Machine perfusion
The crucial step in making in vitro storage of kidneys possible, was the demonstration by Fuhrman in 1943,[17] of a reversible effect of hypothermia on the metabolic processes of isolated tissues.
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.
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.
In 1960 Lapchinsky[26] 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.
Calne[28] challenged the necessity of using continuous perfusion methods by demonstrating that successful 12-hour preservation could be achieved using much simpler techniques.
In 1968 Humphries[37] 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.
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 solution used for these successful cold perfusions imitated the electrolyte composition of intracellular fluids by containing large amounts of potassium and magnesium.
Woods had modified Belzer's 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.
[47] 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.
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.
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 flush solution was designed to imitate intracellular fluid composition and contained mannitol as an impermeable ion to further prevent cell swelling.
Ross's successful solution was similar in electrolyte composition to intracellular fluid with the addition of hypertonic citrate and mannitol.
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.
The structural changes that occur during 72-hour hypothermic storage of previously uninjured kidneys have been described by Mackay[61] who showed how there was progressive vacuolation of the cytoplasm of the cells which particularly affected the proximal tubules.
Woods[62] noted protein casts in the tubules of viable kidneys after 5 day storage, but he did not analyse the urine produced during perfusion.
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.
The importance of this control of cell swelling was demonstrated by McLoughlin[67] who found a significant correlation between canine renal cortical water content and the ability of kidneys to support life after 36-hour storage.
At hypothermia the metabolic needs of the kidney are much reduced but measurable consumption of glucose, fatty acids and ketone bodies occurs.
Pettersson[78] showed that, on a molar basis, glucose and fatty acids were metabolised by hypothermically perfused kidneys at about the same rates.
There was necrosis of capillary loops, occlusion of Bowman's spaces, basement membrane thickening and endothelial cell damage.
Lazarus[89] 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.
[90] Perfusion storage methods can mechanically injury the vascular endothelium of the kidney, which leads to arterial thrombosis or fibrin deposition after reimplantation.
Hill[63] noted that, in human kidneys, fibrin deposition in the glomerulus after reimplantation and postoperative function, correlated with the length of perfusion storage.
Biopsies taken one hour after revascularisation showed platelets and fibrin adherent to any areas of denuded vascular basement membrane.
Light[93] 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[94] 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.
After Cohen found vascular injury with intra renal bleeding after 3 days of perfusion storage,[59] 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.
