CIIYODlOI.OGY

14, 168-178

Perfusion

(1977)

of Rabbit

Kidneys

D. E. PEGG Division

of Cryobiology, Harrow,

with

AND

Clinical Middlesex

The long-term storage of whole organs for subsequent transplantation will almost certainly require the use of subzero temperatures and cryoprotectants. Unfortunately however, the direct application of conventional cryopreservation procedures, involving perfusion with a single initial concentration of a cryoprotectant, followed by slow cooling to -80 or to -196”C, has met with very little success( 1, 5). We have argued that the problems of subzero organ preservation are so complex that the only hope of success lies with a step-by-step analysis of the separate stages that are likely to be found in the final technique (12). The first stage was to develop perfusion conditions that are satisfactory in thcmsclves; although there is certainly scope for improvement, presently available methods are satisfactory for the periods of time needed for the introduction and removal of cryoprotectants (13). The second stage was to identify a suitable cryoprotectant. In work already reported from this laboratory ( ll), rabbit kidneys were perfused with glycerol, ethylene glycol, and dimethyl sulphoxide, and it was found that the vascular resistance assumed a stable low value with glycerol or ethylene glycol at 5”C, and that after 2 hr of perfusion with a 2 M solution of each cryoprotectant, glycerol had penetrated more completely than ethylene glycol. Glycerol seemed to be the cryoprotectant of choice. Since there Received May 12, 1976.

Glycerol

Solutions

at 5°C

M. C. WUSTEhlAN Research

HA1 3tJJ,

Centre, Wutford England

Road,

would be little point in cooling to subzero temperatures before it had been shown that glycerol could be added and removed without damage, the third stage of investigation was to design a suitable method for removing glycerol and then to establish the viability of the kidneys. It was found that when glycerol-perfused kidneys were subsequently perfused with cryoprotectant-free pcrfusatc, thcrc was an increase in weight and a dramatic rise in vascular resistance such that perfusion virtually ceased ( 12). The experiments to be described in this paper concern the development of a technique for adding glycerol to a final concentration of 2 M and then removing it without producing significant impairment of renal function. This was achieved by adding a nonpenetrating solute, mannitol, to the perfusate and employing a controlled slow rate of change of glycerol concentration. hlATERIALS

AND METHODS

1. Animals

New Zealand albino rabbits of either sex weighing 2.5-3.5 kg were used. The animals were anesthetised by the intramuscular injection of 0.3 ml/kg of Hypnorm (Jannsen; fentanyl, 0.2 mg/ml and fluanisone, 10 mg/ml) supplemented when necessary by intravenous Brietal (Lilley; methohexitone sodium, 3 mg/ml). The animals also received 5 mg/kg of chlorpromazine and 5000 units of heparin, both intravenously 5 min prior to nephrectomy. The right kid168

Copyright All rights

Q 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0011-2240

PERFUSION

OF

KIDNEYS

ncy was freed from surrounding tissue, the ureter was severed at the bladder, and the artery was cannulated in uiuo. The vein was then severed and the kidney connected to the perfusion apparatus. Warm ischemia time was less than 5 min.

-~ i.

2. Perfusates The basic perfusion solution contained an extracellular balance of ions similar to that in PF III (13) but the colloid used was dextran, of weight mean molecular weight 150,000 (Dextran 150; Fisons Ltd., Pharmaceutical Division). Details are given in Table 1. In some experiments, as described in Methods, section 4 below, perfusates containing mannitol and/or 2 hl glycerol were used, whiIe in other experiments the glycerol concentration was increased gradually by mixing the mannitolcontaining pelfusate with a concentrated glycerol solution. The composition of these solutions is also given in Table 1. All perfusates were filtered through a 0.2%PM Millipore filter and equilibrated with 5% CO, in oxygen to yield a pH of 7.35 before use.

ii.

IS1 :’ iv.

