15, 18-26
CRYOBIOLOGY
(1978)
Transplantation
of Rabbit Kidneys Perfused Solutions at 10°C
I. A. JACOBSEN,11 2 D. E. PEGG, M. C. WUSTEkIAN,
AND
with
Glycerol
S. M. ROBINSON
Division of Cryobiology, Clinical Research Centre, Watford Road, Hamow, United Kingdom
Experiments previousIy reported from this laboratory showed that rabbit kidneys could be perfused with a 2 M solution of gIycero1 for 2 hr without apparent vascular damage or excessive weight gain (6 ). However, when the glycerol concentration was changed abruptly during glycerolisation and deglycerolisation, a concentration of 1.1 M was the highest that was comparable with unimpaired subsequent function and histology (4). Further studies showed that, if the concentration of glycerol was changed sIowly (30 mM min-l ) and about 100 mmol of mannitol was added to the perfusate, it was possible to increase the glycerol concentration to 2 M without diminishing subsequent function as measured by a normothermic perfusion assay (2, 11). This evidence that the kidney could tolerate exposure to 2 M glycerol was encouraging, since this would permit cooling to -5°C without freezing and should provide a high degree of cryoprotection during subsequent cooling. However, all the experimen,ts with 2 M glycero1 had employed dextran-containing perfusates, and in ~itru methods had been used to assess function: Therefore, it was necessary to Received
February
24,
1977.
1 Supported
by P. Carl Petersen’s Foundation. 2 Present address: Laboratory of Nephrology, Institute of Pathology, University of Odense, Uen-
mark. 18 OOll-2240/78/0151-0018$02.00/O Copyright All rights
Q 1978 by Academic Press, Inc. of reproduction in any form reserved.
confirm these re.suIts by transplantation. Preliminary experiments showed that control kidneys perfused with dextran would not support the life of graft recipients, whereas the gelatin-derived plasma exppander HaemacceI (Hoechst) was a highly satisfactory colloid ( 10). Indeed, rabbit kidneys perfused for 24 hr at 10°C with solutions containing this polypeptide material as the sole colloid have shown excelIent function ,afterwards (9). In addition, it was found that kidneys perfused for 4 hr with a Haemaccel solution containing 2% glycerol (0.22 M) wouId function as well after transplantation as glycerol-free controIs (IO). It was concluded that a Haemaccel perfusate should be suitable for the introduction and removal of gIycero1, and that complete removal of the cryoprotectant prior to transplantation wouId not be necessary. The experiments reported in this paper show that i,t is possible to perfuse rabbit kidneys with glycerol solutions up to a 3 M concentration, with 100% survival and a return to normal renal function. MATERIALS
AND
METBODS
Perfusute. The composition of the perfusion solution used in these experiments (HP-5) is shown in Table 1. It has the same ionic composition as the PF-3 solution used in our earIier studies (7, ll), but the other solutes difFered as follows:
TRANSPLANTATION
OF
GLYCEROL-TREATED TABLE
Composit.iorl
KIDNEYS
19
1
of HP-5
Perfnsate
Component
Component
__----__I
ContcntQ
.--_
Na+ IK+ Ca2+ Mg2+
141 4 appmx 2
3”
ClHCO,sop-
Ma.nniloI Haemaccel Czd~erlicillin
__-.-
111 mmol 17.5 g 100 mg
BPOr2-
TI ?POj__-__-.~_.~-a Contents b AsBurning
of 1 litre. SO(;l, of the t,nt,$ cnlcium
_.-
_.
1.0 be bound.
