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Renal preservation by hypothermic perfusion
CRYOBIOLOGY
11, 238-247
( 1974)
Renal 2. The
Preservation
Influence
by Hypothermic
of Oxygenator
Design
D. E. PEGG, C. J. GREEN Division
of
Cryobiology
and Harrow,
Animal United
The balance of currently available evidence supports the view that the function of stored kidneys is more effectively preserved when continuous hypothermic perfusion is used in preference to initial vascular washout followed by refrigeration at a temperature just above 0°C (4, 12). However, as pointed out in the previous paper of this series (18), there is still doubt as to the best perfusion technique to use: strongly held opinions abound, but evidence to support them is scarce. For this reason we have decided to carry out a systematic study of the influence of some of the more obvious perfusion parameters in a carefully standardized experimental model. In the first series of experiments, perfusion under constant-pressure conditions was compared with the more common technique of constant-flow perfusion: the former technique gave significantly better preservation. In this paper the influence of oxygenation is studied. Several systems of oxygenation have been compared, and an efficient ‘film oxygenator has been used in renal preservation experiments in which rabbit kidneys were perfused for 24 hr at a constant arterial pressure of 40 mmHg using a defined perfusate and a temperature of 5°C. The perfusate was equilibrated with N.JOZ/COP gas mixtures containing either no oxygen, approximately Received November
8, 1973. 238
Copyright A’11 rights
0 1974 by Academic Press, Inc. of reproduction in any form reserved.
AND
Division, Kingdom
Perfusion
and Oxygen
Tension
J. FOREMAN Clinical
Research
Centre,
19% ‘oxygen, or 95% oxygen. Vascular resistance and organ weight were recorded continuously, changes in the composition of the perfusate were monitored, and function was assessedby autografting into an animal subjected to an immediate contralateral ncphrectomy. MATERIALS
AND METHODS
Perfusate. The solution previously designated PFIII was used (17); it contained an “cxtracellular” balance of ions, and colloid osmotic pressure was provided by dextran of mean mol wt 70,000 (45 g/liter: dextran 70, Pharmacia Ltd) and bovine serum albumin (15 g/liter, Armour Pharmaceutical Company Ltd). Glucose (4 m&f), papaverine (30 mg base/liter), and gentamicin (100 mg base/liter) were also included. The measured osmolality was 306 t 2 mOsm/Kg; the solution was filtered sequentially through 5-pm, 1.2-pm, and 0.2pm filters and stored at +4 or -20°C until required for use. Measurement of efficiency of oxygenators. A beaker containing 250 ml of perfusate was bubbled with air at 5°C until stable PO, readings were obtained. A Clark electrode (Beckman 0, Macroelectrode kit) enclosed in a Perspex cuvette was used to measure oxygen tension; a polarizing voltage of 600 mV was applied (22) and the output current read on a Vibron electrometer, Model 33B (Electronic In-
OXYGENATION
IN
RENAL
PERFUSATE
GAS
MIXTURE
PRESERVATION
“30
FLOW
FLOW
FIG. 1. A diagrammatic vertical section through the flat membrane oxygenator. The membrane was held between a supporting matrix, through which the gas flowed, and a Perspex sheet. The perfusate flowed as a thin film between the Perspex sheet and the membrane.
struments Ltd). After equilibration with air the beaker was covered with Parafihn and the solution cquilibratcd with a gas mixture containing 95% oxygen and 5% carbon dioxide using one of the following methods: 1. The gas mixture was bubbled through the perfusate at a flow rate of 1 liter/min through a tube of 2 mm diam. 2. The perfusate was pumped through a flat membrane oxygenator and returned to the reservoir. The construction of the oxygenator is shown in Fig. 1. The membrane was supported from the gas-phase side, and the perfusate passed between the membrane and a Perspex sheet. The flow of gas and liquid was countercurrent, the gas flow rate being 1 liter/mill and the perfusate flow rate 10 or 30 ml/min. The membrane mcasurcd 15 x 20 cm and was made from one of the following materials: Mastic sheet, 0.13-mm thick (Dow Corning) supported on a rigid expanded polyvinyl chloride mesh having 2.0, 3.5, or 7.0 apertures per cm (Expanded Metal Conpany, Ltd); silicone rubber sheet 0.02 mm thick (Sandev Ltd) supported on a soft plastic foam mat (J. Sainsbury Ltd); polyester-reinforced silicone rubber membrane (Esco Ltd) 0.03-0.05 mm thick, supported on a soft plastic foam mat. 3. The perfusate was pumped at 10 or 30 ml/min through a 165-cm length of either silastic tubing (2.0 mm i.d., 3.2 mm o.d.; Dow Corning) or silicone rubber tubing (1.0 mm i.d., 2.0 mm o.d.; Esco Ltd). The lengths of tubing were loosely coiled and enclosed in a glass cylinder through
which the gas mixture flowed at 1 liter/ min. Similar oxygenators have been used by Gimbronc et al. (6), and by Hobbs and Ellis ( 10). 4. The perfusate was pumped at 10 or 30 ml/min through a simple film oxygenator constructed from an inverted 250 ml round-bottomed flask, as illustrntcd in Fig. 2. The gas flow was 1 liter/min. A somcwhat similar oxygenator was described by Love ( 14 ) . In each case, the PO, of samples take!1 from the reservoir was mcasurecl at 5-mi11 intervals, and the rate of transfer of oxygcn to the perfusate was calculated as described below. Operative procedure. The animals were New Zealand albino rabbits of cyther sex weighing 2-3 kg. Anesthesia was induced with 0.1 mg fentanyl and 5 mg haloanisane by intramuscular injection, and maintained with nitrous oxide, oxygen, and halothane. The right kidney was cxciscd, perfused, and autografted 24 hr later using techniques previously described (8). All animals were hydrated with clcxtrose-saline (4.3%. dextrose with O.lS(>, sodium chloride) during the operation (50-100 ml intravenously) and 80 ml was given subcutancously for the first 4 postoperative days. Perfusion. As soon as the kidney had been excised it was perfused slowly with 20 ml of cold perfusate to which 200 units of heparin had been added. It was then transferred to the perfusion apparatus at 5°C and perfused at an arterial pressure of 60 mmHg for 5 min and then at 40 mmHg for 5 min before recirculation of the
240
PEGG,
GREEN
AND
FOREMAN
PERFU OUT-GAS MlXT IN Calculations. Vascular resistance was calculated according to the expression R = P/,F, where P = pressure (mmHg), 7 = viscosity (cp), F = flow (ml/min).
EXCESS PERFUSATE, AND GAS OUT
--PERFUSATE
IN
FIG. 2. The film oxygenator. Perfusate was pumped up the central tube, filmed over the internal surface of the flask, and then flowed into a small reservoir in the neck of the flask. Gas entered the globe of the flask, and left via the overflow tube which also served to return excess perfusate to the reservoir.
venous effluent was started. Perfusion was then continued at 40 mmHg for a total period of 24 hr, and the effluent perfusate was returned to the reservoir. The circuit is illustrated in Fig. 3. The oxygenator pump was a type-MHRE peristaltic pump by Watson Marlow Ltd., and the perfusion pump was a Holter type RL 175, modified as previously described (18) to enable it to be controlled automatically to maintain a constant perfusion pressure. A Gelman 142-mm filter holder, housing a glass fiber prefilter mat ( Gelman type A, 127 mm ) and a 0.22 pm membrane filter (Millipore type GSWP) was used for continuous filtration. Polyamide (nylon) tubing was used throughout the circuit to minimize oxygen loss in the high-tension experiments, and oxygen gain in the low-tension studies ( 16). The gas mixtures all contained 5% carbon dioxide, with either 9570 oxygen (Group I), 95% air ( Group II), or 95% nitrogen (Group III). The measured pH of the perfusate was always 7.30-7.40 at 5°C and the total perfusate volume was 300 ml. Organ weight, perfusion pressure, and perfusate Ilow were monitored continuously as previously described ( 17) using a multichannel chart recorder (Rikadenki, type PHR 12).