3. Apparatus Two perfusion systems were used. The first was a double circuit, similar to one already described (10) which permitted either of the two perfusates to be circulated through the kidney and allowed a change to be made between perfusates without intcrrupting flow and with minimal mixture of the two solutions. The two circuits contained basic perfusate and perfusate with 2 M glycerol, respectively, and were enclosed within a temperature-controlled cabinet at 5°C. In other experiments a feedback control system was used to program the rate of increase and decrease of glycerol concentration during each experiment. The apparatus is a development of one that has previously been described briefly (9) and is shown in Fir. 1. The k&xv was mournted

4.X)

g

170

PEGG AND WUSTEMAN

FIG. 1. The apparatus used to perfuse rabbit kidneys in control groups 1 and 2 and to change the glycerol concentration in experimental groups 5 and 6. For a detailed description see the Methods, section 3.

on a weight transducer ( WTl; Ether, type UF 1) and the venous effluent was allowed to fall into a graduated vessel whence it was aspirated by a pump (P) and returned to a mixing chamber which contained 1 liter of perfusate. By switching off the pump and measuring the time for 10 ml of effluent to be collected, it was possible to measure perfusion flow rate. The mixing chamber was thermostated at 30°C and its contents were kept well-mixed by a reciprocating agitator driven by a motor (M). The pH of the perfusate was maintained at 7.35 by continuously gassing the mixing chamber with 5% CO, in oxygen. Perfusate was continuously circulated through a recording refractometer (Hilger and Watts, type M 550) and returned to the chamber. The electrical output of the refractometer was fed into a process controller where it was compared with a program voltage derived from a linear voltage-ramp generator (Varipot, Rayleigh Instruments Ltd.), and the controller output was used to drive a pump (P) that added concentrated glycerol solution or basic perfusate diluent from a reser-

voir. Since the refractive index of the perfusate was solely dependent on glycerol concentration, and since the relationship between glycerol concentration and refractive index was known (see below), this arrangement made it possible to obtain accurate, linear rates of increase and decrease of glycerol concentration. The total volume of perfusate in the circuit was kept constant by an arrangement in which the “dip stick’ pressure transducer (PTl) operated the solenoid valve (SV) whenever the volume in the reservoir exceeded 1 liter and discharged excess solution to waste. Perfusate for passage through the kidney was aspirated from the refractometer circuit by a pump (P; Watson Marlow, type MHRE) and passed through a 142-mm Gelman filter containing a glass-fiber prefilter mat and a 0.22-pm Millipore membrane (type GSWP), a bubble trap, and a stainless steel paralleltube heat exchanger, to the renal artery cannula. The perfusate temperature was measured just before it entered the cannula by a copper-constantan thermocouple connected to a Comark Thermocouple Multi-

PERFUSION

OF KIDNEYS

WITH

GLYCEROL

171

the mixing chamber. Seven groups, each comprising five kidneys, were studied. Group 0 (freshly isolated controls). These kidneys were not perfused at 5”C, but were immediately assayed for in vitro function as described in Methods, section 6 below. The warm ischemia time was approximately 2 min. GTOU~ 1 (no mannitol controls). These kidneys were perfused for 215 min with the basic pcrfusate without mannitol (i), and glycerol was not added. During the periods in the experiment when glycerol was added and removed in Groups 5 and 6, approximately equivalent volumes of basic perfnsate were’ added to the mixing chamber by hand. GTOU~2 (mannitol controls). This group was treated similarly to Group 1 but the perfusate with mannitol (ii) was used. Group 3 (rapid change of glycerol concent&ion without mannitol). These kidneys were perfused on the double circuit using the basic (i) perfusate and the 2 hf glycerol perfusate without mannitol (iii). For the first 20 min, glycerol-free perfusate was used; between 20 and 115 min the perfusate containing 2 hf glycerol was perfused; between 115 and 215 min the glycerol-free perfusate was again used. Group 4 (rapid change of glycerol concentration with mannitol). The procedure for this group was similar to that for Group 3, but both perfusates contained mannitol (ii and iv). GTOU~5 (glycerol concentration changed at SO mhx mix1 with mannitol perfusate). 4. Glycerolising and Deglycerolizing PTO- These experiments were conducted on the cedures feedback-controlled circuit using perfusates containing mannitol (ii and v). Glycerol Immediately after excision, the kidney concentration was increased at a rate of 80 was attached to the perfusion apparatus nlht min-l starting at 20 min, and decreased and perfused at 60 mm Hg for 10 min; the at a rate of 80 rnhl min-’ starting at 115 venous effluent, which contained considermin. The glycerolizing phase therefore able amounts of blood, was discarded. The lasted 25 min and was followed by a perfusion pressure was then adjusted to 40 plateau phase lasting 75 min, a deglycmm Hg and continued at this le\.el for the crolizing phase lasting 25 min, alid a final remainder of the experiment (205 min), and the effluent perfusate was returned to plateau phase lasting 70 min.

