Glycerol used as a cryoprotectant in subzero preservation of rabbit kidneys

CaYOmOLOGY 12, 123--129 ( 1 9 7 5 )

Glycerol used as a Cryoprotectant in Subzero Preservation of Rabbit Kidneys I. A. J A C O B S E N , E. K E M P , AND H. S T A R K L I N T

Laboratory of Nephrology and Institute of Pathalogy, Odense University Hospital, DK-5000 Odense, Denmark

The recent development of techniques from the available endoeellular eryoprofor detailed immunological diagnosis .and teetants. for specific immunosuppressive treatment The investigation is divided into two (such as enhancement) prior to organ sections. First, an estimation of the highest transplantation has emphasized the need concentration of glycerol in the perfusion for preservation methods which allow the fluid which does not in itself produce tissue storage of donor organs for longer periods damage. Second, the application of glycerol --weeks or months--and ideally in .organ in this concentration to the preservation of kidneys at temperatures below 0°C, with banks. On the basis of already established subsequent transplantation and functional techniques for the storage of cells and investigations. cell suspensions by freezing with eryoMETHODS protectants, attempts have been made using similar techniques to freeze whole isolated Animal Material and Operation Techniques organs, but so far without convincing longThe preservation methods investigated term function after thawing ( 1, 7). were evaluated by renal autografting in In recognition of the considerable techrabbits. nical problems associated with freezing and Rabbits of both sexes weighing between especially with thawing, the present study 2 and 4 kg were used in all experiments. is concerned with the use of eryoproteetants Hypnorm (fluanisone 1.0 mg/ml + fentanyl in supercooling, with the preliminary pur- 0.2 mg/ml) 0.5 ml/kg b,odyweight was pose .of storing isolated kidneys at a temgiven intramuscularly 15 rain before operaperature just below 0°C, partly in order to tion. Anesthesia was initiated and sustained gain experience with the use of cryopro- with intravenous Nembutal (pentobarbiteetants for later freezing experiments, and tone sodium) together with nitrous oxide partly to see whether the low storage tem- and oxygen via an open mask. perature in itself can extend the preservaLeft kidneys were isolated and excised tion period without significant loss of for preservation through an abdominal, viability. transperitoneal midline incision. The renal After disappointing results with dimethyl artery and vein were divided close to the sulphoxide (DMSO) in our hands, glycerol aorta and the inferior vena cava, respecwas chosen (as suggested by others (11)) tively. The ureter was transected 1-2 cm from the bladder. Heparin 400 units and R e c e i v e d N o v e m b e r 5, 1974. 123 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Lasix (furosemide) 4 mg were given intravenously approx 15 min before division of the renal vessels. The kidneys were implanted by anastomosis of the renal artery and vein end-toside with the aorta and the inferior vena cava, respectively, and the ureter was implanted in the bladder over a nylon stent ad modum Dunn (2). Contralateral nephrectomy was performed immediately in all cases.

Biochemistry Blood samples were taken daily from the central artery of the ear and serum creatinine (s-ereatinine) concentration determined by the direct alkaline picrate method. Daily diureses were collected and in the first post-operative day analysed for creatinine for subsequent calculation of endogenous creatinine clearance.

Perfus!ons (1) Flushing with increasing concentrations of glycerol. Three peffusion fluids with increasing glycerol concentrations were used to estimate the highest concentration usable in kidney perfusates. The perfusion fluids were mixtures of a balanced salt solution with an approximated intracellular ion composition (a modified Collins solution (Table 1)) and 5, 10, or 20% glycerol. The function of kidneys flushed with these fluids was TABLE 1 COMPOSITION OF PERFUSION FLUID BEFORI4 ADDITION OF GLYCEROL (MODIFIED COLLINS SOLUTION) K+ Na + H2PO4HPO4 ~C1HCOaGlucose tIeparin Procaine Papaverine

] 15 10 15 43 15 10 25 5000 10 40

mmole/liter minole/liter mmole/liter inmole/liter mmole/liter mmole/liter g/liter enh/liter mg/liter mg/liter

compared with that of kidneys flushed with the modified Collins solution alone. As soon as possible after nephrectomy (warm isehaemia on average 3 rain) each kidney was flushed with the perfusion fluid in question for 10 min ,at a hydrostatic pressure of 100 cm water and +4°C. The kidneys were then stored in a refrigerator for 45 min at +4°C and after 5 min reflushing with the same fluid, in order to obtain a sample for enzyme measurements (not discussed in this paper) reimplanted. Endogenous creatinine clearance in the first postoperative day, as well as the rise in s-creatinine in the same period, were used as measures of graft function in this part of the study. This initial graft function =after perfusion with glycerol containing fluids was eompared with the kidney function after perfusion without glycerol. First-day ereatinine clearance in a group of 10 unilateral nephreetomised rabbits was used as a control. The animals were killed 24 hr after transplantation, tissue samples removed from the grafts for histological examination, and the urine in the bladder removed and pooled with the collected urine for analysis. At least 10 transplantations were carried out in each group. (2) Continuous perfusion with glycerol-

