7.4 by a co2 titrator. woods stressed the importance of hydration of the donor and recipient animals. without the methyl prednisolone, woods found vessel fragility to be a problem when storage times were longer than 48 hours. a major simplification to the techniques of hypothermic perfusion storage was made by johnson and claes in 1972 with the introduction of an albumin based perfusate. this perfusate eliminated the need for the manufacture of the cryoprecipitated and millipore filtered plasma used by belzer. the preparation of this perfusate had been laborious and time-consuming, and there was the potential risk from hepatitis virus and cytotoxic antibodies. the absence of lipo-proteins from the perfusate meant that the membrane oxygenator could be eliminated from the perfusion circuit, as there was no need to avoid a perfusate/air interface to prevent precipitation of lipo-proteins. both workers used the same additives as recommended by belzer. the solution that johnson used was prepared by the blood products laboratory (elstree: england) by extracting heat labile fibrinogen and gamma globulins from plasma to give a plasma protein fraction (ppf) solution. the solution was incubated at 60 °c for 10 hours to inactivate the agent of serum hepatitis. the result was a 45 g/l human albumin solution containing small amounts of gamma and beta globulins which was stable between 0 °c and 30 °c for 5 years. ppf contained 2.2 mmol/l of free fatty acids. johnson's experiments were mainly concerned with the storage of kidneys that had been damaged by prolonged warm injury. however, in a control group of non-warm injured dog kidneys, johnson showed that 24-hour preservation was easily achieved when using a ppf perfusate, and he described elsewhere a survivor after 72 hours perfusion and reimplantation with immediate contralateral nephrectomy. with warm injured kidneys, ppf perfusion gave better results than collins' method, with 6 out of 6 dogs surviving after 40 minutes warm injury and 24-hour storage followed by reimplantation of the kidneys and immediate contralateral nephrectomy. potassium, magnesium, insulin, glucose, hydrocortisone and ampicillin were added to the ppf solution to provide an energy source and to prevent leakage of intracellular potassium. perfusate temperature was 6 °c, pressure 40–80 mm hg, and po2 200–400 mm hg. the ph was maintained between 7.2 and 7.4. claes used a perfusate based on human albumin (kabi: sweden) diluted with saline to a concentration of 45 g/l. claes preserved 4 out of 5 dog kidneys for 96 hours with the kidneys functioning immediately after reimplantation despite immediate contralateral nephrectomies. claes also compared this perfusate with belzer's cryoprecipitated plasma in a control group and found no significant difference between the function of the reimplanted kidneys in the two groups. the only other group besides woods' to report successful seven-day storage of kidneys was liu and humphries in 1973. they had three out of seven dogs surviving, after their kidneys had been stored for seven days followed by reimplantation and immediate contralateral nephrectomy. their best dog had a peak post reimplantation creatinine of 50 mg/l (0.44 mmol/l). liu used well hydrated dogs undergoing a mannitol diuresis and stored the kidneys at 9 °c – 10 °c using a perfusate derived from human ppf. the ppf was further fractionated by using a highly water-soluble polymer (pluronic f-38), and sodium acetyl tryptophanate and sodium caprylate were added to the ppf as stabilisers to permit pasteurisation. to this solution were added human albumin, heparin, mannitol, glucose, magnesium sulphate, potassium chloride, insulin, methyl prednisolone, carbenicillin, and water to adjust the osmolality to 300-310 mosmol/kg. the perfusate was exchanged after 3.5 days storage. perfusate pressure was 60 mm hg or less, at a pump rate of 60 per minute. perfusate ph was 7.12–7.32 (at 37 °c), pco2 27–47 mm hg, and po2 173–219 mm hg. in a further report on this study humphries found that when the experiments were repeated with a new batch of ppf no survivors were obtained, and histology of the survivors from the original experiment showed glomerular hypercellularity which he attributed to a possible toxic effect of the pluronic polymer. joyce and proctor reported the successful use of a simple dextran based perfusate for 72-hour storage of dog kidneys. 10 out of 17 kidneys were viable after reimplantation and immediate contralateral nephrectomy. joyce used non pulsatile perfusion at 4 °c with a perfusate containing dextran 70 (pharmacia) 2.1%, with additional electrolytes, glucose (19.5 g/l), procaine and hydrocortisone. the perfusate contained no plasma or plasma components. perfusate pressure was only 30 cm h2o, ph 7.34-7.40 and po2 250–400 mm hg. this work showed that, for 72-hour storage, no nutrients other than glucose were needed, and low perfusate pressures and flows were adequate. in 1973 sacks showed that simple ice storage could be successfully used for 72-hour storage when a new flushing solution was used for the initial cooling and flush out of the kidney. sacks removed kidneys from well hydrated dogs that were diuresing after a mannitol infusion, and flushed the kidneys with 200 ml of solution from a height of 100 cm. the kidneys were then simply kept at 2 °c for 72 hours without further perfusion. reimplantation was followed by immediate contralateral nephrectomies. the flush solution was designed to imitate intracellular fluid composition and contained mannitol as an impermeable ion to further prevent cell swelling. the osmolality of the solution was 430 mosmol/kg and its ph was 7.0 at 2 °c. the additives that had been used by collins (dextrose, phenoxybenzamine, procaine and heparin) were omitted by sacks. these results have been equalled by ross who also achieved successful 72-hour storage without using continuous perfusion, although he was unable to reproduce collins' or sacks' results using the original collins' or sacks' solutions. ross's successful solution was similar in electrolyte composition to intracellular fluid with the addition of hypertonic citrate and mannitol. no phosphate, bicarbonate, chloride or glucose were present in the solution; the osmolality was 400 mosmol/kg and the ph 7.1. five of 8 dogs survived reimplantation of their kidneys and immediate contralateral nephrectomy, when the kidneys had been stored for 72 hours after having been flushed with