transport rate is sufficient to prevent cell swelling. the rate of cooling of a tissue may also be significant in the production of injury to enzyme systems. francavilla showed that when liver slices were rapidly cooled (immediate cooling to 12 °c in 6 minutes) anaerobic glycolysis, as measured on rewarming to 37 °c, was inhibited by about 67% of the activity that was demonstrated in slices that had been subjected to delayed cooling. however, dog kidney slices were less severely affected by the rapid cooling than were the liver slices. anoxia all cells require atp as an energy source for their metabolic activity. the kidney is damaged by anoxia when kidney cortical cells are unable to generate sufficient atp under anaerobic conditions to meet the needs of the cells. when excising a kidney some anoxia is inevitable in the interval between dividing the renal artery and cooling the kidney. it has been shown by bergstrom that 50% of a dog's kidney's cortical cells atp content is lost within 1 minute of clamping the renal artery, and similar results were found by warnick in whole mice kidneys, with a fall in cellular atp by 50% after about 30 seconds of warm anoxia. warnick and bergstrom also showed that cooling the kidney immediately after removal markedly reduced any further atp loss. when these non warm-injured kidneys were perfused with oxygenated hypothermic plasma, atp levels were reduced by 50% after 24-hour storage and, after 48 hours, mean tissue atp levels were a little higher than this indicating that synthesis of atp had occurred. pegg has shown that rabbit kidneys can resynthesize atp after a period of perfusion storage following warm injury, but no resynthesis occurred in non warm-injured kidneys. warm anoxia can also occur during reimplantation of the kidney after storage. lannon showed, by measurements of succinate metabolism, how the kidney was more sensitive to a period of warm hypoxia occurring after storage than to the same period of warm hypoxia occurring immediately prior to storage. lack of essential nutrients active metabolism of glucose with production of bicarbonate has been demonstrated by pettersson and cohen. pettersson studies were on the metabolism of glucose and fatty acids by kidneys during 6 day hypothermic perfusion storage and he found that the kidneys consumed glucose at 4.4 μmol/g/day and fatty acids at 5.8 μmol/g/day. in cohen's study the best 8 day stored kidneys consumed glucose at the rate of 2.3 μmol/g/day and 4.9 μmol/g/day respectively which made it likely that they were using fatty acids at similar rates to pettersson's dogs' kidneys. the constancy of both the glucose consumption rate and the rate of bicarbonate production implied that no injury was affecting the glycolytic enzyme or carbonic anhydrase enzyme systems. lee showed that fatty acids were the preferred substrate of the rabbit's kidney cortex at normothermic temperatures, and glucose the preferred substrate for the medullary cells which normally metabolise anaerobically. abodeely showed that both fatty acids and glucose could be utilised by the outer medulla of the rabbit's kidney but that glucose was used preferentially. at hypothermia the metabolic needs of the kidney are much reduced but measurable consumption of glucose, fatty acids and ketone bodies occurs. horsburgh showed that lipid is utilised by hypothermic kidneys, with palmitate consumption being 0-15% of normal in the rat kidney cortex at 15 °c. pettersson showed that, on a molar basis, glucose and fatty acids were metabolised by hypothermically perfused kidneys at about the same rates. the cortex of the hypothermic dog kidney was shown by huang to lose lipid (35% loss of total lipid after 24 hours) unless oleate was added to the kidney perfusate. huang commented that this loss could affect the structure of the cell and that the loss also suggested that the kidney was utilising fatty acid. in a later publication huang showed that dog kidney cortex slices metabolised fatty acids, but not glucose, at 10 °c. even if the correct nutrients are provided, they may be lost by absorption into the tubing of the preservation system. lee demonstrated that silicone rubber (a material used extensively in kidney preservation systems) absorbed 46% of a perfusate's oleic acid after 4 hours of perfusion. toxin accumulation abouna showed that ammonia was released into the perfusate during 3 day kidney storage, and suggested that this might be toxic to the kidney cells unless removed by frequent replacement of the perfusate. some support for the use of perfusate exchange during long perfusions was provided by liu who used perfusate exchange in his successful 7 day storage experiments. grundmann also found that 96-hour preservation quality was improved by the use of a double volume of perfusate or by perfusate exchange. however, grundmann's conclusions were based on comparisons with a control group of only 3 dogs. cohen was unable to demonstrate any production of ammonia during 8 days of perfusion and no benefit from perfusate exchange; the progressive alkalinity that occurred during perfusion was shown to be due to bicarbonate production. toxic damage from the perfusate certain perfusates have been shown to have toxic effects on kidneys as a result of the inadvertent inclusion of