An essential preliminary to the development of kidney storage and transplantation was the work of Alexis Carrel in developing methods for vascular anastomosis. Carrel went on to describe the first kidney transplants, which were performed in dogs in 1902; Ullman independently described similar experiments in the same year. In these experiments kidneys were transplanted without there being any attempt at storage.
The crucial step in making in vitro storage of kidneys possible, was the demonstration by Fuhrman in 1943, of a reversible effect of hypothermia on the metabolic processes of isolated tissues. Prior to this, kidneys had been stored at normal body temperatures using blood or diluted blood perfusates, but no successful reimplantations had been made. Fuhrman showed that slices of rat kidney cortex and brain withstood cooling to 0.2 °C for one hour at which temperature their oxygen consumption was minimal. When the slices were rewarmed to 37 °C their oxygen consumption recovered to normal.
The beneficial effect of hypothermia on ischaemic intact kidneys was demonstrated by Owens in 1955 when he showed that, if dogs were cooled to 23-26 °C, and their thoracic aortas were occluded for 2 hours, their kidneys showed no apparent damage when the dogs were rewarmed. This protective effect of hypothermia on renal ischaemic damage was confirmed by Bogardus who showed a protective effect from surface cooling of dog kidneys whose renal pedicles were clamped in situ for 2 hours. Moyer demonstrated the applicability of these dog experiments to the human, by showing the same effect on dog and human kidney function from the same periods of hypothermic ischaemia.
It was not until 1958 that it was shown that intact dog kidneys would survive ischaemia even better if they were cooled to lower temperatures. Stueber showed that kidneys would survive in situ clamping of the renal pedicle for 6 hours if the kidneys were cooled to 0-5 °C by being placed in a cooling jacket, and Schloerb showed that a similar technique with cooling of heparinised dog kidneys to 2-4 °C gave protection for 8 hours but not 12 hours. Schloerb also attempted in vitro storage and auto-transplantation of cooled kidneys, and had one long term survivor after 4 hours kidney storage followed by reimplantation and immediate contralateral nephrectomy. He also had a near survivor, after 24-hour kidney storage and delayed contralateral nephrectomy, in a dog that developed a late arterial thrombosis in the kidney.
These methods of surface cooling were improved by the introduction of techniques in which the kidney's vascular system was flushed out with cold fluid prior to storage. This had the effect of increasing the speed of cooling of the kidney and removed red cells from the vascular system. Kiser used this technique to achieve successful 7 hours in vitro storage of a dog kidney, when the kidney had been flushed at 5 °C with a mixture of dextran and diluted blood prior to storage. In 1960 Lapchinsky confirmed that similar storage periods were possible, when he reported eight dogs surviving after their kidneys had been stored at 2-4 °C for 28 hours, followed by auto-transplantation and delayed contralateral nephrectomy. Although Lapchinsky gave no details in his paper, Humphries reported that these experiments had involved cooling the kidneys for 1 hour with cold blood, and then storage at 2-4 °C, followed by rewarming of the kidneys over 1 hour with warm blood at the time of reimplantation. The contralateral nephrectomies were delayed for two months.
Humphries developed this storage technique by continuously perfusing the kidney throughout the period of storage. He used diluted plasma or serum as the perfusate and pointed out the necessity for low perfusate pressures to prevent kidney swelling, but admitted that the optimum values for such variables as perfusate temperature, Po, and flow, remained unknown. His best results, at this time, were 2 dogs that survived after having their kidneys stored for 24 hours at 4-10 °C followed by auto-transplantation and delayed contralateral nephrectomy a few weeks later.
Calne challenged the necessity of using continuous perfusion methods by demonstrating that successful 12-hour preservation could be achieved using much simpler techniques. Calne had one kidney supporting life even when the contralateral nephrectomy was performed at the same time as the reimplantation operation. Calne merely heparinised dog kidneys and then stored them in iced solution at 4 °C. Although 17-hour preservation was shown to be possible in one experiment when nephrectomy was delayed, no success was achieved with 24-hour storage.
The next advance was made by Humphries in 1964, when he modified the perfusate used in his original continuous perfusion system, and had a dog kidney able to support life after 24-hour storage, even when an immediate contralateral nephrectomy was performed at the same time as the reimplantation. In these experiments autogenous blood, diluted 50% with Tis-U-Sol solution at 10 °C, was used as the perfusate. The perfusate pressure was 40 mm Hg and perfusate pH 7.11-7.35 (at 37 °C). A membrane lung was used for oxygenation to avoid damaging the blood.
In attempting to improve on these results Manax investigated the effect of hyperbaric oxygen, and found that successful 48-hour storage of dog kidneys was possible at 2 °C without using continuous perfusion, when the kidneys were flushed with a dextran/Tis-U-Sol solution before storage at 7.9 atmospheres pressure, and if the contralateral nephrectomy was delayed till 2 to 4 weeks after reimplantation. Manax postulated that hyperbaric oxygen might work either by inhibiting metabolism or by aiding diffusion of oxygen into the kidney cells, but he reported no control experiments to determine whether other aspects of his model were more important than hyperbaria.