Expressed in these terms, the normal resistance of a single rabbit kidney is approximately 0.5. Resistance change during perfusion was expressed as the ratio of the final resistance to the initial value after stabilization. Weight gain during perfusion was expressed as a percentage of the initial weight of the freely drained, unperfused kidney. In calculating the efficiency of the various oxygenators tested it was assumed that the oxygen tension of the perfusate in the reservoir increased linearly with time over the period required to reach half the maximum value in the reservoir; from the slope of the best straight line, and an assumed oxygen solubility of 0.043 ml Ol/ml at 1 atmosphere at 5°C (25), an oxygen-transfer rate was calculated in ml/min. Chemical estimations. Perfusate samples were taken before the kidney was placed on the perfusion circuit and after 2, 4, 18, and 24 hr. Each sample was analyzed for
FIG. 3. The perfusion circuit. Fluid from the reservoir was pumped through the oxygenator as illustrated in Fig. 2. Oxygenated perfusate was then pumped through a 0.22-pm filter, and a bubble trap, to the arterial cannula. A pressure transducer was attached to the cannula which was tied into the renal artery, and the kidney was suspended from a weight ‘transducer. The venous effluent was allowed to return to the reservoir.
OXYGENATION
IN
RENAL
total osmolality by the freezing point depression method ( Fiske osmometer ), for sodium and potassium by emission flame photometry (EEL flame photometer), for glucose by the glucose oxidase method, and for lactate, alanine aminotransfcrase, and aspartate aminotransferase by the standard enzymatic techniques using the Boehringer kit methods. Assessment of function. Blood urea and serum creatinine levels were measured at 2- to 3-day intervals using urease and alkaline picrate methods, respectively. For each animal that survived for I4 days, an index of renal function was obtained by integrating the blood urea value with respect to time between Day zero and Day 14. Normal values (mean f SD) for rabbits unoperated upon were Blood urea 44 f 12 mg/lOO ml Serum crcstininc 2.1 +Z 0.2 mg/lOO 1111 14
.i 0
BU rlt 0.65 + 0.35 x 10” mgjlO0
ml X days
Surviving animals were killed at 2-3 mo. The kidneys of all animals were subjected to histological examination after fixation in formol-saline and staining by the hematoxylin/eosin and periodic acid-Schiff techniques. RESULTS
The oxygen transfer rates ‘obtained with the different oxygenators arc shown in Table 1: no significant differences were observed between the two perfusate flow rates tested. It was found that the efficiency of the 0.13-mm membrane was very dependent on the membrane support used, a 12-fold difference being observed between the most efficient grid (the coarsest) and the least efficient. The thinner membranes were too fragile to be used with any support other than the soft, but relatively dense, plastic foam mat, and with this support they were not notably efficient. The much simpler tubing oxygenators were also less efficient than the best flat-membrane
“41
PRESERVATION
Derke
Oxygen tranvfer rate (ml/min)
Membrane oxygenators 1 0.13 mm Silastic, coarse-grid 2 0.13 mm Silastic, medium grid 3 0.13 mm Silastic, fine grid 4 0.02 mm silicone ruhher, foam suppor1 5 0.04 mm reinforced silicone rubber, foam support 6 165 cm Silastic tube 7 16.5 cm Esco silicone rubber t,uhe Film oxygenator Bubble oxygenator
0.36 0.17 0.03 ().I;, 0.11 0.07 0.09 0.21 0.38
system; they could have been improved by using longer tubes, but the high resistance of this fine tubing presents difficulties at flows of lo-20 ml/ min. The bubble oxygenator was the most efficient of all, but the film oxygenator was reasonably etficient, was simple, and avoided the problem of foaming; it was, therefore, used in the subsequent renal perfusion experiments. Ten kidneys were perfused and transplanted in each of the three experimental groups. Table 2 shows that the groups were comparable with respect to the delay before starting continuous perfusion, the duration of perfusion, and the time taken to complete the anastomoses. The preperfusion interval of 11-12 min included less than 3 miu warm time, approximately 4
Preperfusion time (min)
GroupI (95% Oz)
11.9
f
(95C7” air)
11.4
Grmp III (95’70 N*)
I1.Y
Perfusion time W)
0.8
Postperfusion time (min)
24.03
i
0.02
23.i
*
1.3
zko.5
24.05
f
0.09
22.9
*
1.4
zk 0.6
24.03
f
0.02
28.4
*
1.4
Oroup II
242
PEGG,
GREEN
AND
min to flush the kidney with cold heparinized perfusate, and then about 5 min to transport the cooled kidney to the perfusion apparatus, attach it to the equipment and record its initial weight. The behavior ‘of the three experimental groups during perfusion is shown in Table 3. The only significant difference is in the measured oxygen tensions which were close to the expected values, although it was not possible to obtain zero values for the oxygen tension in Group III, even though nylon tubing was used throughout. The pH, measured at 5”C, was similar in all the experiments. Vascular resistance exhibited fluctuations, as previously described (18) with a general tendency to increase slightly during perfusion. One kidney in Group I exhibited an unusually large increase in resistance ( x 13.9), but this kidney functioned well after transplantation; if this experiment is excluded, the mean _t 1 SE for Group I becomes 1.74 r+ 0.30. No matter which value is taken, however, there is no significant difference between the increase in resistance that occurred in the three groups. Nor is there any difference between the weight gained by kidneys in the three groups, and of the various compounds that were estimated in the perfusate (see Methods) only the lactate concentration showed any significant distinction between groups, being higher in Group III (0.7 4 0.1 mhl) than in the oxygenated kidneys (0.4 k 0.1 mM) (P < 0.05).
The function of the perfused kidneys was assessed by survival, the maximum blood urea and serum creatinine concentrations recorded, and by the integral of blood urea concentration with respect to time in those animals that survived for 14 days or longer (Table 4). More animals survived in the groups in which the perfusate was oxygenated (see Fig. 4) but the difference is not significant (x’ = .9524, 0.5 > P > 0.25). Moreover, the renal function of survivors in the nitrogen group, as measured by the urea integral, was better than that in the oxygenated groups, although the difference was not significant at the 5% level (P < 0.3 and < 0.1 when compared with Groups I and II, respectively ). There were no significant differences between the maximum blood urea and serum creatinine levels in the experimental groups. Moreover, when the 10 kidneys giving best function were selected it was found that four were from Group I, two from Group II, and four from Group III, which reveals no benefit from oxygenation. Histological examination of the kidneys also failed to reveal significant differences between the experimental groups. There were two acute tubular necroses in each for six of the acute group, accounting deaths shown in Table 4 and Fig. 4; the two additional early deaths in Group III were caused by total renal infarction (1) and septicemia ( 1). Each experimental group also included two kidneys that
PO2
P”
Group
FOREMAN
Weight win (7%)
(mrn”g)
R&;sgc
I
(96%
02)
7.35 f
0.01
676 rt 4
4X
*
.i
x3.0
(X1.7
‘1 Value
Group II (95% nir)
7.37 *
0.02
137 f
Group III (03% Nz)
7.35 f
0.01
Of1
obtained
when
one very
high
result
is excluded
5
40 *
f
1.2
*0.3p
4
x1.7
rt 0.2
42 zk 3
x2.1
f
: see the text.