meter, type 160 C. About two-thirds of the flow was returned to the reservoir through an adjustable bypass in order to reduce the dead-time between the perfusion pump and the kidney; the bypass also provided a point from which perfusate samples could be collected. The perfusion pump was controlled from a pressure transducer (Ether, type UP 4; PT2) connected to a T-piece on the arterial cannula, such that the arterial pressure was kept constant at 60 or 40 mm Hg ( 14). Industrial methylated ethanol (IMS) at 3°C from a solid CO, cooling unit (8) was pumped through the heat exchanger to cool the perfusate to 5°C. The heat exchanger and the kidney were enclosed in a “Styrofoam” insulated box that was cooled by the external surface of the heat exchanger itself. Kidney weight and renal arterial pressure were recorded continually on strip-chart recorders ( Rikadenki, type B 34). Temperature and flow were recorded manually at 5-min intervals. Throughout the periods during which glycerol was added or removed, independent measurcmcnts of glycerol concentration were made on samples taken at 5-min intervals from the mixing chamber and the arterial cannula. An AbbC refractometer (Hilger and Watts, type M 46) thermostatically controlled at 30°C and calibrated with solutions of ‘Analar’ glycerol prepared in weight/volume terms was used for this purpose. The program of increasing glyccrol concentration was stopped when the mixing chamber concentration reached 2 hf.

172

PEGG

AND

WUSTEMAN

Group 6 (glycerol concentration changed at 30 mM min-l with mannitol perfusate). These experiments were also carried out with the feedback-controlled circuit using the perfusates containing mannitol (ii and v), but the glycerol concentration was increased at a rate of 30 mM min-l starting at 20 min and decreased at the same rate starting at 115 min. The glycerolizing phase lasted approximately 65 min and was followed by a plateau phase lasting 30 mm, a deglycerolizing phase lasting 65 min, and a final plateau phase of 35 min.

I- ‘\\ \

‘\

5. Calculations Renal vascular resistance was calculated by dividing the pressure measured in the renal artery cannula (mm Hg) by the product of the perfusate flow rate (ml min-I) and the perfusate viscosity (cp) measured with a cone and plate viscometer ( Wells-Brookfield) at 5°C. The variation in viscosity as the glycerol concentration was changed was taken into account. Changes in vascular resistance during the various phases of perfusion were related to the resistance immediately before the phase in question: thus, the minimum resistance which occurred during glycerolization was expressed as the ratio of that resistance to the resistance at the end of the initial 20min period that preceded glycerolization; the maximum resistance which occurred during deglycerolization was expressed as the ratio of that resistance to the resistance when glycerolization was complete; and the final resistance was the ratio of the resistance at the completion of each phase of the experiment to the resistance at the end of the preceding phase. The overall resistance ratio referred to the change in resistance from the beginning of glycerolization to the end of deglycerolization. Kidney weight was related to the weight of the blood-filled, freely drained kidney immeditncly prior to perfusion, and weight changes during perfusion were expressed as

;,,, l’,> -‘II T,:t l”‘,IJ FIG. 2. Typical vascular resistance patterns in rabbit kidneys from csperimental groups 3 (top), 4, 5, and 6 (bottom). Including mannitol and slowing the rate of change of glycerol concentration minimized the changes in vascular resistance.

ratios in the same way as the corresponding resistance changes. 6. Assay of Viability Renal function was assayed by normothermic perfusion using a technique that has been described in detail elsewhere (4) although the per&sate used here included a different colloid. Briefly, the technique consisted of perfusion at 110 mm Hg with a solution containing carbohydrate and fatty acid substrates (vi in Table l), sufficient dissolved oxygen to raise the partial pressure to 600-650 mm Hg, and appropriate tracers for the measurement of glomerular