containing peffusate and subzero preservation. On the basis of the results of the transplantation experiments described above, a group of kidneys was stored at subzero temperature using the following preservation method. Immediately after nephrectomy (average warm ischaemia 3 rain) the left kidneys were washed out with the modified Collins solution (Table 1) for 10 rain at a hydrostatic pressure of 100 cm water and +4°C. The kidneys were then perfused continuously for 2hr at 5-7°C with glycerolcontaining perfusion fluid to ensure an adequate distribution of glycerol in the tissue water.

SUBZERO KIDNEY PRESERVATION The kidney was placed in a reservoir containing perfusion fluid and connected to the perfusion system through a canula. During perfusion, the fluid was cooled to 5-7°C and oxygenated by surface diffusion from an atmosphere containing 99% oxygen and 1% carbon dioxide, which gave oxygen tensions varying between 150 and 400 mm Hg. Fluid was pumped from the reservoir by a peristaltic pump (Gambro or Watson-Marlow MHRE 100) through a filter (Leukopak leucocyte filter, Fenwal Laboratories or Acropor 200 (0.2 /~m) + Type A glass fibre filter, Gelman Instrument Company) and an air trap back to the renal artery. Venous etqquent was allowed to flow freely into the reservoir. Perfusion pressure was measured as close as possible to the renal artery using a pressure transducer (P 23 Db, Statham Inc.). Some of the kidneys were perfused with pulsatile flow (Gambro Roller Pumb) at a systolic pressure of 40-50 mm Hg, and amplitude 3-5 mm Hg. The perfusion pressure was held constant during the whole period of perfusion. In the remaining experiments nonpulsatile flow was used (Watson-Marlow Ftow Inducer), perfusion pressures being held constant at 40 mm Hg using a feed back control unit (10). Two slightly different glycrol-containing perfusion fluids were used (Table 2). Both consisted of a balanced salt solution with a n approximated intracellular ion composition. Bovine serum albumin was used as a colloid. Both fluids contained 10% glycerol, heparin, papaverin, glucose and in some experiments penicillin and hydrocortisone were added, pH of the perfusion fluids was adjusted to 7.0-7.2 (37°C) before use. Flow rates with the perfusion pressure used ranged from 0.7 to 1.7 ml per g kidney per rain. After continuous perfusion the kidneys were stored by simple hypothermia at I°C for 19-22 hr. They were then reperfused for 5 min at a hydrostatic pressure of 100

125 TABLE 2

PERFUSION FLUIDS FOR CONTINUOUS PERFUSION

Odense XII a Electrolyte solution shown in Table 1 Bovine albumin (Stayne Laboratories Ltd.) Glycerol Glucose Heparin P~paverine Procaine

1000 ml 35 g 100 g 25 g 5000 IU 40 mg 10 mg

Odense XII b Electrolyte solution shown 1000 ml in Table 1 Bovine albumin, fraction V 45 g (Sigma Chemical Company) Glycerol 100 g Glucose 12.5 g Heparin 10000 IU Papaverine 40 mg Penicillin 1.25 million IU Hydrocortisone 125 mg em water and +4°C with the glycerolcontaining perfusion fluid (but without albumin), immediately prior to implantation, (this reperfusion was made in order to ,obtain samples for enzyme measurements, not discussed in this article). S-creatinine was determined daily as a measure of kidney function. RESULTS

(1) Flushing with Increasing concentrations of Glycerol ( a ) Graft [unction. Four groups of 10, 12, 12, and 10 kidneys were flushed with fluids containing respectively 0, 5, 10, and 20% glycerol (Table 3). The mean endogenous creatinine clearance in the first postoperative day of the group of kidneys perfused without glycerol was 2.0 ml/min, corresponding to approximately 50% of the "physiological" first-day clearance of unilateral nephrectomised controis. Creatinine clearance in the groups perfused with 5 and 10% glycerol was 1.3 ml/minute in both eases, which is not significantly different from the group without

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JACOBSEN, KEMP AND STARKLINT TABLE 3 FUNCTION IN THE ~'IRST POSTOPERATIVE DAY OF I4~IDNEYS PERFUSED WITH FLUIDS CONTAINING INCREASING CONCENTRATIONS OF GLYCEROL Group

No. of kidneys

Glycerol content in perfusate fluid (w/v %)

Mean s-creatinine increase 0~mole/liter)

Mean endogenous creatinine clearance (ml/min)