(i) HaemacceI (Hoechst) was the only coll,oid; (ii) sufficient mannitol was added to raise the final osmolality to 400 mosmol/ kg; (iii) the small amount of glucose (4 m;M) was omi.tted; (iv) papaverine was omitted because the kidneys for these experiments were first flushed with a cold washout solution containing papaverine, and further vasodiletion was considered to be unnecessary; (v) gentamicin was rcplaced by carbenicillin, since gcntamicin is nephrotoxic in some species (5 ), although we have never seen any evidence of such an effect in isolated rabbit kidneys (1). The solution was prepared by mixing equal volumes of Raemaccel infusion fluid and a stock solution containing (grams per litre) : NaCI, 5.06; KCl, 0.19; NaHCOa, 4.20; NaaHPOa, 0.17; NaHzP04, 0.12; MgS04* 7Hz0, 0.49; mannitol, 40.40; carbenicillin, 0.20. A ooncentrated gIycero1 soIution was also prepared by dissolving the salts of the stock solution in a minimum quantity of water (approxima,tely 100 ml) and bringing it up to 1 litre with Analar glycerol. After dilution with Haemaccel infusion fluid, this gave a glycerol concentration of approximately 450 g/litre. Each solution was fiRered through a 0.22-pm MiIIipore filter and was equilibrated with 5% CO2 in oxylgen to produce a pH of 7.35 -+ 0.05 before use. Animals. Now ZraJ:md albino rabbits of clitllcr sc’x weighing 2-3 kg were subjeotc:d -_ to the same anacsthctic, surgical, nud fluid
management techniques as in earlier experiments (3, 10). Briefly, anaesthesia was induced with fentanyl and haIoanisone and was maintained with nitrous oxide/oxygen, suppIemented by intravenous methohexi,tone as necessary. Dextrose-saline was administered throughout the operation, and a brisk diuresis was induced with mannitol and fruscmide. Chlorpromazine and heparin were injected just before excising the left kidney. The incision was cIosed, and nalorphine was administered to accelerate recovery. The rabbit was then kept on a warmed blanket until the second (autograft) operation. Anaesthesia for this procedure was similar, but no chlorpromazine or hcparin w‘as given. The right kidney was excised and discarded, and the perfused kidney was transplanted using end-to-side anastomosis of the renal vesseIs to the aorta and inferior vena cava. The ureter was implanted into the bladder. As the clamps were released further dextrose-saline, mannitol, and frusemide were administered. FinalIy, 25 mg of ampiciIIin was injected, nalorphine was used once more to restore consciousness, and the animal was kept on a heated blanket until fully recovered. Perfusion appara&s. Minor changes were made in the apparatus described previously (11) (see Fig. 1); the heat exchanger was a plastic blood-warming bag (Fenwal Lab#oratories) sandwiched between two hollow copper cooling platcls througJ1 which industrial &l~ulol at 9°C was circulated; :L b11b111~ trap was j~~cludetl between the hcat
20
JACOBSEN RESERVOIR SOLUTION
ET AL. OF GLYCEROL OR DILUENT
-22pm
WT
PT
FILTER
FIG. I.. Schematic diagram of the perfusion apparatus. PTl and PTZ: pressure transducers; WTl: weight transducer: M: stirrer motor: P: pump; SV: solenoid valve; TC; thermocouple; IMS: induitrial methylated spirit (ethanol j .
exchanger and the cannuIa as an additional precaution against air embolism; the kidney was suspended from a weight transducer mounted outside the cooled enclosure; the sample paint on the bypass was eliminated, and samples were taken from the distal end of the bl-pass tube; temperature was continuously recorded using a copper-constantan thermocouple with an eIectronic cold junction compensator (0mega Engineering Type MCJ-T) and a three-channel chart recorder (Rikadenki Type 1334). With this apparatus the glycerol concentration in the mixing chamber was continuously measured by monitoring its refractive index, and the necessary quantities of concentrated glycerol solution or plain perfusntc were added automatically to make the measured glycerol concentration agree with that dictated by an electronic programme. Perfusate of the
correct composition was then cooled to the desired temperature ( 10°C ) in the heat exchanger and was pumped through the kidney at a controIled renal arteria1 pressure of 40 mm Hg; a feedback control loop from the arterial pressure transducer to the perfusion pump ensured stable contro1 of perfusion pressure ( 8). Experimental procedure. Within 2 min ,of clamping the Ieft renaI artery, the excised left kidney was perfused at a pressure of 60 mm Hg with 100 ml of WFS PD washout solution at 4°C ( 13). This is a balanced salt solution with a high magnesium concentration (36 mM) t,o which glucose (167 mmol/litre) ,dextran 40 (50 g/ Iitre Rheomacrodex, Pharmacia), and papaverine (30 mg/litre) have been added. Its osmolaIity is 400 mosmol/kg. Immediately after blood washout, the kidney was attached to the perfusion apparatus