PERFUSION

OF

KIDNEYS

filtration rate (GFR) by inulin clearance, and protein leakage. Perfusate and urine flow rates were measured by timed volumetric collections, and samples of each fluid were collected for the following estimations: [14C]hydroxymethyl inulin, by liquid scintillation spectrometry (Packard Model human serum 3375 counter ) ; ‘““I-labeled albumin, by gamma spectrometry (Packard Model 3002 counter); sodium concentration, by emission flame photometry (EEL flame photometer); and glucose, by the glucose oxidase method (Boehringer Mannheim reagent kit). These measurements permitted the calculation of in&n and albumin clearance, and the tubular reabsorption of sodium and glucose was calc&ted as follows : Filtered

load

Excrctcd Rcabsorbcd

= GIl’R x pcrfusatc conccnt,ration

load

= Urine flo\v X Urine concenbration

load = Filttlrcd load - cxcrct (~1 load

Pcrccntagc rclabsorption

=

In conformity with previous practice protein leakage was measured by the ratio of albumin clearance to inulin clearance. In this study the functions measured after 1 hr of normothermic perfusion were used to compare the four experimental groups with the two control groups and the group of freshly-isolated rabbit kidneys. RESULTS

1. Changes in Vascular Resistance Typical vascular resistance patterns are shown in Fig. 2 and detailed results in Table 2. Sudden changes in glycerol concentration (Groups 3 and 4) produced marked changes in vascular resistance, a reduction on addition and an increase on removal, but these were greatly reduced by the inclusion of mannitol in the perfusate. Slowing the rate of addition and removal of

WITH

GLYCEROL

173

glycerol produced further improvements, but in every case, the final vascular resistance was considerably greater than that recorded at the beginning of the experiment. When the glycerol-treated kidneys were compared with control kidneys it was found that there was a significant decrease in resistance during glycerolization in all cases (P < 0.001) but that the decrease was less when mannitol was present (P < 0.05) and was reduced still further when the rate of addition was reduced to 30 mM mill-l (P < 0.01). The maximum vascular resistance during deglycerolization was also strikingly affected by the inclusion of mannitol in the pelfusates; there was a significant increase in every case (P < 0.001) but it was much greater in the absence of mannitol than in its presence (P < 0.001). Although it was observed that slowing the rate of removal reduced the resistance changes still further, the differences were not statistically significant at the 5% level. The final vascular resistance at 215 min was increased in all the glycerol-treated kidneys (P < 0.05) but this was much greater in the absence of mannitol than in its prcsciicc (P < 0.01); there was no significant difference between any of the matmitol-perfused groups.

2. Weight

Changes

All the kidneys gained weight during the perfusions. This was greatest in Group 3 in which the glycerol concentration was changed abruptly in the absence of mannitol and was least in Group 6 in which the glycerol concentration was changed at 30 m&f min-l in the presence of mannitol, but in no case was the weight gain significantly different from that observed in the control perfusions (Groups 1 and 2).

3. Functional

Comparisons

These results are shown in Table 3. There were no significant differences in GFR between any of the perfused kidneys,

.a For

groups

0.93 zko.11

0.90 ztO.OG

0.78 10.07

0.69 zto.04

0.40 iO.OG

0.33 zto.02

0.66 -10.0.5

0.64 Eko.03

5.

0.8G fO.10

0.81 *0.07

Section

1.10 rto.09

Glycerolization

Changes

0.95 zto.04

Final

Weight

Resistance

and

hIinimum

see the )Iethods,

Control perfused without mannitol Control perfllsed with mannil 01 Rapid dG/dt without mannitol Rapid dG/dt with malmitol 80 mM mill+ glycerol with manuilol 30 mu min-’ glycerol with mannitol