0.4
showed fairly severe chronic renal damage in the form ‘of significant arcas of fibrosis with calcification and dilated tubules; one of these animals (in Group I ) died at 18 days, but the other five survived with adcquate renal function until sacrificed at 2 mo or later. One other late death occurred, from septicemia, at 4; wk in an animal in Group II. All the other animals survived until sacrificed at 2-3 mo, and their kidneys were either completely normal histologically, or showed minimal changes, typically a few small infarcts or mild tubular dilatation. In previous experiments ( 18 ) using 95% oxygen and a membrane oxygenator, the survival rate was S/10, the mean urea integral was 1.60 * 0.12 x 10” mg/lOO ml x
FIG.
4. Graph
showing
the postoperative
survival
!).2 f
2-l
9.0 *
l..-l
9.2 f
1.8
days, and the highest serum creatininc level 7.12 0.6 mg/lOO ml. These results do not differ significantly from those obtained in Group I of the present series of experiments, or from the pooled results of all three groups. Comparisons with previous results can be influenced by unrecognized factors and must, therefore, bc examined particularly critically: in the prescnt case, the two sets of experiments were done sequentially without interruptimon by the same workers, using the same cquipment and animals from the same breeding colony. In these circumstances the comparison is believed to be valid. As in the experiments previously dcscribed (18), an attempt was made to discover whcthcr any of the measurements
of animals
in the three
experimental
groups.
244
PEGG,
GREEN
made during perfusion might be able to predict the posttransplantation function of individual kidneys. As before, the results were combined and regrouped into those in which the animal died in the first 14 days and those in which the animal survived longer than 2 wk. Comparisons were made of absolute resistance, resistance change, and weight gain of kidneys during perfusion, and the measurements of the biochemical parameters in the perfusate as listed in the Methods section. Only the glucose level showed a significant difference, 4.44 * 0.06 mM in the survivors, and 4.67 5 0.09 mM in those dying, giving a value for P = 0.05. The mean potassium concentration in the survivors was lower than in the animals that died, but not significantly so. DISCUSSION
These experiments produced some unexpected results, which must lead to a reexamination ‘of several aspects of current practice and dogma in organ preservation: our failure to find any benefit from membrane oxygenation, or from oxygenation at all, leads us to question the expense and complexity of the perfusion techniques and equipment in current use. Moreover, the c’emonstration that a continuous supply of oxygen has no effect on the subsequent function of transplanted kidneys removes one of the central arguments f,or perfusion; but it remains true that the best results obtained to date have been obtained by continuous perfusion techniques (3) and we do not advocate that these methods be abandoned at the present time. The majority of workers using continuous perfusion have employed membrane oxygenators, through which they have passed 95% oxygen (12), 33% oxygen ( 23), ‘or air (2). The oxygen tensions produced have not always been reported, and when they have, they have sometimes been recorded at a higher temperature than the preservation temperature, making interpre-
AND
FOREMAN
tation very difficult: the experiments reported in this paper show how variable the gas-exchange properties of oxygenators can be, especially membrane oxygenators, so that the oxygen tensions actually achieved remain in some doubt. Recent years have seen significant advances in the design of membrane oxygenators, and these are admirably reviewed by Drinker (5). It is certainly possible to construct efficient units, even with relatively thick membranes of silicone rubber, providing satisfactory supporting matrices are used, so that adequate flow of gas in close contact with the membrane is achieved. The justification ‘of membrane oxygenation rests on two premises: (1) that gas/ liquid interfaces must be avoided and (2) that a continuous supply of ‘oxygen is beneficial. There is some evidence to support the first premise; it is known that the exposure of blood to gas/liquid interfaces causes protein denaturation (26) and platelets are also damaged in these circumstances (24); such effects have been implicated in the pathogenesis of organ dysfunction after prolonged use of a disc oxygenator in heart-lung bypass ( 1). Similar effects can also ‘occur in isolated organ perfusion, even when the fresh perfusate contains neither blood nor plasma: it was found in this laboratory that when rabbit kidneys were perfused using a bubble oxygenator and a bloodless (dextran) perfusate which was recirculated, that the vascular resistance increased ( 15). The same effect could be produced in the absence of recirculation by the addition of small quantities of autologous blood and it was deduced that sufficient blood can remain in a kidney to cause vascular damage when it is gradually released during the course of a perfusion. This effect could be abolished either by using a membrane oxygenator or by including a filter of pore size < 1 pm (15). These results support the suggestion that vascular damage is primarily due to denatured protein and platelets, and they
OXYGENATION
IN
RENAL
also explain why a filter of sufficiently fine pore size can prevent such damage. In the present experiments, as in most perfusion systems, a fine filter was inch&d. There is also direct evidence for the adequacy of film oxygenation in hypothermic renal perfusion; Scott et al. (21), and Johnson et al. (12) have for many years successfully used 3 film ‘oxygenator in renal preservation, and more recently, Clnes and Rlohme (3) and Proctor and Jones (19) have found gas/ liquid surface oxygenation satisfactory in renal preservation. It seems clear, thcrefore, that membrane oxygenation is not cssential for successful preservation by hypothermic perfusion. What of the second premise: is oxygenation necessary at all? One of the reasons given by Humphries when he introduced hypothermic perfusion for renal prescrvation was that the continuous flow of perfusate would provide the organ with its admittedly minimal requirement for oxygen (11). Relzcr has also assumed that oxpgenation is essential (2). The oxygen consumption of dog kidneys is reported to bc approximately 5% of normal at 5°C (13) which would be only about .06 ml/min for a 15-g kidney. It has been reported that there is an arterovenous oxygen differcncc in perfusate flowing through kidneys at 8°C (Claes, G.; Personal communication), but it is difficult to make accurate measurements of oxygen consumption by perfused kidneys; if the PO2 of fluid flowing through the cortical vessels exceeds the partial pressure of oxygen in the surrounding medium, then oxygen will be lost by diffilsion, and an A-V difference created. NeverthcIcss, some oxygen probably is consumed by respiration even at S”C, and the fact that more lactate was produced in Group III of the present experiments supports this conclusion. It does not, however, follow that oxygen must be provided for masimum viability. We have already shown that excellent preservation can bc obtained with rabbit kidneys by Collins’ technique
PRESERVATIOS
Lz-45