Controls" 1 2 3 4

10 10 12 12 10

-0 5 10 20

36 289 323 275 525

5.5 2.0 1.3 1.3 0.3

" Unilateral nephrectomy. glycerol (p > 0.10). Conversely, the function of kidneys in Group 4 (perfused with 2070 glycerol) was significantly poorer, corresponding to a mean creatinine clearance of 0.3 ml/min (p < 0.01). In agreement with these observations, it can be seen in Table 3 that the mean s-creatinine increase in the first postoperative day in Groups 1, 2, and 3 is between 275 and 323 ~mole/liter without significant differences (p > 0.10); while the mean increase in the group perfused with 20% glycerol is 525 #mole/liter which is significantly higher than in the o.ther groups (p < 0.01). (b) Histology. In the group perfused without glycerol slight changes were seen in the most peripheral part .of the cortex. These changes consisted of a narrow subcapsular zone of tubuli with atrophic epithelium together with slight dystrophic calcifications. In the groups perfused with glycerolcontaining perfusion fluids more pronounced changes were seen. Thus in the group perfused with 5% glycerol the zone with atrophic tubulus epithelium extended deeper into the cortex, and in one case total cortical necrosis was seen. Small elongated infarctions were seen in two cases. The changes in the kidneys perfused with 10% glycerol could not be distinguished quantitatively or qualitatively from those in the kidneys perfused with 5%. In contrast to the other groups, serious

damage was seen in the groups perfused with 2070 glycerol. Thus, in four cases there was cortical necrosis, which in one kidney was combined with extensive infarction of the medulla. In this case (as in all others) the deepest part of the papilla was unaffected. The remaining kidneys in this group showed more or less extensive infarctions of both .cortex and medulla. The histological findings agree with the results of the functional evaluations. The conclusion drawn from the described results is that glycerol contents up to 10% give preservation as good as the described modification of Collins fluid, while the initial graft function after preservation with 20% glycerol is significantly poorer.

(2) Subzero Preservation On the basis of this conclusion, perfusion fluids containing 107o glyerol were used in kidney preservation including storage below 0°C. The perfusion fluids described in Table 2 have an osmolality of 1340 mosm/kg with a corresponding freezing point of -2.49°C. According to earlier .observations (9) 2 hr perfusion at the temperature used (5°C) gives rise to equilibration of the glycerol in the perfusate with 75% of the tissue water. Thus there will be sufficient glycerol diffusion into the kidney's fluid phase for the organ to be co.oled to I°C without the risk of ice crystal formation, so that the rate of cooling is not critical.

SUBZERO KIDNEY PRESERVATION A total of 20 kidneys were preserved and transplanted according to the described procedure. All the kidneys were deeply cyanotic after revaseularisation but resumed after a few minutes a normal eolour and ~one. All the kidneys also produced a certain quantity of urine during operation. Fifteen of these grafts had no postoperative lifesustaining function. At autopsy six showed thromboses of the renal vessels or ureteral obstruction with eoagulated blood in the whole length of the ureter. No mechanical explanation for absence of function was found in the remaining nine .cases. Seven .of the nonfunctioning kidneys were examined histologically and severe ischaemie damage in the form of extensive infarctions of both cortex and medulla was seen. Five kidneys had life-sustaining function after subzero preservation and transplantation. Perfnsion parameters for these kidneys (given in Table 4) were no different from the parameters in the larger group of nonfunctioning kidneys. As shown in Fig. 1, s-ereatinine increased slightly after excision of the left kidney. Transplantation and eontralateral nephreetomy was followed, in the first two to six postoperative days, by a considerable increase in s-ereatinine which then fell indicating graft function. Two of the animals died in severe gastroenteritis with decreasing s-ereatinine on the 12th and 14th postoperative days, respectively. Two survived with only slightly increased s-creatinine until sacrificed three months after transplantation, and the last rabbit survived with slightly increased screatinine until four weeks after when total blockage of the ureter occurred. Four .of the surviving kidneys were exeamined histologically and showed moderate to severe calcification in both cortex and medulla. Considerable interstitial fibrosis was observed in connexion with

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reduction of function. This result agrees with other investigations (3, 6). The tissue-damaging effect of high glycerol concentrations is probably related to excessive osmotic changes (intracellular dehydration) resulting from the contact of cells with the strongly hyperosmotic perfusion fluid with cell shrinkage and increased membrane permeability to follow

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calcification. No infarctions were seen. A few glomeruli in areas with fibrosis were destroyed, but in general the glomeruli appeared normal in light microscopic examination. DISCUSSION