particular chemicals in their formulation. collins showed that the procaine included in the formulation of his flush fluids could be toxic, and pegg has commented how toxic materials, such as pvc plasticizers, may be washed out of perfusion circuit tubing. dvorak showed that the methyl-prednisolone addition to the perfusate that was thought to be essential by woods might in some circumstances be harmful. he showed that with over g of methyl-prednisolone in 650 ml of perfusate (compared with 250 mg in 1 litre used by woods) irreversible haemodynamic and structural changes were produced in the kidney after 20 hours of perfusion. there was necrosis of capillary loops, occlusion of bowman's spaces, basement membrane thickening and endothelial cell damage. washout of essential substrates the level of nucleotides remaining in the cell after storage was thought by warnick to be important in determining whether the cell would be able to re-synthesize atp and recover after rewarming. frequent changing of the perfusate or the use of a large volume of perfusate has the theoretical disadvantage that broken down adenine nucleotides may be washed out of the cells and so not be available for re-synthesis into atp when the kidney is rewarmed. injury to nuclear dna nuclear dna is injured during cold storage of kidneys. lazarus showed that single stranded dna breaks occurred within 16 hours in hypothermically stored mice kidneys, with the injury being inhibited a little by storage in collins' or sacks' solutions. this nuclear injury differed from that seen in warm injury when double stranded dna breaks occurred. mechanical injury to the vascular system perfusion storage methods can mechanically injury the vascular endothelium of the kidney, which leads to arterial thrombosis or fibrin deposition after reimplantation. hill noted that, in human kidneys, fibrin deposition in the glomerulus after reimplantation and postoperative function, correlated with the length of perfusion storage. he had taken biopsies at revascularisation from human kidneys preserved by perfusion or ice storage, and showed by electron microscopy that endothelial disruption only occurred in those kidneys that had been perfused. biopsies taken one hour after revascularisation showed platelets and fibrin adherent to any areas of denuded vascular basement membrane. a different type of vascular damage was described by sheil who showed how a jet lesion could be produced distal to the cannula tied into the renal artery, leading to arterial thrombosis approximately 1 cm distal to the cannula site. post reimplantation injury there is evidence that immunological mechanisms may injure hypothermically perfused kidneys after reimplantation if the perfusate contained specific antibody. cross described two pairs of human cadaver kidneys that were perfused simultaneously with cryoprecipitated plasma containing type specific hla antibody to one of the pairs. both these kidneys suffered early arterial thrombosis. light described similar hyperacute rejection following perfusion storage and showed that the cryoprecipitated plasma used contained cytotoxic igm antibody. this potential danger of using cryoprecipitated plasma was demonstrated experimentally by filo who perfused dog kidneys for 24 hours with specifically sensitised cryoprecipitated dog plasma and found that he could induce glomerular and vascular lesions with capillary engorgement, endothelial swelling, infiltration by polymorphonuclear leucocytes and arterial thrombosis. immunofluorescent microscopy demonstrated specific binding of igg along endothelial surfaces, in glomeruli, and also in vessels. after reimplantation, complement fixation and tissue damage occurred in a similar pattern. there was some correlation between the severity of the histological damage and subsequent function of the kidneys. many workers have attempted to prevent kidneys rewarming during reimplantation but only cohen has described using a system of active cooling. measurements of lysosomal enzyme release from kidneys subjected to sham anastomoses, when either in or out of the cooling system, demonstrated how sensitive kidneys were to rewarming after a period of cold storage, and confirmed the effectiveness of the cooling system in preventing enzyme release. a further factor in minimising injury at the reimplantation operations may have been that the kidneys were kept at 7 °c within the cooling coil, which was within a degree of the temperature used during perfusion storage, so that the kidneys were not subjected to the greater changes in temperature that would have occurred if ice cooling had been used. dempster described using slow release of the vascular clamps at the end of kidney reimplantation operations to avoid injuring the kidney, but other workers have not mentioned whether or not they used this manoeuvre. after cohen found vascular injury with intra renal bleeding after 3 days of perfusion storage, a technique of slow revascularisation was used for all subsequent experiments, with the aim of giving the intra- renal vessels time to recover their tone sufficiently to prevent full systolic pressure being applied to the fragile glomerular vessels. the absence of gross vascular injury in his later perfusions may be attributable to the use of this manoeuvre.