for 24 and even for 48 hr (9), and Sacks et (~1. have preserved dog kidneys for 72 hr with a similar solution (20): in neither technique is a continuous Supply of oxygen provided. The present results, obtained in a ~\iell-standardized cxpcrimental system show that oxygen tension has no effect on the preservation of rabbit kidneys by hypothcrmic perfusion: a moderate oxygen tension is without benefit, and a high oxygen tension is not harmful, as some have suggested might be the case (11). These findings must be confirmed by others, and in different animals, but on our evidence it does stem that the basic premise that a continuous supply ,of ox):gcii is bcncficial is at fauIt. If oxygen is unnecessary, what other function might perfusion be performing? Does perfusion have any value at all? The available evidence still suggests that it does; the longest prescrvntion periods so far reported in the most commonl\- used cxpcrimcntal animal, the dog, have been with perfusion techniques, and Sacks’ results, especially with ischcmic injury are still significantly worse than Claes’ rrsults (3, 20). This sort of evidence is not uuequivocal, however, and direct comparisons of both techniques by the same group are urgently needed; when this was done for PF III perfusate, perfusion was superior to washout (9). SIlould it bc confirmed that perfusion does indeed provide the best preservation, then it would seem that perfusion equipment can now be greatly simplificd. In the absence of any need for oxygenation, the only reason for retaining a gas exchanger wouId be to regulate the pH of a CO-/bicarbonate buffer system. However, if the latter were to be replaced by one of Good’s buffers (7) such as HEPES (N-2-hydroxyethylpipernzine-N’2ethanesulfonnte), the gas phase could be dispcnscd with, the oxygenator and its heavy gas bottles elminated, and the equipmcnt made lighter, nlore compact, and less expensive. The inclusion of a submicron
246
PEGG,
GREEN
filter would seem to be mandatory in such circumstances, but such filters are inexpensive, light, and compact. A disappointing feature of the present series of experiments was the failure of any of the monitored features adequately to predict subsequent function. In the preceding study (18), the concentration of potassium in the perfusate was found to correlate with subsequent function: in the present study the mean potassium levels were somewhat higher in the survivors and slightly lower in the nonsurvivors than previously, and no separation could be achieved on this basis. As previously, the perfusate glucose concentration was found to correlate inversely with function, and it seems likely that the additional glucose is derived from the breakdown of dextran by lysosomal I-glucosidasc released from damaged renal cells. However, the separation was less clear-cut than previously because the glucose levels in the nonsurvivors were considerably lower than in the earlier experiments. It seems clear that parameters other than those included in the present study will be needed for a useful predictive test. SUMMARY
Rabbit kidneys have been perfused for 24 hr at 5°C and tested by autotransplantation with immediate contralateral nephrectomy. The perfusate contained an extracellular balance of ions with dextran and bovine serum albumin. The circuit included a nonpulsatile pump, a 0.22-pm membrane filter, and an oxygenator. The efficiency of membrane, film, and bubble oxygenators was compared. Flat membrane oxygenators were found to be more efficient than tubing oxygenators, and although the bubble oxygenator was the most efficient it created problems with foaming. A simple film oxygenator provided the best compromise. Ten kidneys were perfused and transplanted in each of three experimental
AND
FOREMAN
groups; a film oxygenator was used to provide partial pressures of oxygen of 676 mmHg, 137 mmHg, and 9 mmHg, respectively. There were no significant differences in behavior during perfusion, function after transplantati’on, or histology of transplanted kidneys in the three groups. The similarity of the results using a film oxygenator with those previously obtained with a membrane oxygenator indicates that complex membrane oxygenators arc not necessary for preservation in our system. Moreover, the similarity of the results with oxygen tensions from 9 mmHg to 676 mmHg shows that deliberate oxygenation is also unnecessary. REFERENCES 1. Allerdyce, I). B., Yoshida, S. H., and Ashmore, P. G. The importance of microembolism in the pathogcnesis of organ dysfunction caused by prolonged USC of the pump oxygenator. J. Thoruc. Cardiovasc. Surg. 52, 706-715 ( 1966). 2. Belzer, F. O., Ashby, B. S., and Dun&y, J. E. 24-hour and 7%hour preservation of canine kidneys. Lancet 2, 536-539 ( 1967). 3. Claes, G., and Blohme, I. Experimental and clinical results of continuous albumin perfusion of kidneys. In “Organ Preservation” (D. E. Pcgg, Ed.), pp. 51-57. ChurchillLivingstone, Edinburgh, 1973. 4. Claes, G., Blohmc, I., and Gelin, L-E. Clinical kidney preservation with and without continuous perfusion. Proc. Eur. Did. Transpkunt Ass. 8, 307311 (1971). 5. Drinker, P. A. Progress in membrane oxygcnator design. Anaesthesiology 37, 242-260 (1972). 6. Gimbrone, M. A., Aster, R. II., Cotran, R. S., Corkery, J., Jandl, J. H., and Folkman, J. Preservation of vascular integrity in organs perfused in vitro with platelet-rich medium. Nature (London) 222, 33-36 (1969). 7. Good, N. E., Winget, G. D., Winter, W., Connolly, T. N., Izawa, S., and Singh, R. M. M. Hydrogen ion buffers for biological research. Biochemistry 5, 467477 ( 1966 ). 8. Green, C. J. Rabbit renal autografts as an organ preservation model. Lab. Anim. 7, l-11 (1973).
OXYGENATION
IN
RENAL
9. Green, C. J., and Pegg, D. E. A comparison of non-perfusion and perfusion methods for preservation of rabbit kidneys. In “Organ Preservation” (D. E. Pcgg, Ed.), pp. 1629. Churchill-Livingtonc, Edinburgh, 1973. 10. llolhs, K. E. IT., and Ellis, hf. The present status of subzero organ prcscrvation with special reference to the rat hcalt. In “Organ Prcscrvation” (1~. E. Pegg, Ed.), pp. 123-136. Churchill-Livingstonc, Edinburgh, 1973. 11. Humphries, A. L., Russell, R., Ostafin, J. Goodrich, S. ht., and Mnretz, W. H. Successful reimplantation of canine kidney after 24 hr storage. SurgertJ 54, 136-143 (1963). 12. Johnson, R. W. G., Anderson, M., Morley, A. R., Taylor, R. M. R., and Swinney, J. Twenty-four hour preservation of kidneys injured by prolonged warm ischacmia. 7’mns~Jlantation 13, 174-179 ( 1972). 13. Levy, N. M. Oxygen consumption and blood flow in the hypothermic perfused kidney. Amcr. J. Physiol. 197, 1111-1114 (1959). 14. Love, J. W. A low-priming volume hcarthmg machine for use in thr laboratory. 1. 7’11orac. Cnrtliocosc. Slug. 45, 62%627 (1963). 15. Pegg, 1). E. Vascular resistance of the isnlated rabbit kidney. Crryohiology 8, 431440 (1971). 10. I’cgg, 1). E., Fuller, B. J., Foreman, J., nntl Green, C. J. The choice of plastic tubing for organ perfusion espcrimcnts. CryolTiology 9, 569-571 ( 1972). 17. Pcgg, D. E., and Green, C. J. Renal prescrvation by hypothermic perfusion using a defined perfusion fluid. Cr!yohiolog!y 9, 420428 ( 1972).
I’RESER\‘A’I’ION
247
18. Prgg, I). E., ant1 Green, C. J. Renal prcscrvation by hypothermic perfusion 1. The importance of presslirc~-control. Cr!yobiology 10, S&66 (1973). 19. Proctor, E., and Jones, G. R. N. In vitro asscssnicnt of preserved dog hearts, with xomc rcfcrenct: to preserved dog kidneys. In “Organ l’reservation” (1). E. I’egg, Ed.), pp. 216-224. Chilrchill-Livingstonc, Edinburgh, 1973. 20. Sacks, S. A., Petritsch, P. II., and Kaufman, J. J. Canine kidney preservation using a new pcrfusate. Ltrncct 2, 1024-1028 ( 1973). 21. Scott, D. F., Morley, A. R., and Swinncy, J. Canine renal preservation following hypothcrmic perfusion and subsequent function. Brit. J. Surg. 56, 688-691 (1969). 22. Severinghaus, J. W. Electrodes for blood and gas PCO, Paz and blood pH. Acta Anaesthcsiol. Scard. SuppI. 11, 207-220 ( 1962). 23. Stephenson, T. P., O’Donoghuc, E. P. N., Hendry, W. F., and Wickham, J. E. A. Prcscrvation of human kidneys for transplantation: Preliminary results with a Gambro perfusion machine. Brit. Afcd. J. 1, 379381 (1973). 24. Swank, R. L., Hirsch, H., Brewer, hl., and Hisser], W. Effect of glass wool filtration on blood during circulation. extracorporeal Surg. Gyrwcol. Obstct. 117, 547-552 (1963). 25. Umbreit, W. W., Burris, R. H., and Stauffer, J. F. “Manomctric Tcchniqllrs,” p. .5. Burgess, Minneapolis, 1964. 26. Wright, E. S., Sarkozy, E., Harpur, E. R., Dobell, A. R. C., and Murphy, D. R. Plasma protein clmaturation in extracorporeal circulation. J. Thornc. Cardiot~n.rc. hi-g. 44, 550556 (1962).
