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Preservation of kidneys in vitro
Preservation
of Kidneys
in Vitro*
Value of Hypothermia LAWRENCE E. STEVENS, M.D., FREDERICK C. SWENSON, M.D., AND JAN S. FREEMAN, M.D.,
Salt Lake City, Utah
From the Department of Surgery, University of Utah College of Medicine, Salt Lake City, Utah. This study was supported in part by U.S.P.H.S. Grant HE10253-01.
METHOD A sterile system (Fig. 1 and 2) was devised for the perfusion of the isolated organs in this study. The perfusate was circulated through the system by a coronary perfusion pump (model 1420t) with a 370 degree single-arm De Bakey roller which delivered a pulsatile flow (fluctuating 25 mm. Hg between systolic and diastolic levels) at adjustable rates. The renal artery of the isolated organ was cannulated with a tapered plastic catheter (model U 510)t which was connected to the forward flow line from the pump. The arterial pressure was monitored with an anaeroid gauge, and this pressure was maintained at 110 f 10 nun. Hg by varying the rate of the pump. The renal vein was cannulated with a catheter similar to that used in the artery. The renal vein pressure was maintained at 6 em. Hz0 by elevation of the venous line and by venting the tubing. Effluent venous perfusate passed through a glass wool filter, then through a bubble (model U 340$) (02 flow at 3 to 4 L./min.), and finally back to the pump for recirculation. The ureter was cannulated with plastic tubing and the kidney placed in a sterile gIass container. All urinary flow was returned to the perfusate, except for those samples taken at intervals during the study. Temperatures of the perfused kidney were controlled with a heat exchanger in the arterial line and by refrigeration of the box and tubing in the case of the hypothermic group. The perfusate was made up of autologous whole blood diluted with equal parts of a balanced salt solutiont with the addition of 100 mg. of procaine, 100 mg. of heparin, 2 million units of aqueous penicillin, and 0.72 gm./L. of glucose.
HE NEED for preserving organs becomes pressing as clinical programs for renal homotransplantation expand. Suitable methods of preservation would make possible the widespread use of organs from cadaveric donors and would allow sufficient time for tissue typing, pretransplant preparation of the recipient, and pretreatment of the isolated organ prior to implantation. Two methods of preserving kidneys have met recently with success for periods up to twentyfour hours, and certainly warrant additional investigation. The first of these methods of preservation utilizes pressurized oxygenation of the organ and has been described by Manax et al. [1] and Nakamoto, Straffon, and Kolff [Z]. The second method of preservation has featured isolated perfusion of the organ, as described by Humphries et al. [3] and Cleveland et al. [PI. Both of these methods include cooling of the organ, which parallels the clinical observations of a number of authors [&7] that cooling of the organ is advantageous for the short interval between excision of the organ from the donor and implantation within the recipient. This study was undertaken to confirm the value of hypothermia in the prolonged perfusion of organs and to evaluate how much protection the isolated kidney derives from the cooling process.
T more
* Presented at the Eighteenth
t Med-Science Electronics, St. Louis, Missouri. $ Travenol Laboratories, Inc., Los Angeles, California.
Annual Meeting of the Southwestern April 18-21, 1966. 788
Surgical Congress, Las Vegas, Nevada,
American Journal
of Surgery
Preservation
of Kidneys
in Vitro
ZR RESULTS
FIG. 1. The isolated perfusion
system used in the study.