TRANSPLANTATION
OF GLYCEROL-TREATED
and was perfused with HP-5 soIution at 10°C using a pressure of 40 mm Hg. After 20 min, the programmed increase in glycerol concentration at a rate of 30 mM min-i was started. The glycerol concentration in the mixing chamber and at the sample point was checked at lo-min intervals using an Abbe! refractometer (Hilger and Watts Type M 46), and the programme was stopped when the concentration reached 2, 3, or 4 M in the mixing chamber. The cannu1.a concentration lagged by approximateIy 5 min. After 30 min, the programme of decreasing glqzerol concentration was started at the same rate and was continued until 5 litre of pIain perfusate had been added. The residual glycerol concentration ‘was always
CRYOBIOLOGY
(1978)
Transplantation
of Rabbit Kidneys Perfused Solutions at 10°C
I. A. JACOBSEN,11 2 D. E. PEGG, M. C. WUSTEkIAN,
AND
with
Glycerol
S. M. ROBINSON
Division of Cryobiology, Clinical Research Centre, Watford Road, Hamow, United Kingdom
Experiments previousIy reported from this laboratory showed that rabbit kidneys could be perfused with a 2 M solution of gIycero1 for 2 hr without apparent vascular damage or excessive weight gain (6 ). However, when the glycerol concentration was changed abruptly during glycerolisation and deglycerolisation, a concentration of 1.1 M was the highest that was comparable with unimpaired subsequent function and histology (4). Further studies showed that, if the concentration of glycerol was changed sIowly (30 mM min-l ) and about 100 mmol of mannitol was added to the perfusate, it was possible to increase the glycerol concentration to 2 M without diminishing subsequent function as measured by a normothermic perfusion assay (2, 11). This evidence that the kidney could tolerate exposure to 2 M glycerol was encouraging, since this would permit cooling to -5°C without freezing and should provide a high degree of cryoprotection during subsequent cooling. However, all the experimen,ts with 2 M glycero1 had employed dextran-containing perfusates, and in ~itru methods had been used to assess function: Therefore, it was necessary to Received
February
24,
1977.
1 Supported
by P. Carl Petersen’s Foundation. 2 Present address: Laboratory of Nephrology, Institute of Pathology, University of Odense, Uen-
mark. 18 OOll-2240/78/0151-0018$02.00/O Copyright All rights
Q 1978 by Academic Press, Inc. of reproduction in any form reserved.
confirm these re.suIts by transplantation. Preliminary experiments showed that control kidneys perfused with dextran would not support the life of graft recipients, whereas the gelatin-derived plasma exppander HaemacceI (Hoechst) was a highly satisfactory colloid ( 10). Indeed, rabbit kidneys perfused for 24 hr at 10°C with solutions containing this polypeptide material as the sole colloid have shown excelIent function ,afterwards (9). In addition, it was found that kidneys perfused for 4 hr with a Haemaccel solution containing 2% glycerol (0.22 M) wouId function as well after transplantation as glycerol-free controIs (IO). It was concluded that a Haemaccel perfusate should be suitable for the introduction and removal of gIycero1, and that complete removal of the cryoprotectant prior to transplantation wouId not be necessary. The experiments reported in this paper show that i,t is possible to perfuse rabbit kidneys with glycerol solutions up to a 3 M concentration, with 100% survival and a return to normal renal function. MATERIALS
AND
METBODS
Perfusute. The composition of the perfusion solution used in these experiments (HP-5) is shown in Table 1. It has the same ionic composition as the PF-3 solution used in our earIier studies (7, ll), but the other solutes difFered as follows:
TRANSPLANTATION
OF
GLYCEROL-TREATED TABLE
Composit.iorl
KIDNEYS
19
1
of HP-5
Perfnsate
Component
Component
__----__I
ContcntQ
.--_
Na+ IK+ Ca2+ Mg2+
141 4 appmx 2
3”
ClHCO,sop-
Ma.nniloI Haemaccel Czd~erlicillin
__-.-
111 mmol 17.5 g 100 mg
BPOr2-
TI ?POj__-__-.~_.~-a Contents b AsBurning
of 1 litre. SO(;l, of the t,nt,$ cnlcium
_.-
_.
1.0 be bound.