ross's solution; but ross was unable to achieve 7 day storage with this technique even when delayed contralateral nephrectomy was used. the requirements for successful 72-hour hypothermic perfusion storage have been further defined by collins who showed that pulsatile perfusion was not needed if a perfusate pressure of 49 mm hg was used, and that 7 °c was a better temperature for storage than 2 °c or 12 °c. he also compared various perfusate compositions and found that a phosphate buffered perfusate could be used successfully, so eliminating the need for a carbon dioxide supply. grundmann has also shown that low perfusate pressure is adequate. he used a mean pulsatile pressure of 20 mm hg in 72-hour perfusions and found that this gave better results than mean pressures of 15, 40, 50 or 60 mm hg. successful storage up to 8 days was reported by cohen using various types of perfusate – with the best result being obtained when using a phosphate buffered perfusate at 8 °c. inability to repeat these successful experiments was thought to be due to changes that had been made in the way that the ppf was manufactured with higher octanoic acid content being detrimental. octanoic acid was shown to be able to stimulate metabolic activity during hypothermic perfusion and this might be detrimental. nature of kidney preservation injury structural injury the structural changes that occur during 72-hour hypothermic storage of previously uninjured kidneys have been described by mackay who showed how there was progressive vacuolation of the cytoplasm of the cells which particularly affected the proximal tubules. on electron microscopy the mitochondria were seen to become swollen with early separation of the internal cristal membranes and later loss of all internal structure. lysosomal integrity was well preserved until late, and the destruction of the cell did not appear to be caused by lytic enzymes because there was no more injury immediately adjacent to the lysosomes than in the rest of the cell. woods and liu – when describing successful 5 and 7 day kidney storage - described the light microscopic changes seen at the end of perfusion and at post mortem, but found few gross abnormalities apart from some infiltration with lymphocytes and occasional tubular atrophy. the changes during short perfusions of human kidneys prior to reimplantation have been described by hill who also performed biopsies 1 hour after reimplantation. on electron microscopy hill found endothelial damage which correlated with the severity of the fibrin deposition after reimplantation. the changes that hill saw in the glomeruli on light microscopy were occasional fibrin thrombi and infiltration with polymorphs. hill suspected that these changes were an immunologically induced lesion, but found that there was no correlation between the severity of the histological lesion and the presence or absence of immunoglobulin deposits. there are several reports of the analysis of urine produced by kidneys during perfusion storage. kastagir analysed urine produced during 24-hour perfusion and found it to be an ultrafiltrate of the perfusate, scott found a trace of protein in the urine during 24-hour storage, and pederson found only a trace of protein after 36 hours perfusion storage. pederson mentioned that he had found heavy proteinuria during earlier experiments. woods noted protein casts in the tubules of viable kidneys after 5 day storage, but he did not analyse the urine produced during perfusion. in cohen's study there was a progressive increase in urinary protein concentration during 8 day preservation until the protein content of the urine equalled that of the perfusate. this may have been related to the swelling of the glomerular basement membranes and the progressive fusion of epithelial cell foot processes that was also observed during the same period of perfusion storage. mechanisms of injury the mechanisms that damage kidneys during hypothermic storage can be sub-divided as follows: injury to the metabolic processes of the cell caused by: cold anoxia when the kidney is warm both before and after the period of hypothermic storage. failure to supply the correct nutrients. toxin accumulation in the perfusate. toxic damage from the storage fluid. washout of essential substrates from the kidney cells. injury to nuclear dna. mechanical injury to the vascular system of the kidney during hypothermic perfusion. post reimplantation injury. cold at normal temperatures pumping mechanisms in cell walls retain intracellular potassium at high levels and extrude sodium. if these pumps fail sodium is taken up by the cell and potassium lost. water follows the sodium passively and results in swelling of the cells. the importance of this control of cell swelling was demonstrated by mcloughlin who found a significant correlation between canine renal cortical water content and the ability of kidneys to support life after 36-hour storage. the pumping mechanism is driven by the enzyme system known as na+k+- activated atpase and is inhibited by cold. levy found that metabolic activity at 10 °c, as indicated by oxygen consumption measurements, was reduced to about 5% of normal and, because all enzyme systems are affected in a similar way by hypothermia, atpase activity is markedly reduced at 10 °c. there are, however, tissue and species differences in the cold sensitivity of this atpase which may account for the differences in the ability of tissues to withstand hypothermia. martin has shown that in dog kidney cortical cells some atpase activity is still present at 10 °c but not at 0 °c. in liver and heart cells activity was completely inhibited at 10 °c and this difference in the cold sensitivity of atpase correlated with the greater difficulty in controlling cell swelling during hypothermic storage of liver and heart cells. a distinct atpase is found in vessel walls, and this was shown by belzer to be completely inhibited at 10 °c, when at this temperature kidney cortical cells atpase is still active. these experiments were performed on aortic endothelium, but if the vascular endothelium of the kidney has the same properties, then vascular injury may be the limiting factor in prolonged kidney storage. willis has shown how hibernators derive some of their ability to survive low temperatures by having a na+k+-atpase which is able to transport sodium and potassium actively across their cell membranes, at 5 °c, about six times faster than in non-hibernators; this