A marked improvement in storage times was achieved by Belzer in 1967 when he reported successful 72-hour kidney storage after returning to the use of continuous perfusion using a canine plasma based perfusate at 8-12 °C. Belzer found that the crucial factor in permitting uncomplicated 72-hour perfusion was cryoprecipitation of the plasma used in the perfusate to reduce the amount of unstable lipo-proteins which otherwise precipitated out of solution and progressively obstructed the kidney's vascular system. A membrane oxygenator was also used in the system in a further attempt to prevent denaturation of the lipo-proteins because only 35% of the lipo-proteins were removed by cryo-precipitation. The perfusate comprised 1 litre of canine plasma, 4 mEq of magnesium sulphate, 250 mL of dextrose, 80 units of insulin, 200,000 units of penicillin and 100 mg of hydrocortisone. Besides being cryo-precipitated, the perfusate was pre-filtered through a 0.22 micron filter immediately prior to use. Belzer used a perfusate pH of 7.4-7.5, a Po of 150–190 mm Hg, and a perfusate pressure of 50–80 mm Hg systolic, in a machine that produced a pulsatile perfusate flow. Using this system Belzer had 6 dogs surviving after their kidneys had been stored for 72 hours and then reimplanted, with immediate contralateral nephrectomies being performed at the reimplantation operations.
Belzers use of hydrocortisone as an adjuvant to preservation had been suggested by Lotkes work with dog kidney slices, in which hydrocortisone improved the ability of slices to excrete PAH and oxygen after 30 hour storage at 2-4 °C; Lotke suggested that hydrocortisone might be acting as a lysosomal membrane stabiliser in these experiments. The other components of Belzers model were arrived at empirically. The insulin and magnesium were used partially in an attempt to induce artificial hibernation, as Suomalainen found this regime to be effective in inducing hibernation in natural hibernators. The magnesium was also provided as a metabolic inhibitor following Kamiyamas demonstration that it was an effective agent in dog heart preservation. A further justification for the magnesium was that it was needed to replace calcium which had been bound by citrate in the plasma.
Belzer demonstrated the applicability of his dog experiments to human kidney storage when he reported his experiences in human renal transplantation using the same storage techniques as he had used for dog kidneys. He was able to store kidneys for up to 50 hours with only 8% of patients requiring post operative dialysis when the donor had been well prepared.
In 1968 Humphries reported 1 survivor out of 14 dogs following 5 day storage of their kidneys in a perfusion machine at 10 °C, using a diluted plasma medium containing extra fatty acids. However, delayed contralateral nephrectomy 4 weeks after reimplantation was necessary in these experiments to achieve success, and this indicated that the kidneys were severely injured during storage.
In 1969 Collins reported an improvement in the results that could be achieved with simple non perfusion methods of hypothermic kidney storage. He based his technique on the observation by Keller that the loss of electrolytes from a kidney during storage could be prevented by the use of a storage fluid containing cations in quantities approaching those normally present in cells. In Collins model, the dogs were well hydrated prior to nephrectomy, and were also given mannitol to induce a diuresis. Phenoxybenzamine, a vasodilator and lysozomal enzyme stabiliser, was injected into the renal artery before nephrectomy. The kidneys were immersed in saline immediately after removal, and perfused through the renal artery with 100-150 mL of a cold electrolyte solution from a height of 100 cm. The kidneys remained in iced saline for the rest of the storage period. The solution used for these successful cold perfusions imitated the electrolyte composition of intracellular fluids by containing large amounts of potassium and magnesium. The solution also contained glucose, heparin, procaine and phenoxybenzamine. The solutions pH was 7.0 at 25 °C. Collins was able to achieve successful 24-hour storage of 6 kidneys, and 30 hour storage of 3 kidneys, with the kidneys functioning immediately after reimplantation, despite immediate contralateral nephrectomies. Collins emphasised the poor results obtained with a Ringers solution flush, in finding similar results with this management when compared with kidneys treated by surface cooling alone. Liu reported that Collins solution could give successful 48-hour storage when the solution was modified by the inclusion of amino acids and vitamins. However, Liu performed no control experiments to show that these modifications were crucial.
Difficulty was found by other workers in repeating Belzers successful 72-hour perfusion storage experiments. Woods was able to achieve successful 48-hour storage of 3 out of 6 kidneys when he used the Belzer additives with cryoprecipitated plasma as the perfusate in a hypothermic perfusion system, but he was unable to extend the storage time to 72 hours as Belzer had done. However, Woods later achieved successful 3 and 7 days storage of dog kidneys. Woods had modified Belzers perfusate by the addition of 250 mg of methyl prednisolone, increased the magnesium sulphate content to 16.2 mEq and the insulin to 320 units. Six of 6 kidneys produced life sustaining function when they were reimplanted after 72 hours storage despite immediate contralateral nephrectomies; 1 of 2 kidneys produced life sustaining function after 96 hours storage, 1 of 2 after 120 hours storage, and 1 of 2 after 168 hours storage. Perfusate pressure was 60 mm Hg with a perfusate pump rate of 70 beats per minute, and perfusate pH was automatically maintained at 7.4 by a CO 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.
Johnsons 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 Po 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 Po 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 HO, pH 7.34-7.40 and Po 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. Rosss 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 Rosss 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.