Twentv-four kidneys were removed from dogs and perfused-in the system just described. One half the organs (group A) were perfused at normal temperatures (38’ c.) and the other half (group B) at hypothermic temperatures (10’ c.). In several instances it was possible to pair the two kidneys of one animal, placing one in group A and the contralateral organ in group B. The organs were perfused as long as renal arterial and urinary flows could be maintained, and the length of this perfusion period was recorded. Urine secretion and arterial flow rates were recorded, and determinations were made of perfusate and urine electrolyte values. Estimation of oxygen utilization by the perfused organ was made by comparing differences in color between the arterial and venous lines. Glucose utilization was estimated by recording the amount of glucose added during perfusion to keep the urinary glucose at a I+ value (Urostix@) or greater, and the change in weight of the organ during the perfusion interval was recorded. Histologic studies of the perfused organ were made from biopsy specimens of the kidney taken at varying stages of the perfusion. Observations were made regarding the time of appearance and the degree of hemolysis in the perfusate solution. Vol. 112. November
1966
Comparisons between the two groups of perfused kidneys revealed several significant differences. The mean length of the perfusion period was eighteen hours for group A and forty-eight hours for group B. Rates of arterial flow were lower by half in the cooled kidneys (group B) for the first twelve hours, which was reflected as an increased resistance, but continued at this adequate level many hours after arterial blockage forced cessation of perfusion in group A. (Fig. 3.) Diuresis occurred in both groups of organs but appeared later and was less marked in group B. (Fig. 3.) The urine potassium excretion values were high at first in the normothermic group but rapidly fell to the perfusate potassium levels within six hours. In contrast, the potassium values in the urine from organs in group B, although lower at first, persisted above the perfusate levels for intervals beyond thirty hours. (Fig. 4.) Interstitial edema and hemorrhage appeared in the glomeruli and tubules after four hours of perfusion, as revealed in the histologic sections of organs from group A, and grew progressively more severe, while such changes began only after twelve hours in group B and did not reach the severity of the changes in group A even after forty-eight hours of perfusion. (Fig. 5, 6, and 7.) Perfusate hemolysis occurred in both groups of organs but appeared after ten hours of perfusion in group A and after eighteen hours in group B.
FIG. 2. The cannulated, perfusion box.
isolated kidney in the sterile
Stevens,
730
E
Swenson,
and Freeman
0 0
3
6
9
12 15 16 HOURS OFPERFUSION
FIG.
21
3. Resistance and urine flow in group A (normothermic) pothermic) organs.
Appreciable color difference (light red versus dark red) was noted between the perfusate in the arterial and in the venous catheters in organs under perfusion from group A, but no arteriovenous color difference was evident in those organs of group B after cooling. The kidneys from group A required 0.06 gm. of glucose per hour to keep the urine glucose 1 + or greater during perfusion, whereas those organs from group B required no additional glucose during the entire period of perfusion to maintain a l+ glycosuria. COMMENTS
The temperature of 10’~. was selected for the hypothermic organs of this study (group B) because of favorable flow characteristics at this temperature in pilot studies of our circuit before beginning the present study. At temperatures below 5’~. sludging and freezing of the perfusate occurred in the refrigerated coils while, in contrast, free flow of the perfusate continued for long periods at 10’~. The rates of reaction of most tissue enzymatic systems are known to be markedly that slowed at ~O’C., and we anticipated prolonged preservation of an organ might well be as effective at this temperature as at lower temperatures. The arterial pressure in the perfusion circuit of 110 mm. Hg was arbitrarily selected as a value representing the lower margin of normal
24
27
30
and group B (hy-
for the dog’s kidney. Edema in the perfused organ seems to depend, in part, upon renal arterial pressures, and lower pressures seemed to minimize the tendency toward edema. The level of 6 cm. Hz0 that was used for the renal venous pressure in our circuit falls within the normal range in the dog and was therefore selected for use in our system. The reason for such marked diuresis (Fig. 3) in both groups is not clear, but two possible explanations are advanced : 1. Early metabolism of the antidiuretic hormone (ADH), which was present in the perfusate from the autologous whole blood, may have released the organ from ADH control after a short period. 2. The lower osmolality of the dilute perfusate may have prevented reabsorption of filtered water from the distal tubule at normal rates. Our data do not yield evidence that one or both of these possibilities are operative. The ultimate test for any method of preserving organs is, of course, whether an organ preserved by that method can function sufficiently well upon implantation to maintain good health for the animal. Observations of function were not made after implantation of the organs perfused in this study, but rather the organs were perfused as long as possible until arterial and urinary blockage occurred. Implantation studies utilizing the system American
Journal
of Stm!ery
Preservation
of Kidneys
x31
in V&o
HOURS OF PERFUSiON
FIG. 4. Rates of excretion of sodium and potassium during the study.