11, 238-247
( 1974)
Renal 2. The
Preservation
Influence
by Hypothermic
of Oxygenator
Design
D. E. PEGG, C. J. GREEN Division
of
Cryobiology
and Harrow,
Animal United
The balance of currently available evidence supports the view that the function of stored kidneys is more effectively preserved when continuous hypothermic perfusion is used in preference to initial vascular washout followed by refrigeration at a temperature just above 0°C (4, 12). However, as pointed out in the previous paper of this series (18), there is still doubt as to the best perfusion technique to use: strongly held opinions abound, but evidence to support them is scarce. For this reason we have decided to carry out a systematic study of the influence of some of the more obvious perfusion parameters in a carefully standardized experimental model. In the first series of experiments, perfusion under constant-pressure conditions was compared with the more common technique of constant-flow perfusion: the former technique gave significantly better preservation. In this paper the influence of oxygenation is studied. Several systems of oxygenation have been compared, and an efficient ‘film oxygenator has been used in renal preservation experiments in which rabbit kidneys were perfused for 24 hr at a constant arterial pressure of 40 mmHg using a defined perfusate and a temperature of 5°C. The perfusate was equilibrated with N.JOZ/COP gas mixtures containing either no oxygen, approximately Received November
8, 1973. 238
Copyright A’11 rights
0 1974 by Academic Press, Inc. of reproduction in any form reserved.
AND
Division, Kingdom
Perfusion
and Oxygen
Tension
J. FOREMAN Clinical
Research
Centre,
19% ‘oxygen, or 95% oxygen. Vascular resistance and organ weight were recorded continuously, changes in the composition of the perfusate were monitored, and function was assessedby autografting into an animal subjected to an immediate contralateral ncphrectomy. MATERIALS
AND METHODS
Perfusate. The solution previously designated PFIII was used (17); it contained an “cxtracellular” balance of ions, and colloid osmotic pressure was provided by dextran of mean mol wt 70,000 (45 g/liter: dextran 70, Pharmacia Ltd) and bovine serum albumin (15 g/liter, Armour Pharmaceutical Company Ltd). Glucose (4 m&f), papaverine (30 mg base/liter), and gentamicin (100 mg base/liter) were also included. The measured osmolality was 306 t 2 mOsm/Kg; the solution was filtered sequentially through 5-pm, 1.2-pm, and 0.2pm filters and stored at +4 or -20°C until required for use. Measurement of efficiency of oxygenators. A beaker containing 250 ml of perfusate was bubbled with air at 5°C until stable PO, readings were obtained. A Clark electrode (Beckman 0, Macroelectrode kit) enclosed in a Perspex cuvette was used to measure oxygen tension; a polarizing voltage of 600 mV was applied (22) and the output current read on a Vibron electrometer, Model 33B (Electronic In-
OXYGENATION
IN
RENAL
PERFUSATE
GAS
MIXTURE
PRESERVATION
“30
FLOW
FLOW
FIG. 1. A diagrammatic vertical section through the flat membrane oxygenator. The membrane was held between a supporting matrix, through which the gas flowed, and a Perspex sheet. The perfusate flowed as a thin film between the Perspex sheet and the membrane.
struments Ltd). After equilibration with air the beaker was covered with Parafihn and the solution cquilibratcd with a gas mixture containing 95% oxygen and 5% carbon dioxide using one of the following methods: 1. The gas mixture was bubbled through the perfusate at a flow rate of 1 liter/min through a tube of 2 mm diam. 2. The perfusate was pumped through a flat membrane oxygenator and returned to the reservoir. The construction of the oxygenator is shown in Fig. 1. The membrane was supported from the gas-phase side, and the perfusate passed between the membrane and a Perspex sheet. The flow of gas and liquid was countercurrent, the gas flow rate being 1 liter/mill and the perfusate flow rate 10 or 30 ml/min. The membrane mcasurcd 15 x 20 cm and was made from one of the following materials: Mastic sheet, 0.13-mm thick (Dow Corning) supported on a rigid expanded polyvinyl chloride mesh having 2.0, 3.5, or 7.0 apertures per cm (Expanded Metal Conpany, Ltd); silicone rubber sheet 0.02 mm thick (Sandev Ltd) supported on a soft plastic foam mat (J. Sainsbury Ltd); polyester-reinforced silicone rubber membrane (Esco Ltd) 0.03-0.05 mm thick, supported on a soft plastic foam mat. 3. The perfusate was pumped at 10 or 30 ml/min through a 165-cm length of either silastic tubing (2.0 mm i.d., 3.2 mm o.d.; Dow Corning) or silicone rubber tubing (1.0 mm i.d., 2.0 mm o.d.; Esco Ltd). The lengths of tubing were loosely coiled and enclosed in a glass cylinder through
which the gas mixture flowed at 1 liter/ min. Similar oxygenators have been used by Gimbronc et al. (6), and by Hobbs and Ellis ( 10). 4. The perfusate was pumped at 10 or 30 ml/min through a simple film oxygenator constructed from an inverted 250 ml round-bottomed flask, as illustrntcd in Fig. 2. The gas flow was 1 liter/min. A somcwhat similar oxygenator was described by Love ( 14 ) . In each case, the PO, of samples take!1 from the reservoir was mcasurecl at 5-mi11 intervals, and the rate of transfer of oxygcn to the perfusate was calculated as described below. Operative procedure. The animals were New Zealand albino rabbits of cyther sex weighing 2-3 kg. Anesthesia was induced with 0.1 mg fentanyl and 5 mg haloanisane by intramuscular injection, and maintained with nitrous oxide, oxygen, and halothane. The right kidney was cxciscd, perfused, and autografted 24 hr later using techniques previously described (8). All animals were hydrated with clcxtrose-saline (4.3%. dextrose with O.lS(>, sodium chloride) during the operation (50-100 ml intravenously) and 80 ml was given subcutancously for the first 4 postoperative days. Perfusion. As soon as the kidney had been excised it was perfused slowly with 20 ml of cold perfusate to which 200 units of heparin had been added. It was then transferred to the perfusion apparatus at 5°C and perfused at an arterial pressure of 60 mmHg for 5 min and then at 40 mmHg for 5 min before recirculation of the
240
PEGG,
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PERFU OUT-GAS MlXT IN Calculations. Vascular resistance was calculated according to the expression R = P/,F, where P = pressure (mmHg), 7 = viscosity (cp), F = flow (ml/min).
EXCESS PERFUSATE, AND GAS OUT
--PERFUSATE
IN
FIG. 2. The film oxygenator. Perfusate was pumped up the central tube, filmed over the internal surface of the flask, and then flowed into a small reservoir in the neck of the flask. Gas entered the globe of the flask, and left via the overflow tube which also served to return excess perfusate to the reservoir.
venous effluent was started. Perfusion was then continued at 40 mmHg for a total period of 24 hr, and the effluent perfusate was returned to the reservoir. The circuit is illustrated in Fig. 3. The oxygenator pump was a type-MHRE peristaltic pump by Watson Marlow Ltd., and the perfusion pump was a Holter type RL 175, modified as previously described (18) to enable it to be controlled automatically to maintain a constant perfusion pressure. A Gelman 142-mm filter holder, housing a glass fiber prefilter mat ( Gelman type A, 127 mm ) and a 0.22 pm membrane filter (Millipore type GSWP) was used for continuous filtration. Polyamide (nylon) tubing was used throughout the circuit to minimize oxygen loss in the high-tension experiments, and oxygen gain in the low-tension studies ( 16). The gas mixtures all contained 5% carbon dioxide, with either 9570 oxygen (Group I), 95% air ( Group II), or 95% nitrogen (Group III). The measured pH of the perfusate was always 7.30-7.40 at 5°C and the total perfusate volume was 300 ml. Organ weight, perfusion pressure, and perfusate Ilow were monitored continuously as previously described ( 17) using a multichannel chart recorder (Rikadenki, type PHR 12).