Treatment

definitions

Experimental

NUlllber

Resistance

0.9SS 10.012

0.999 *0.001

0.920 f0.035

0.930 10.023

0.999 fO.OO1

0.976 f0.020

hlinimum

Weight

in Perfused

1.013 zko.014

1.043 f0.014

7.7 fl.O

7.1 f2.1

13.1 f2.7

1.040 f0.017

1.03 +0.03

1.05 zto.04

.5. 1 f1.G

2.70 zto.::o

3.2:: f0.63

20.9 f2.1

0.9G zko.04

O.SG zko.07

Final

Dcglycerolization

the Introdlxtion

Resistance

during

iVaximum

2

29.0 f0.9

Kidneys

TABLE

1.091 f0.050

1.024 fO.O1O

1.028 f0.031

Final

Rabbit

1.0:32 &0.013

l.OlG 1O.OOG

1.031 10.00s

1.036 f0.021

1.033 10.023

hlaximum

\L eight

and Removal

1.030 10.016

0.992 zto.017

1.048 f0.009

1.030 SzO.024

1.033 f0.025

1.010 r!ro.o03

Final

-

of Glycerol” Overall

3.37 f0.93

2.04 10.21

2.79 f0.40

19.j *3..i

O.&Y zko.13

0.95 10.11

Resistance

Weight

7.042 ztO.016

1.036 10.02s

1.090 *0.021

1.125 &0.005

1.03 10.035

1.03s f0.033

ratios

PERFUSION

OF

KIDNEYS

WITH

175

GLYCEROL

I?, t? -!=i 00 ii

176

PEGG AND WUSTEMAN

and in fact only Group 3 showed a signifi- solutions shut down completely when subcantly lower GFR than freshly isolated kid- sequently perfused with glycerol-free meneys. There were, however, important difdium (12) and Halasz and his colleagues ferences in the other more sensitive func(6) showed that dog kidneys perfused with tional indices especially in the measure- glycerol solutions exhibited “outflow block” ments of protein retention. A low value for when their blood supply was restored, unthe ratio of albumin to inulin clearance in- doubtedly an osmotic effect. The influence dicates a high degree of retention, whereas of perfusate osmolality on vascular resista ratio of I indicates that albumin is as ance is well known and is considered to be freely filtered as inulin; Group 3 gave a due to effects on the hydration of the cndoratio that was very close to 1. The control thelial cells and perivascular tissues which perfusions gave values that were not sig- control the dimensions of the vascular channificantly greater than those recorded for nels. Recent evidence that capillary basefreshly isolated kidneys, and both Groups 1 ment membranes are remarkably rigid (7) and 2 and the freshly isolated kidneys were suggeststhat the main effect is on the endosimilar to the Group 6 kidneys. The addi- thelial cells. The inclusion of a nontoxic tion of mamritol (Group 4) produced a sig- solute, mannitol, which penetrates cell nificant improvement in protein retention, membranes only slowly (2) reduces the and slowing the rate of addition and re- osmotic flux during deglycerolization. Slowmoval of glycerol (Group 5 and 6) gave ing the rate of change of glycerol concenfurther significant improvements (P < tration limits cell volume changes still fur0.05 ) . ther by reducing the time lag between All the perfused kidneys showed a sig- outward diffusion of glycerol and inward diffusion of solvent (water). The fact that nificant reduction in glucose reabsorption, the lowest value being in Group 3 and the vascular resistance was still elevated at the greatest in Group 6; the difference between end of the 215min perfusion period sugGroup 3 and Group 6 was significant (P < gests incomplete removal of glycerol, but 0.01) and Group 6 was similar to the per- the possibility of permanent vascular damage cannot be completely excluded. This fused controls (Groups 1 and 2). Tubular reabsorption of sodium showed a similar remains to be established by ultrastructural picture; again the inclusion of mannitol in studies. Important though the effects on vascular the perfusate increased the subsequent reabsorptive function, and again Group 6 resistance are, the effects of glycerolization showed similar function to the perfused on function are crucial. The normothermic controls although it was significantly de- perfusion technique used to assay function pressed with respect to freshly isolated kid- in these experiments has been shown to be sensitive to renal ischemia (15) and has neys (P < 0.02). proved to be reliable in selecting perfusatrs for renal preservation (3). In the present DISCUSSION study it was found that the perfused conIt is widely recognized that a major trol kidneys (Groups 1 and 2) functioned probIem to be overcome before the cryo- less we11 than the freshly isolated kidneys, preservation of whole organs will be pos- indicating that the perfusion technique itsible, lies with the osmotic effects of in- self was to some extent damaging. It is still troducing and removing cryoprotective important to seek improved methods of compounds. Vascular resistance is pro- perfusion. foundly influenced by such osmotic pheThe effects on vascular resistance of the different techniques of adding and removnomena. It has already been reported that ing glycerol correlated well with the effects rabbit kidneys perfused with 2 M glycerol

