneys.

Measurement by HPLC of desferrioxamine-available iron in rabbit kidneys to assess the effect of ischaemia on the distribution of iron within the total pool.

A method for the determination of desferrioxamine-available iron in tissue fractions is described which involves incubation with desferrioxamine, extraction of desferrioxamine and its iron-bound form, ferrioxamine, and quantitation of these two forms of the drug by reversed-phase hplc analysis. Chelatable iron levels in the 1-10 microMolar region could be accurately and reproducibly measured using this technique. The desferrioxamine-available iron levels in both the cortex and medulla of rabbit kidneys were significantly elevated (up to 2-fold) after the organs had been subjected to 2 hours warm ischaemia or 24 hours cold storage at 0 degrees C in hypertonic citrate solution. There was no change in the total iron content of the tissues under these circumstances and thus a redistribution of intracellular iron to more available pools had presumably taken place as a result of ischaemia. This redistribution of iron may be an important factor in the initiation of peroxidative damage to cell membranes upon reperfusion of the organ with oxygen.

Increased susceptibility to lipid peroxidation in rabbit kidneys: A consequence of warm ischaemia and subsequent reperfusion

Rabbit kidneys were clamped and rendered warm ischaemic (WI) in situ for 60 and 120 min. They were then either removed immediately after the ischaemic insult or after reperfusion with blood for 60 min or 24 hr. Homogenates were assayed for phospholipid-Schiff base fluorescence (Ex. 360 nm, Em. 435 nm) and for diene conjugate formation by u.v. spectrophotometry (240 nm) as indices of lipid peroxidation. No alteration in tissue levels of Schiff base was evident immediately after WI but when the homogenates were incubated at 37 degrees C for 90 min, the rate of peroxidation was significantly elevated compared to controls (P less than 0.02 after WI of 60 min and P less than 0.001 after 120 min of WI). These values were still further elevated after reperfusion with blood for 60 min and 24 hr (P less than 0.001). Diene conjugates were raised after WI alone and further still after reperfusion. Thus an early index of lipid peroxidation (diene conjugation) suggested peroxidative damage during the warm ischaemic period itself, whilst detection of Schiff bases was only possible after in vitro incubation of the tissue. Both indices of oxygen-derived free radical damage were increased after reperfusion in vivo with blood and may relate to the degree of tissue damage sustained during ischaemia and reflow.

Allopurinol Inhibits Lipid Peroxidation in Warm Ischaemic and Reperfused Rabbit Kidneys

Rabbit kidneys were subjected to 120 min of warm ischaemia or to 120 min of warm ischaemia followed by 60 min reperfusion with blood in vivo before being removed, homogenised and incubated at 37 degrees C for 90 min. Lipid extracts were obtained and monitored for Schiff base (fluorescence emission 400-450 nm, excited at 360 nm), thiobarbituric acid (TBA)-reactive material (emission 553 nm, excited at 515 nm) and diene conjugates (absorbance at 237 nm). Samples removed before incubation were assayed for reduced glutathione (GSH) and oxidised glutathione (GSSG) to provide an index of glutathione redox activity (GSH:GSSG). Allopurinol injected systemically i.v. (a) 15 mins before kidneys were clamped, (b) 15 mins before they were reperfused or (c) as two injections (before clamping and before reperfusion) significantly inhibited these biochemical markers of lipid peroxidation. Administration before reperfusion had a markedly more pronounced effect than when allopurinol was given before warm ischaemia only. It is concluded that allopurinol is probably effective because of its ability to inhibit xanthine oxidase and consequently lipid peroxidation during reperfusion rather than by preventing loss of purine nucleotides from hypoxic cells during ischaemia.

Iron Redistribution and Lipid Peroxidation in the Cold Ischaemic Kidney

Oxygen-derived free radicals may play an important role in the damage which occurs to organs subjected to extended periods of cold storage followed by reperfusion with oxygenated blood upon transplantation into the recipient1. One damaging radical-mediated process is the peroxidation of membrane-bound polyunsaturated fatty acids and we have previously demonstrated significant elevations in lipid peroxidation markers in kidneys subjected to cold storage and autotransplantation2. Iron is required for the initiation of lipid peroxidation3 which may be the consequence of highly reactive OH. radical formation from O 2 .- and H2O2 via the Haber-Weiss reaction or direct attack on polyunsaturated fatty acids by iron complexes with oxygen4. In addition, iron also catalyses the decomposition of lipid hydroperoxides (LOOH) to alkoxy (LO.) and peroxy (LOO.) radicals which stimulate the chain reaction of lipid peroxidation4. In order to minimize the likelihood of these damaging reactions, iron is transported and stored in specific proteins. However, a small pool of iron exists in the cell as low molecular weight chelates5 which are able to exert catalytic activity.