Expressed in these terms, the normal resistance of a single rabbit kidney is approximately 0.5. Resistance change during perfusion was expressed as the ratio of the final resistance to the initial value after stabilization. Weight gain during perfusion was expressed as a percentage of the initial weight of the freely drained, unperfused kidney. In calculating the efficiency of the various oxygenators tested it was assumed that the oxygen tension of the perfusate in the reservoir increased linearly with time over the period required to reach half the maximum value in the reservoir; from the slope of the best straight line, and an assumed oxygen solubility of 0.043 ml Ol/ml at 1 atmosphere at 5°C (25), an oxygen-transfer rate was calculated in ml/min. Chemical estimations. Perfusate samples were taken before the kidney was placed on the perfusion circuit and after 2, 4, 18, and 24 hr. Each sample was analyzed for
FIG. 3. The perfusion circuit. Fluid from the reservoir was pumped through the oxygenator as illustrated in Fig. 2. Oxygenated perfusate was then pumped through a 0.22-pm filter, and a bubble trap, to the arterial cannula. A pressure transducer was attached to the cannula which was tied into the renal artery, and the kidney was suspended from a weight ‘transducer. The venous effluent was allowed to return to the reservoir.
OXYGENATION
IN
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total osmolality by the freezing point depression method ( Fiske osmometer ), for sodium and potassium by emission flame photometry (EEL flame photometer), for glucose by the glucose oxidase method, and for lactate, alanine aminotransfcrase, and aspartate aminotransferase by the standard enzymatic techniques using the Boehringer kit methods. Assessment of function. Blood urea and serum creatinine levels were measured at 2- to 3-day intervals using urease and alkaline picrate methods, respectively. For each animal that survived for I4 days, an index of renal function was obtained by integrating the blood urea value with respect to time between Day zero and Day 14. Normal values (mean f SD) for rabbits unoperated upon were Blood urea 44 f 12 mg/lOO ml Serum crcstininc 2.1 +Z 0.2 mg/lOO 1111 14
.i 0
BU rlt 0.65 + 0.35 x 10” mgjlO0
ml X days
Surviving animals were killed at 2-3 mo. The kidneys of all animals were subjected to histological examination after fixation in formol-saline and staining by the hematoxylin/eosin and periodic acid-Schiff techniques. RESULTS
The oxygen transfer rates ‘obtained with the different oxygenators arc shown in Table 1: no significant differences were observed between the two perfusate flow rates tested. It was found that the efficiency of the 0.13-mm membrane was very dependent on the membrane support used, a 12-fold difference being observed between the most efficient grid (the coarsest) and the least efficient. The thinner membranes were too fragile to be used with any support other than the soft, but relatively dense, plastic foam mat, and with this support they were not notably efficient. The much simpler tubing oxygenators were also less efficient than the best flat-membrane
“41
PRESERVATION
Derke
Oxygen tranvfer rate (ml/min)
Membrane oxygenators 1 0.13 mm Silastic, coarse-grid 2 0.13 mm Silastic, medium grid 3 0.13 mm Silastic, fine grid 4 0.02 mm silicone ruhher, foam suppor1 5 0.04 mm reinforced silicone rubber, foam support 6 165 cm Silastic tube 7 16.5 cm Esco silicone rubber t,uhe Film oxygenator Bubble oxygenator
0.36 0.17 0.03 ().I;, 0.11 0.07 0.09 0.21 0.38
system; they could have been improved by using longer tubes, but the high resistance of this fine tubing presents difficulties at flows of lo-20 ml/ min. The bubble oxygenator was the most efficient of all, but the film oxygenator was reasonably etficient, was simple, and avoided the problem of foaming; it was, therefore, used in the subsequent renal perfusion experiments. Ten kidneys were perfused and transplanted in each of the three experimental groups. Table 2 shows that the groups were comparable with respect to the delay before starting continuous perfusion, the duration of perfusion, and the time taken to complete the anastomoses. The preperfusion interval of 11-12 min included less than 3 miu warm time, approximately 4
Preperfusion time (min)
GroupI (95% Oz)
11.9
f
(95C7” air)
11.4
Grmp III (95’70 N*)
I1.Y
Perfusion time W)
0.8
Postperfusion time (min)
24.03
i
0.02
23.i
*
1.3
zko.5
24.05
f
0.09
22.9
*
1.4
zk 0.6
24.03
f
0.02
28.4
*
1.4
Oroup II
242
PEGG,
GREEN
AND
min to flush the kidney with cold heparinized perfusate, and then about 5 min to transport the cooled kidney to the perfusion apparatus, attach it to the equipment and record its initial weight. The behavior ‘of the three experimental groups during perfusion is shown in Table 3. The only significant difference is in the measured oxygen tensions which were close to the expected values, although it was not possible to obtain zero values for the oxygen tension in Group III, even though nylon tubing was used throughout. The pH, measured at 5”C, was similar in all the experiments. Vascular resistance exhibited fluctuations, as previously described (18) with a general tendency to increase slightly during perfusion. One kidney in Group I exhibited an unusually large increase in resistance ( x 13.9), but this kidney functioned well after transplantation; if this experiment is excluded, the mean _t 1 SE for Group I becomes 1.74 r+ 0.30. No matter which value is taken, however, there is no significant difference between the increase in resistance that occurred in the three groups. Nor is there any difference between the weight gained by kidneys in the three groups, and of the various compounds that were estimated in the perfusate (see Methods) only the lactate concentration showed any significant distinction between groups, being higher in Group III (0.7 4 0.1 mhl) than in the oxygenated kidneys (0.4 k 0.1 mM) (P < 0.05).
The function of the perfused kidneys was assessed by survival, the maximum blood urea and serum creatinine concentrations recorded, and by the integral of blood urea concentration with respect to time in those animals that survived for 14 days or longer (Table 4). More animals survived in the groups in which the perfusate was oxygenated (see Fig. 4) but the difference is not significant (x’ = .9524, 0.5 > P > 0.25). Moreover, the renal function of survivors in the nitrogen group, as measured by the urea integral, was better than that in the oxygenated groups, although the difference was not significant at the 5% level (P < 0.3 and < 0.1 when compared with Groups I and II, respectively ). There were no significant differences between the maximum blood urea and serum creatinine levels in the experimental groups. Moreover, when the 10 kidneys giving best function were selected it was found that four were from Group I, two from Group II, and four from Group III, which reveals no benefit from oxygenation. Histological examination of the kidneys also failed to reveal significant differences between the experimental groups. There were two acute tubular necroses in each for six of the acute group, accounting deaths shown in Table 4 and Fig. 4; the two additional early deaths in Group III were caused by total renal infarction (1) and septicemia ( 1). Each experimental group also included two kidneys that
PO2
P”
Group
FOREMAN
Weight win (7%)
(mrn”g)
R&;sgc
I
(96%
02)
7.35 f
0.01
676 rt 4
4X
*
.i
x3.0
(X1.7
‘1 Value
Group II (95% nir)
7.37 *
0.02
137 f
Group III (03% Nz)
7.35 f
0.01
Of1
obtained
when
one very
high
result
is excluded
5
40 *
f
1.2
*0.3p
4
x1.7
rt 0.2
42 zk 3
x2.1
f
: see the text.