PERFUSION

OF KIDNEYS

on function. The addition of 100 m&f mannitol to the basic perfusate (Group 2) did not affect subsequent function, which confirms that mannitol itself is nontoxic. When glycerol was introduced at a rate of 30 mM/min to a final concentration of 2 hf and then removed at the same rate, all in the presence of 100 mM mannitol (Group 6), the kidneys functioned as well as the control perfused kidneys, showing that glycerol is also nontoxic under these conditions. It is probably more important to remove glycerol slowly than it is to add it slowly, since cells are more sensitive to swelling than to shrinkage, but in these experiments no attempt was made to assess the importance of rate of change of glycerol concentration of the two phases separately. This study did not include any measurements of glycerol permeation in the kidneys, although we know from an earlier study that perfusion with 2 M glycerol for 2 hr at 5°C gives 73% equilibration (11). The question of equilibration is of some importance, not only because it affects the degree of cryoprotection provided, but also because incomplete penetration could have some bearing on the observed lack of glycerol toxicity. The possibility has already been mentioned that glycerol may not have been completely removed at the end of the hypothermic perfusion. Plleasurements of equilibration and washout using [14C]glycerol will be needed to answer these questions. The resuIts reported here provide the basis for the next stage in the development of a technique for subzero renal preservation; this will be to perfuse kidneys by the method developed here, using autotransplantation with immediate contralateral nephrectomy to assessviability and [‘“Clglycerol to measure cryoprotectant permeation. SUhZM4RY

The long-term preservation of whole organs will almost certainly require the use

WITH

GLYCEROL

177

of subzero temperatures and cryoprotectants. An essential part of such a technique is the ability to add a cryoprotectant in adequate concentration and subsequently to remove it without damage to the organ. In this study rabbit kidneys have been perfused with solutions containing 3% dextran and 2 ;\I gl!-wrol at 5”C, and their function has been measured after removal of the glycerol. The assay technique involved the measurement of glomerular filtration rate, protein leakage, and tubular reabsorption of sodium and glucose. The results indicate that the inclusion in the perfusate of an impermeant solute (mannitol) and limitation of the rate of change of glycerol concentration (to 30 mhi min-I) permits rabbit kidneys to retain a degree of functiou similar to that found in perfused control kidneys, although somewhat reduced in comparison with freshly isolated kidneys. REFERENCES 1. Dietzman, R. II., Rebelo, A. E., Graham, E. F., Crabo, B. G., and Lillehci, R. C. Longterm functional success following freezing of canine kidneys. Surgmj 74, 181-188 (1973). 2. Flares, J., DiBona, D. R., Frega, N., and Leaf, A. Cell volume regulation and ischaemic tissue damage. J. Membrane Bid. 10, 331343 (1972). 3. Fuller, B. J., and Pegg, D. E. The assessment of renal preservation by normothermic bloodless perfusion. Cryobiology 13, l77184 (1976). 4. Fuller, B. J., Wusteman, hl. C., and Pegg, D. E. The measurement of renal function by isolated perfusion at 37°C. In Vitro 2) CSSR 3(2), 26-34 (1974). 5. Halasz, N. A., Roscnficld, H. A., Orloff, XI. J., and Seifrlt, L. N. Whole organ preservation II. Freezing studies. Sttrgq 61, 417-421

(1967). 6. Halnsz, N. -4.. Seifert, L. N., and Orloff, hi. J. Whole organ preservation I. Organ perfusion stud&. Surgery 60, 368-372 (1966). 7. Murphy, M. E., and Johnson, P. C. Possible contribution of basement mernlxa~~o to the structural rigidity of blood capillaries. Microvascular Research 9, 242-245 ( 1975).

PEGG

178

AND

8. Pegg, D. E. Close tolerance cooling apparatus for cryobiological studies. Lab. Pruct. 15, 772-773 ( 1966). 9. Pegg, D. E. An apparatus for the simultaneous programmed control of dimethyl sulphoxide concentration and temperature in an organ Cryobiology 4, 249 perfusion system.

(1968). 10. Pegg, D. E. Some effects of dextran and of bovine serum albumin on the isolated perfused rabbit kidney. Cryobiology 6, 419424 (1970). 11. Pegg, D. E. Perfusion of rabbit kidneys with cryoprotective agents. Cryobiology 9, 411419 (1972).

WUSTEMAN 12. Pegg, D. E. Theory and experiments towards subzero organ preservation. In “Organ Preservation” (Pegg, D. E., Ed.) pp. 108-121. Churchill-Livingstone, 1973. 13. Pegg, D. E., and Green, C. J. Renal preservation by hypothermic perfusion using a defined perfusion fluid. Cryobiology 9, 420428 (1972). 14. Pegg, D. E., and Green, C. J. Renal prcservation by hypothermic perfusion. 1. The importance of pressure-control. Cryobiology 10, 56-66 ( 1973). 15. Wusteman, ht. C. Effect of warm ischaemia on in citro function of rabbit kidneys. Submitted for publication.