5 FIG. 5. Histologic FIG. 6. Appearance
6
appearance of a kidney from group A (normothermic) of the contralateral
described are now in progress reported later.
hours of hypothermic
and will be
SUMMARY AND CONCLUSIONS
The effect of hypothermia upon function and survival of the dog’s kidney under the conditions of isolated perfusion was studied. Comparisons between kidneys perfused at normal temperatures (38”c.) and hypothermic temperatures (1O’C.) reveal significant preser1966
after twenty-three
kidney from group B (hypothermic)
FIG. 7. Same kidney as in Figure 6 after forty-eight
Vol. 112, November
7 hours of perfusion
after twenty-four
hours of perfusion.
perfusion.
vation of function and prolongation of perfusion in the organs protected by hypothermia. We conclude that hypothermia is an effective tool in the preservation of organs. We wish to thank Dr. De Witt Hunter, Director of Clinical Laboratories at the University of Utah Medical Center, Salt Lake City, Utah, for his valuable assistance in this study. The technical assistance
Acknowledgment:
732
Stevens,
Swenson,
and Freeman
of Mr. Gordon Van Ballegooie is also gratefully acknowledged. 4. REFERENCES
1. MANAX, W. G., BLOCH, H. H., LONGERBEAM,J. K., and LILLEHEI, R. C. Successful 24-hour in vitro preservation of canine kidneys by the combined use of hyperbaric oxygenation and hypothermia. Surgery, 56: 275, 1964. 2. NAKAMOTO,S., STRAFFON, R. A., and KOLFF, W. J. Human renal homotransplantations with cadaver kidneys. J.A.M.A., 192: 102, 1965. 3. HUMPHRIES, A. L., JR., RUSSEL, R., OSTAFIN, J.,
5.
6. 7.
GOODRICH,S. M., and MORETZ, W. H. Successful reimplantation of canine kidney after twenty-four hours storage. Surgery, 54: 136, 1963. CLEVELAND, R. J., LEE, H. M., PROUT, G. R., and HUME, D. M. Preservation of the cadaver kidney for renal homotransplantation in man. Surg. Gynec. & Obst., 119: 991, 1964. DEMPSTER, W. J., KOUNTZ, S. L., and JOVANIC, M. Simple kidney storage technique. &it. M. J., 1: 407, 1964. STARZL, T. E. Experience in Renal Homotransplantation. Philadelphia, 1965. n’. B. Saunders Co. CALNE, R. Y., PEGG, E. D., PRYSE-DAVIES, J., and BROWN, F. L. Renal preservation by ice cooling. &it. M. J., 2: 651, 1963.
American Journal
of Sur~cry
of Kidneys
in Vitro*
Value of Hypothermia LAWRENCE E. STEVENS, M.D., FREDERICK C. SWENSON, M.D., AND JAN S. FREEMAN, M.D.,
Salt Lake City, Utah
From the Department of Surgery, University of Utah College of Medicine, Salt Lake City, Utah. This study was supported in part by U.S.P.H.S. Grant HE10253-01.
METHOD A sterile system (Fig. 1 and 2) was devised for the perfusion of the isolated organs in this study. The perfusate was circulated through the system by a coronary perfusion pump (model 1420t) with a 370 degree single-arm De Bakey roller which delivered a pulsatile flow (fluctuating 25 mm. Hg between systolic and diastolic levels) at adjustable rates. The renal artery of the isolated organ was cannulated with a tapered plastic catheter (model U 510)t which was connected to the forward flow line from the pump. The arterial pressure was monitored with an anaeroid gauge, and this pressure was maintained at 110 f 10 nun. Hg by varying the rate of the pump. The renal vein was cannulated with a catheter similar to that used in the artery. The renal vein pressure was maintained at 6 em. Hz0 by elevation of the venous line and by venting the tubing. Effluent venous perfusate passed through a glass wool filter, then through a bubble (model U 340$) (02 flow at 3 to 4 L./min.), and finally back to the pump for recirculation. The ureter was cannulated with plastic tubing and the kidney placed in a sterile gIass container. All urinary flow was returned to the perfusate, except for those samples taken at intervals during the study. Temperatures of the perfused kidney were controlled with a heat exchanger in the arterial line and by refrigeration of the box and tubing in the case of the hypothermic group. The perfusate was made up of autologous whole blood diluted with equal parts of a balanced salt solutiont with the addition of 100 mg. of procaine, 100 mg. of heparin, 2 million units of aqueous penicillin, and 0.72 gm./L. of glucose.