0.4
showed fairly severe chronic renal damage in the form ‘of significant arcas of fibrosis with calcification and dilated tubules; one of these animals (in Group I ) died at 18 days, but the other five survived with adcquate renal function until sacrificed at 2 mo or later. One other late death occurred, from septicemia, at 4; wk in an animal in Group II. All the other animals survived until sacrificed at 2-3 mo, and their kidneys were either completely normal histologically, or showed minimal changes, typically a few small infarcts or mild tubular dilatation. In previous experiments ( 18 ) using 95% oxygen and a membrane oxygenator, the survival rate was S/10, the mean urea integral was 1.60 * 0.12 x 10” mg/lOO ml x
FIG.
4. Graph
showing
the postoperative
survival
!).2 f
2-l
9.0 *
l..-l
9.2 f
1.8
days, and the highest serum creatininc level 7.12 0.6 mg/lOO ml. These results do not differ significantly from those obtained in Group I of the present series of experiments, or from the pooled results of all three groups. Comparisons with previous results can be influenced by unrecognized factors and must, therefore, bc examined particularly critically: in the prescnt case, the two sets of experiments were done sequentially without interruptimon by the same workers, using the same cquipment and animals from the same breeding colony. In these circumstances the comparison is believed to be valid. As in the experiments previously dcscribed (18), an attempt was made to discover whcthcr any of the measurements
of animals
in the three
experimental
groups.
244
PEGG,
GREEN
made during perfusion might be able to predict the posttransplantation function of individual kidneys. As before, the results were combined and regrouped into those in which the animal died in the first 14 days and those in which the animal survived longer than 2 wk. Comparisons were made of absolute resistance, resistance change, and weight gain of kidneys during perfusion, and the measurements of the biochemical parameters in the perfusate as listed in the Methods section. Only the glucose level showed a significant difference, 4.44 * 0.06 mM in the survivors, and 4.67 5 0.09 mM in those dying, giving a value for P = 0.05. The mean potassium concentration in the survivors was lower than in the animals that died, but not significantly so. DISCUSSION
These experiments produced some unexpected results, which must lead to a reexamination ‘of several aspects of current practice and dogma in organ preservation: our failure to find any benefit from membrane oxygenation, or from oxygenation at all, leads us to question the expense and complexity of the perfusion techniques and equipment in current use. Moreover, the c’emonstration that a continuous supply of oxygen has no effect on the subsequent function of transplanted kidneys removes one of the central arguments f,or perfusion; but it remains true that the best results obtained to date have been obtained by continuous perfusion techniques (3) and we do not advocate that these methods be abandoned at the present time. The majority of workers using continuous perfusion have employed membrane oxygenators, through which they have passed 95% oxygen (12), 33% oxygen ( 23), ‘or air (2). The oxygen tensions produced have not always been reported, and when they have, they have sometimes been recorded at a higher temperature than the preservation temperature, making interpre-
AND
FOREMAN
tation very difficult: the experiments reported in this paper show how variable the gas-exchange properties of oxygenators can be, especially membrane oxygenators, so that the oxygen tensions actually achieved remain in some doubt. Recent years have seen significant advances in the design of membrane oxygenators, and these are admirably reviewed by Drinker (5). It is certainly possible to construct efficient units, even with relatively thick membranes of silicone rubber, providing satisfactory supporting matrices are used, so that adequate flow of gas in close contact with the membrane is achieved. The justification ‘of membrane oxygenation rests on two premises: (1) that gas/ liquid interfaces must be avoided and (2) that a continuous supply of ‘oxygen is beneficial. There is some evidence to support the first premise; it is known that the exposure of blood to gas/liquid interfaces causes protein denaturation (26) and platelets are also damaged in these circumstances (24); such effects have been implicated in the pathogenesis of organ dysfunction after prolonged use of a disc oxygenator in heart-lung bypass ( 1). Similar effects can also ‘occur in isolated organ perfusion, even when the fresh perfusate contains neither blood nor plasma: it was found in this laboratory that when rabbit kidneys were perfused using a bubble oxygenator and a bloodless (dextran) perfusate which was recirculated, that the vascular resistance increased ( 15). The same effect could be produced in the absence of recirculation by the addition of small quantities of autologous blood and it was deduced that sufficient blood can remain in a kidney to cause vascular damage when it is gradually released during the course of a perfusion. This effect could be abolished either by using a membrane oxygenator or by including a filter of pore size < 1 pm (15). These results support the suggestion that vascular damage is primarily due to denatured protein and platelets, and they
OXYGENATION
IN
RENAL
also explain why a filter of sufficiently fine pore size can prevent such damage. In the present experiments, as in most perfusion systems, a fine filter was inch&d. There is also direct evidence for the adequacy of film oxygenation in hypothermic renal perfusion; Scott et al. (21), and Johnson et al. (12) have for many years successfully used 3 film ‘oxygenator in renal preservation, and more recently, Clnes and Rlohme (3) and Proctor and Jones (19) have found gas/ liquid surface oxygenation satisfactory in renal preservation. It seems clear, thcrefore, that membrane oxygenation is not cssential for successful preservation by hypothermic perfusion. What of the second premise: is oxygenation necessary at all? One of the reasons given by Humphries when he introduced hypothermic perfusion for renal prescrvation was that the continuous flow of perfusate would provide the organ with its admittedly minimal requirement for oxygen (11). Relzcr has also assumed that oxpgenation is essential (2). The oxygen consumption of dog kidneys is reported to bc approximately 5% of normal at 5°C (13) which would be only about .06 ml/min for a 15-g kidney. It has been reported that there is an arterovenous oxygen differcncc in perfusate flowing through kidneys at 8°C (Claes, G.; Personal communication), but it is difficult to make accurate measurements of oxygen consumption by perfused kidneys; if the PO2 of fluid flowing through the cortical vessels exceeds the partial pressure of oxygen in the surrounding medium, then oxygen will be lost by diffilsion, and an A-V difference created. NeverthcIcss, some oxygen probably is consumed by respiration even at S”C, and the fact that more lactate was produced in Group III of the present experiments supports this conclusion. It does not, however, follow that oxygen must be provided for masimum viability. We have already shown that excellent preservation can bc obtained with rabbit kidneys by Collins’ technique
PRESERVATIOS
Lz-45