In this study an attempt was made to use a penetrating cryoproteetant in a simple system for the storage of kidneys at a temperature just below 0°C. Glycerol was chosen as eryoprotectant because with the technique employed it was adequately distributed throughout the kidney. Unlike DMSO, glycerol does not damage capillary membranes with resulting increases in perfusion resistance and kidney weight (9). In addition, studies in our laboratory (8), using L D H release as an index of tissue damage during preservation, have shown that enzyme release during perfusion with glycerol is signifleantly lower than during perfusion with DMSO. In the experimental transplantation of glyeerol-treated kidneys it was found that fluids containing 10~o glycerol eaused a reduction of kidney function of the same size as preservation with a modified Collins fluid using the same technique; while 20~o glycerol resulted in a more pronounced

This phenomenon could possibly be avoided by gradually increasing the glycerol concentration in the perfusion fluid during equilibration [as suggested by Farrant (4) ]. Only a small number of the kidneys which had been perfused with 107o glycerol over a longer period and stored at subzero temperature, had an immediate life-sustaining function. A characteristic of all the subzero preserved kidneys has been, that bloodflow immediately after revaseularisation was greatly reduced, resulting in missing or greatly reduced pulsation of the renal artery and a cyanotic miseolouration of the kidneys' surface. After a few minutes the kidneys resumed a normal eolour and tone, and pulsation could be observed in the renal artery. The increased resistance to flow may be explained by the extreme intraeellular overhydration which follows the equilibration over the cell membrane of the preserved kidney's strongly hypertonie intraeellular phase with the normotonie plasma of the animal. This explanation is supported by the observation of increasing kidney size and tone immediately after revascularisation. Quantitatively, the result of the present study (5 functioning kidneys out of 20) do not differ convincingly from the results of freezing experiments undertaken by Halasz (7), in whieh 4 out of 38 kidneys functioned after freezing with 10~0 glycerol to minus 50°C. This could indicate that loss of viability occurs mainly during distribution and elimination of the eryoproteetant used.

SUBZERO KIDNEY PRESERVATION In conclusion it is our impression that glycerol must be added and eliminated gradually, if it is to be used with greater success in organ preservation at subzero temperatures. SUMMARY Perfusion fluids containing different concentrations of glycerol were examined using autotransplantation of kidneys in rabbits. The isolated left kidney was flushed with the perfusion fluid in question for 10 rain, stored for 45 min in a refrigerator at + 4 ° C , and finally reperfused for 5 min with the same fluid immediately before transplantation. Graft function was estimated by measuring serum creatinine and creatinine clearance in the first 24 hr after transplantation. Three perfusion fluids eontaining 5, 10, and 20% of glycerol were examined. The function of kidney grafts flushed with fluids containing up to 10% glycerol did not differ significantly from the function of kidneys preserved with a modified Collins solution using the same procedure; whereas perfusion with fluids containing 20% glycerol resulted in a large increase in serum creatinine and a smaller creatinine clearance. Following these results, a group of kidneys were perfused continuously with a fluid containing 10% glycerol for 2 hr, stored b y simple hypothermia at - I ° C for 19-22 hr, and then transplanted. Five out of 20 animals survived. Serum ereatinine showed a temporary increase after transplantation but returned subsequently to almost normal.

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ACKNOWLEDGMENT Supported by The Danish Medical Research Counsil. REFERENCES 1. Dietzman, R. H., Rebelo, A. E., Graham, E. F., Grabo, B. G., and Lillehei, R. C. Long-term functional success following freezing of canine kidneys. Surgery 74, 181-189 (1973). 2. Dunn, D. Department of Surgery, University of Cambridge. Personal communication. 3. Egedahl, R. H., and Harris, R. In vivo tolerance of canine kidneys to glycerol perfusions. Transplant. Bu'll. 6, 110-112

(1959). 4. Farrant, J. Mechanism of cell damage during freezing and thawing and its prevention. Nature (London) 205, 1284 (1965). 5. Farrant, J. and Woolgar, A. E. Human red cells under hypertonie conditions; A system for investigating freezing damage. Cryobiology 9, 9-15 ( 1972 ). 6. Halasz, N. A., Seifert, L. N., and Orloff, M. J. Whole organ preservation. I Organ perfusion studies. Surgery 60, 368-372 (1966). 7. Halasz, N. A., Rosenfield, H. A., Orloff, M. J., and Seifert, L. N. Whole organ preservation. II Freezing studies. Surgery 61, 417421 (1967). 8. Kemp, E., Jacobsen, I. A., and Amtrup, F. LDH-release into kidney perfusates as a parameter for viability of isolated and preserved kidneys. 11th Annual meeting of The International Society for Cryobiology, London 1974. 9. Pegg, D. E. Perfusion of rabbit kidneys with cryoproteetive agents. Cryobiology 9, 411419 (1972). 10. Pegg, D. E., and Green, C. J. Renal preservation by hypothermic perfusion. Cryobiology 10, 56-66 (1973). 11. Pegg, D. E. Theory and experiments towards subzero organ preservation. "Organ Preservation," pp. 108-118, Churchill-Livingstone, London, 1973.