HE NEED for preserving organs becomes pressing as clinical programs for renal homotransplantation expand. Suitable methods of preservation would make possible the widespread use of organs from cadaveric donors and would allow sufficient time for tissue typing, pretransplant preparation of the recipient, and pretreatment of the isolated organ prior to implantation. Two methods of preserving kidneys have met recently with success for periods up to twentyfour hours, and certainly warrant additional investigation. The first of these methods of preservation utilizes pressurized oxygenation of the organ and has been described by Manax et al. [1] and Nakamoto, Straffon, and Kolff [Z]. The second method of preservation has featured isolated perfusion of the organ, as described by Humphries et al. [3] and Cleveland et al. [PI. Both of these methods include cooling of the organ, which parallels the clinical observations of a number of authors [&7] that cooling of the organ is advantageous for the short interval between excision of the organ from the donor and implantation within the recipient. This study was undertaken to confirm the value of hypothermia in the prolonged perfusion of organs and to evaluate how much protection the isolated kidney derives from the cooling process.
T more
* Presented at the Eighteenth
t Med-Science Electronics, St. Louis, Missouri. $ Travenol Laboratories, Inc., Los Angeles, California.
Annual Meeting of the Southwestern April 18-21, 1966. 788
Surgical Congress, Las Vegas, Nevada,
American Journal
of Surgery
Preservation
of Kidneys
in Vitro
ZR RESULTS
FIG. 1. The isolated perfusion
system used in the study.
Twentv-four kidneys were removed from dogs and perfused-in the system just described. One half the organs (group A) were perfused at normal temperatures (38’ c.) and the other half (group B) at hypothermic temperatures (10’ c.). In several instances it was possible to pair the two kidneys of one animal, placing one in group A and the contralateral organ in group B. The organs were perfused as long as renal arterial and urinary flows could be maintained, and the length of this perfusion period was recorded. Urine secretion and arterial flow rates were recorded, and determinations were made of perfusate and urine electrolyte values. Estimation of oxygen utilization by the perfused organ was made by comparing differences in color between the arterial and venous lines. Glucose utilization was estimated by recording the amount of glucose added during perfusion to keep the urinary glucose at a I+ value (Urostix@) or greater, and the change in weight of the organ during the perfusion interval was recorded. Histologic studies of the perfused organ were made from biopsy specimens of the kidney taken at varying stages of the perfusion. Observations were made regarding the time of appearance and the degree of hemolysis in the perfusate solution. Vol. 112. November
1966
Comparisons between the two groups of perfused kidneys revealed several significant differences. The mean length of the perfusion period was eighteen hours for group A and forty-eight hours for group B. Rates of arterial flow were lower by half in the cooled kidneys (group B) for the first twelve hours, which was reflected as an increased resistance, but continued at this adequate level many hours after arterial blockage forced cessation of perfusion in group A. (Fig. 3.) Diuresis occurred in both groups of organs but appeared later and was less marked in group B. (Fig. 3.) The urine potassium excretion values were high at first in the normothermic group but rapidly fell to the perfusate potassium levels within six hours. In contrast, the potassium values in the urine from organs in group B, although lower at first, persisted above the perfusate levels for intervals beyond thirty hours. (Fig. 4.) Interstitial edema and hemorrhage appeared in the glomeruli and tubules after four hours of perfusion, as revealed in the histologic sections of organs from group A, and grew progressively more severe, while such changes began only after twelve hours in group B and did not reach the severity of the changes in group A even after forty-eight hours of perfusion. (Fig. 5, 6, and 7.) Perfusate hemolysis occurred in both groups of organs but appeared after ten hours of perfusion in group A and after eighteen hours in group B.
FIG. 2. The cannulated, perfusion box.
isolated kidney in the sterile
Stevens,
730
E
Swenson,
and Freeman
0 0
3
6
9
12 15 16 HOURS OFPERFUSION
FIG.