for 24 and even for 48 hr (9), and Sacks et (~1. have preserved dog kidneys for 72 hr with a similar solution (20): in neither technique is a continuous Supply of oxygen provided. The present results, obtained in a ~\iell-standardized cxpcrimental system show that oxygen tension has no effect on the preservation of rabbit kidneys by hypothcrmic perfusion: a moderate oxygen tension is without benefit, and a high oxygen tension is not harmful, as some have suggested might be the case (11). These findings must be confirmed by others, and in different animals, but on our evidence it does stem that the basic premise that a continuous supply ,of ox):gcii is bcncficial is at fauIt. If oxygen is unnecessary, what other function might perfusion be performing? Does perfusion have any value at all? The available evidence still suggests that it does; the longest prescrvntion periods so far reported in the most commonl\- used cxpcrimcntal animal, the dog, have been with perfusion techniques, and Sacks’ results, especially with ischcmic injury are still significantly worse than Claes’ rrsults (3, 20). This sort of evidence is not uuequivocal, however, and direct comparisons of both techniques by the same group are urgently needed; when this was done for PF III perfusate, perfusion was superior to washout (9). SIlould it bc confirmed that perfusion does indeed provide the best preservation, then it would seem that perfusion equipment can now be greatly simplificd. In the absence of any need for oxygenation, the only reason for retaining a gas exchanger wouId be to regulate the pH of a CO-/bicarbonate buffer system. However, if the latter were to be replaced by one of Good’s buffers (7) such as HEPES (N-2-hydroxyethylpipernzine-N’2ethanesulfonnte), the gas phase could be dispcnscd with, the oxygenator and its heavy gas bottles elminated, and the equipmcnt made lighter, nlore compact, and less expensive. The inclusion of a submicron
246
PEGG,
GREEN
filter would seem to be mandatory in such circumstances, but such filters are inexpensive, light, and compact. A disappointing feature of the present series of experiments was the failure of any of the monitored features adequately to predict subsequent function. In the preceding study (18), the concentration of potassium in the perfusate was found to correlate with subsequent function: in the present study the mean potassium levels were somewhat higher in the survivors and slightly lower in the nonsurvivors than previously, and no separation could be achieved on this basis. As previously, the perfusate glucose concentration was found to correlate inversely with function, and it seems likely that the additional glucose is derived from the breakdown of dextran by lysosomal I-glucosidasc released from damaged renal cells. However, the separation was less clear-cut than previously because the glucose levels in the nonsurvivors were considerably lower than in the earlier experiments. It seems clear that parameters other than those included in the present study will be needed for a useful predictive test. SUMMARY
Rabbit kidneys have been perfused for 24 hr at 5°C and tested by autotransplantation with immediate contralateral nephrectomy. The perfusate contained an extracellular balance of ions with dextran and bovine serum albumin. The circuit included a nonpulsatile pump, a 0.22-pm membrane filter, and an oxygenator. The efficiency of membrane, film, and bubble oxygenators was compared. Flat membrane oxygenators were found to be more efficient than tubing oxygenators, and although the bubble oxygenator was the most efficient it created problems with foaming. A simple film oxygenator provided the best compromise. Ten kidneys were perfused and transplanted in each of three experimental
AND
FOREMAN
groups; a film oxygenator was used to provide partial pressures of oxygen of 676 mmHg, 137 mmHg, and 9 mmHg, respectively. There were no significant differences in behavior during perfusion, function after transplantati’on, or histology of transplanted kidneys in the three groups. The similarity of the results using a film oxygenator with those previously obtained with a membrane oxygenator indicates that complex membrane oxygenators arc not necessary for preservation in our system. Moreover, the similarity of the results with oxygen tensions from 9 mmHg to 676 mmHg shows that deliberate oxygenation is also unnecessary. REFERENCES 1. Allerdyce, I). B., Yoshida, S. H., and Ashmore, P. G. The importance of microembolism in the pathogcnesis of organ dysfunction caused by prolonged USC of the pump oxygenator. J. Thoruc. Cardiovasc. Surg. 52, 706-715 ( 1966). 2. Belzer, F. O., Ashby, B. S., and Dun&y, J. E. 24-hour and 7%hour preservation of canine kidneys. Lancet 2, 536-539 ( 1967). 3. Claes, G., and Blohme, I. Experimental and clinical results of continuous albumin perfusion of kidneys. In “Organ Preservation” (D. E. Pcgg, Ed.), pp. 51-57. ChurchillLivingstone, Edinburgh, 1973. 4. Claes, G., Blohmc, I., and Gelin, L-E. Clinical kidney preservation with and without continuous perfusion. Proc. Eur. Did. Transpkunt Ass. 8, 307311 (1971). 5. Drinker, P. A. Progress in membrane oxygcnator design. Anaesthesiology 37, 242-260 (1972). 6. Gimbrone, M. A., Aster, R. II., Cotran, R. S., Corkery, J., Jandl, J. H., and Folkman, J. Preservation of vascular integrity in organs perfused in vitro with platelet-rich medium. Nature (London) 222, 33-36 (1969). 7. Good, N. E., Winget, G. D., Winter, W., Connolly, T. N., Izawa, S., and Singh, R. M. M. Hydrogen ion buffers for biological research. Biochemistry 5, 467477 ( 1966 ). 8. Green, C. J. Rabbit renal autografts as an organ preservation model. Lab. Anim. 7, l-11 (1973).
OXYGENATION
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9. Green, C. J., and Pegg, D. E. A comparison of non-perfusion and perfusion methods for preservation of rabbit kidneys. In “Organ Preservation” (D. E. Pcgg, Ed.), pp. 1629. Churchill-Livingtonc, Edinburgh, 1973. 10. llolhs, K. E. IT., and Ellis, hf. The present status of subzero organ prcscrvation with special reference to the rat hcalt. In “Organ Prcscrvation” (1~. E. Pegg, Ed.), pp. 123-136. Churchill-Livingstonc, Edinburgh, 1973. 11. Humphries, A. L., Russell, R., Ostafin, J. Goodrich, S. ht., and Mnretz, W. H. Successful reimplantation of canine kidney after 24 hr storage. SurgertJ 54, 136-143 (1963). 12. Johnson, R. W. G., Anderson, M., Morley, A. R., Taylor, R. M. R., and Swinney, J. Twenty-four hour preservation of kidneys injured by prolonged warm ischacmia. 7’mns~Jlantation 13, 174-179 ( 1972). 13. Levy, N. M. Oxygen consumption and blood flow in the hypothermic perfused kidney. Amcr. J. Physiol. 197, 1111-1114 (1959). 14. Love, J. W. A low-priming volume hcarthmg machine for use in thr laboratory. 1. 7’11orac. Cnrtliocosc. Slug. 45, 62%627 (1963). 15. Pegg, 1). E. Vascular resistance of the isnlated rabbit kidney. Crryohiology 8, 431440 (1971). 10. I’cgg, 1). E., Fuller, B. J., Foreman, J., nntl Green, C. J. The choice of plastic tubing for organ perfusion espcrimcnts. CryolTiology 9, 569-571 ( 1972). 17. Pcgg, D. E., and Green, C. J. Renal prescrvation by hypothermic perfusion using a defined perfusion fluid. Cr!yohiolog!y 9, 420428 ( 1972).
I’RESER\‘A’I’ION
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18. Prgg, I). E., ant1 Green, C. J. Renal prcscrvation by hypothermic perfusion 1. The importance of presslirc~-control. Cr!yobiology 10, S&66 (1973). 19. Proctor, E., and Jones, G. R. N. In vitro asscssnicnt of preserved dog hearts, with xomc rcfcrenct: to preserved dog kidneys. In “Organ l’reservation” (1). E. I’egg, Ed.), pp. 216-224. Chilrchill-Livingstonc, Edinburgh, 1973. 20. Sacks, S. A., Petritsch, P. II., and Kaufman, J. J. Canine kidney preservation using a new pcrfusate. Ltrncct 2, 1024-1028 ( 1973). 21. Scott, D. F., Morley, A. R., and Swinncy, J. Canine renal preservation following hypothcrmic perfusion and subsequent function. Brit. J. Surg. 56, 688-691 (1969). 22. Severinghaus, J. W. Electrodes for blood and gas PCO, Paz and blood pH. Acta Anaesthcsiol. Scard. SuppI. 11, 207-220 ( 1962). 23. Stephenson, T. P., O’Donoghuc, E. P. N., Hendry, W. F., and Wickham, J. E. A. Prcscrvation of human kidneys for transplantation: Preliminary results with a Gambro perfusion machine. Brit. Afcd. J. 1, 379381 (1973). 24. Swank, R. L., Hirsch, H., Brewer, hl., and Hisser], W. Effect of glass wool filtration on blood during circulation. extracorporeal Surg. Gyrwcol. Obstct. 117, 547-552 (1963). 25. Umbreit, W. W., Burris, R. H., and Stauffer, J. F. “Manomctric Tcchniqllrs,” p. .5. Burgess, Minneapolis, 1964. 26. Wright, E. S., Sarkozy, E., Harpur, E. R., Dobell, A. R. C., and Murphy, D. R. Plasma protein clmaturation in extracorporeal circulation. J. Thornc. Cardiot~n.rc. hi-g. 44, 550556 (1962).