(i) HaemacceI (Hoechst) was the only coll,oid; (ii) sufficient mannitol was added to raise the final osmolality to 400 mosmol/ kg; (iii) the small amount of glucose (4 m;M) was omi.tted; (iv) papaverine was omitted because the kidneys for these experiments were first flushed with a cold washout solution containing papaverine, and further vasodiletion was considered to be unnecessary; (v) gentamicin was rcplaced by carbenicillin, since gcntamicin is nephrotoxic in some species (5 ), although we have never seen any evidence of such an effect in isolated rabbit kidneys (1). The solution was prepared by mixing equal volumes of Raemaccel infusion fluid and a stock solution containing (grams per litre) : NaCI, 5.06; KCl, 0.19; NaHCOa, 4.20; NaaHPOa, 0.17; NaHzP04, 0.12; MgS04* 7Hz0, 0.49; mannitol, 40.40; carbenicillin, 0.20. A ooncentrated gIycero1 soIution was also prepared by dissolving the salts of the stock solution in a minimum quantity of water (approxima,tely 100 ml) and bringing it up to 1 litre with Analar glycerol. After dilution with Haemaccel infusion fluid, this gave a glycerol concentration of approximately 450 g/litre. Each solution was fiRered through a 0.22-pm MiIIipore filter and was equilibrated with 5% CO2 in oxylgen to produce a pH of 7.35 -+ 0.05 before use. Animals. Now ZraJ:md albino rabbits of clitllcr sc’x weighing 2-3 kg were subjeotc:d -_ to the same anacsthctic, surgical, nud fluid
management techniques as in earlier experiments (3, 10). Briefly, anaesthesia was induced with fentanyl and haIoanisone and was maintained with nitrous oxide/oxygen, suppIemented by intravenous methohexi,tone as necessary. Dextrose-saline was administered throughout the operation, and a brisk diuresis was induced with mannitol and fruscmide. Chlorpromazine and heparin were injected just before excising the left kidney. The incision was cIosed, and nalorphine was administered to accelerate recovery. The rabbit was then kept on a warmed blanket until the second (autograft) operation. Anaesthesia for this procedure was similar, but no chlorpromazine or hcparin w‘as given. The right kidney was excised and discarded, and the perfused kidney was transplanted using end-to-side anastomosis of the renal vesseIs to the aorta and inferior vena cava. The ureter was implanted into the bladder. As the clamps were released further dextrose-saline, mannitol, and frusemide were administered. FinalIy, 25 mg of ampiciIIin was injected, nalorphine was used once more to restore consciousness, and the animal was kept on a heated blanket until fully recovered. Perfusion appara&s. Minor changes were made in the apparatus described previously (11) (see Fig. 1); the heat exchanger was a plastic blood-warming bag (Fenwal Lab#oratories) sandwiched between two hollow copper cooling platcls througJ1 which industrial &l~ulol at 9°C was circulated; :L b11b111~ trap was j~~cludetl between the hcat
20
JACOBSEN RESERVOIR SOLUTION
ET AL. OF GLYCEROL OR DILUENT
-22pm
WT
PT
FILTER
FIG. I.. Schematic diagram of the perfusion apparatus. PTl and PTZ: pressure transducers; WTl: weight transducer: M: stirrer motor: P: pump; SV: solenoid valve; TC; thermocouple; IMS: induitrial methylated spirit (ethanol j .
exchanger and the cannuIa as an additional precaution against air embolism; the kidney was suspended from a weight transducer mounted outside the cooled enclosure; the sample paint on the bypass was eliminated, and samples were taken from the distal end of the bl-pass tube; temperature was continuously recorded using a copper-constantan thermocouple with an eIectronic cold junction compensator (0mega Engineering Type MCJ-T) and a three-channel chart recorder (Rikadenki Type 1334). With this apparatus the glycerol concentration in the mixing chamber was continuously measured by monitoring its refractive index, and the necessary quantities of concentrated glycerol solution or plain perfusntc were added automatically to make the measured glycerol concentration agree with that dictated by an electronic programme. Perfusate of the
correct composition was then cooled to the desired temperature ( 10°C ) in the heat exchanger and was pumped through the kidney at a controIled renal arteria1 pressure of 40 mm Hg; a feedback control loop from the arterial pressure transducer to the perfusion pump ensured stable contro1 of perfusion pressure ( 8). Experimental procedure. Within 2 min ,of clamping the Ieft renaI artery, the excised left kidney was perfused at a pressure of 60 mm Hg with 100 ml of WFS PD washout solution at 4°C ( 13). This is a balanced salt solution with a high magnesium concentration (36 mM) t,o which glucose (167 mmol/litre) ,dextran 40 (50 g/ Iitre Rheomacrodex, Pharmacia), and papaverine (30 mg/litre) have been added. Its osmolaIity is 400 mosmol/kg. Immediately after blood washout, the kidney was attached to the perfusion apparatus
TRANSPLANTATION
OF GLYCEROL-TREATED
and was perfused with HP-5 soIution at 10°C using a pressure of 40 mm Hg. After 20 min, the programmed increase in glycerol concentration at a rate of 30 mM min-i was started. The glycerol concentration in the mixing chamber and at the sample point was checked at lo-min intervals using an Abbe! refractometer (Hilger and Watts Type M 46), and the programme was stopped when the concentration reached 2, 3, or 4 M in the mixing chamber. The cannu1.a concentration lagged by approximateIy 5 min. After 30 min, the programme of decreasing glqzerol concentration was started at the same rate and was continued until 5 litre of pIain perfusate had been added. The residual glycerol concentration ‘was always