21
3. Resistance and urine flow in group A (normothermic) pothermic) organs.
Appreciable color difference (light red versus dark red) was noted between the perfusate in the arterial and in the venous catheters in organs under perfusion from group A, but no arteriovenous color difference was evident in those organs of group B after cooling. The kidneys from group A required 0.06 gm. of glucose per hour to keep the urine glucose 1 + or greater during perfusion, whereas those organs from group B required no additional glucose during the entire period of perfusion to maintain a l+ glycosuria. COMMENTS
The temperature of 10’~. was selected for the hypothermic organs of this study (group B) because of favorable flow characteristics at this temperature in pilot studies of our circuit before beginning the present study. At temperatures below 5’~. sludging and freezing of the perfusate occurred in the refrigerated coils while, in contrast, free flow of the perfusate continued for long periods at 10’~. The rates of reaction of most tissue enzymatic systems are known to be markedly that slowed at ~O’C., and we anticipated prolonged preservation of an organ might well be as effective at this temperature as at lower temperatures. The arterial pressure in the perfusion circuit of 110 mm. Hg was arbitrarily selected as a value representing the lower margin of normal
24
27
30
and group B (hy-
for the dog’s kidney. Edema in the perfused organ seems to depend, in part, upon renal arterial pressures, and lower pressures seemed to minimize the tendency toward edema. The level of 6 cm. Hz0 that was used for the renal venous pressure in our circuit falls within the normal range in the dog and was therefore selected for use in our system. The reason for such marked diuresis (Fig. 3) in both groups is not clear, but two possible explanations are advanced : 1. Early metabolism of the antidiuretic hormone (ADH), which was present in the perfusate from the autologous whole blood, may have released the organ from ADH control after a short period. 2. The lower osmolality of the dilute perfusate may have prevented reabsorption of filtered water from the distal tubule at normal rates. Our data do not yield evidence that one or both of these possibilities are operative. The ultimate test for any method of preserving organs is, of course, whether an organ preserved by that method can function sufficiently well upon implantation to maintain good health for the animal. Observations of function were not made after implantation of the organs perfused in this study, but rather the organs were perfused as long as possible until arterial and urinary blockage occurred. Implantation studies utilizing the system American
Journal
of Stm!ery
Preservation
of Kidneys
x31
in V&o
HOURS OF PERFUSiON
FIG. 4. Rates of excretion of sodium and potassium during the study.
5 FIG. 5. Histologic FIG. 6. Appearance
6
appearance of a kidney from group A (normothermic) of the contralateral
described are now in progress reported later.
hours of hypothermic
and will be
SUMMARY AND CONCLUSIONS
The effect of hypothermia upon function and survival of the dog’s kidney under the conditions of isolated perfusion was studied. Comparisons between kidneys perfused at normal temperatures (38”c.) and hypothermic temperatures (1O’C.) reveal significant preser1966
after twenty-three
kidney from group B (hypothermic)
FIG. 7. Same kidney as in Figure 6 after forty-eight
Vol. 112, November
7 hours of perfusion
after twenty-four
hours of perfusion.
perfusion.
vation of function and prolongation of perfusion in the organs protected by hypothermia. We conclude that hypothermia is an effective tool in the preservation of organs. We wish to thank Dr. De Witt Hunter, Director of Clinical Laboratories at the University of Utah Medical Center, Salt Lake City, Utah, for his valuable assistance in this study. The technical assistance
Acknowledgment:
732
Stevens,
Swenson,
and Freeman
of Mr. Gordon Van Ballegooie is also gratefully acknowledged. 4. REFERENCES
1. MANAX, W. G., BLOCH, H. H., LONGERBEAM,J. K., and LILLEHEI, R. C. Successful 24-hour in vitro preservation of canine kidneys by the combined use of hyperbaric oxygenation and hypothermia. Surgery, 56: 275, 1964. 2. NAKAMOTO,S., STRAFFON, R. A., and KOLFF, W. J. Human renal homotransplantations with cadaver kidneys. J.A.M.A., 192: 102, 1965. 3. HUMPHRIES, A. L., JR., RUSSEL, R., OSTAFIN, J.,
5.
6. 7.
GOODRICH,S. M., and MORETZ, W. H. Successful reimplantation of canine kidney after twenty-four hours storage. Surgery, 54: 136, 1963. CLEVELAND, R. J., LEE, H. M., PROUT, G. R., and HUME, D. M. Preservation of the cadaver kidney for renal homotransplantation in man. Surg. Gynec. & Obst., 119: 991, 1964. DEMPSTER, W. J., KOUNTZ, S. L., and JOVANIC, M. Simple kidney storage technique. &it. M. J., 1: 407, 1964. STARZL, T. E. Experience in Renal Homotransplantation. Philadelphia, 1965. n’. B. Saunders Co. CALNE, R. Y., PEGG, E. D., PRYSE-DAVIES, J., and BROWN, F. L. Renal preservation by ice cooling. &it. M. J., 2: 651, 1963.
American Journal
of Sur~cry