JOURNAL
OF SURGICAL
RESEARCH
Sachs
20,
103-106 (1976)
I Solution
and 7% Mannitol
for Renal W. A. STERLING,
Solution
Preservation
M.D.,’ B. DATNOW, M.D. AND A. G. DIETHELM,
M.D.
Departments of Surgery and Surgical Pathology Birmingham VA Hospital and University of Alabama Medical Center, Birmingham, Alabama 35294 Submitted August 5, 1975
Presently there are two chief methods of preserving cadaver kidneys prior to transplantation. The most common method in the United States today is by machine preservation as first described by Belzer and his colleagues in 1967 [2]. Another method is by flush-out of kidneys with hypothermic solutions of extracellular or intracellular electrolyte composition. Most recent experience with flush-out and cold storage is with so-called Collins [5] or Sachs [lo] solutions, which have intracellular electrolyte compositions. Currently, there is uncertainty regarding the optimal method to preserve cadaver kidneys. Reports of immediate kidney failure, both in dogs [7] and man [6] due to cytotoxic antibody in the perfusate during pulsatile preservation as well as human kidney failures at one month and one year presumably related to pulsatile preservation [3, 41 have appeared. Canine kidneys [5, lo] and human kidneys [8] have been successfully preserved by cold storage following flush-out with hypothermic solutions of intracellular electrolyte composition; however, a recent report describes failure of this method to preserve canine kidneys for 24 hr [9]. Although most of our experience in human renal preservation is with pulsatile perfusion, which immediately follows core cooling with an extra-cellular solution or by the so-called combined method, namely, 1 to 9 hr of core cooling ‘Present address: William A. Sterling, M.D., Department of Surgery, University of New Mexico, School of Medicine, and Department of Surgery, Veterans Administration Hospital Albuquerque, New Mexico 87108.
and cold storage followed by pulsatile perfusion [ll], we have preserved canine and human kidneys successfully using Sachs I solution. In addition, to test the assumption that the beneficial effects of intracellular wash-out solutions are due to prevention of cellular swelling by high concentrations of substances that are poorly permeable and osmotically active, rather than to the high potassium content [l], 24 hr canine kidney preservations were attempted using a solution of distilled water to which mannitol was added to approximate the osmolality of Sachs I solution. The present report describes results of 24 hr Sachs I preservations of canine kidneys compared with 24 hour pulsatile preservation as well as the failure of flush-out with 7% mannitol to preserve canine kidneys for 24 hr. MATERIALS AND METHODS Cryoprecipitated pooled dog plasma was prepared and machine preservations were carried out as described previously [ 111.The Sachs I solution was prepared by adding 50 g of 25% mannitol to commercially prepared kidney preservation solution after removing approximately 100 cm3 from the one liter bottle in order to accommodate the volume of mannitol. The mannitol solution was prepared by adding 25% mannitol to distilled water to reach an osmolality of approximately 400 mM. This was a 7% solution. Eighteen mongrel dogs of both sexes weighing 15 kg to 20 kg were divided into three groups. Anesthesia was by intravenous nembutal. During surgery, dogs received
103 Copyright 01976 by Academic Press, Inc. All rights of reproduction in any form reserved.
104
JOURNAL OF SURGICAL
RESEARCH, VOL. 20, NO. 2, FEBRUARY
1000 to 1500 cm3 of 5% dextrose in normal saline. There was no attempt to use the “notouch” technique of nephrectomy. Five minutes before nephrectomy, 12.5 g of mannitol was given intravenously. Group I consisted of six dogs. Kidneys from these dogs were flushed with a balanced pH extracellular solution then placed directly on the preservation machine. Group II consisted of six dogs whose kidneys were removed and flushed with 100 to 150 ml of Sachs I solution, then immersed in the solution and stored at 5°C. Group III consisted of six dogs whose kidneys were flushed with 100 to 150 ml of the 7% mannitol solution then immersed in this solution and stored at 5°C. Following 24 hr of preservation, dogs in each group were again anesthetized and given 1000 to 1500 cm3 of 5% dextrose in saline while kidneys were reimplanted. Mannitol(12.5 g) was administered intravenously while the ureteroneocytostomy was being done. Following contralateral nephrectomy, dogs were placed in their cages and given food and water ad lib. Blood samples for serum creatinine levels were drawn at one day, one week and two weeks. RESULTS Table I gives serum creatinine values for the three intervals in each group. One dog in Group I and one dog in Group II died of uremia. In addition, one dog in Group I was sacrificed after one week due to infection of the wound and dehiscence. Five dogs in Group III died of uremia. All other animals lived. Grossly all of the kidneys in Group III, except in dog #2 appeared mottled and cyanotic at the time of reimplantation, and failed to make urine. Biopsies of these kidneys at one hour after reimplantation (Fig. 1) showed necrotizing glomerulonephritis with marked congestion as well as hydropic degeneration and necrosis of the tubules with intraluminal deposits of eosinophilic material. At autopsy, the kidneys were very large with both venous and arterial occlusion.
1976
TABLE 1 Results Following Preservation by Pulsatile Flow, Flush with SachsI and Flush with 7% Mannitol Dog
SERUM CREATININE IN mg/lOOml 1 Day 1 Week 2 Weeks Group I-Pulsatile preservation 4.7 8.0 7.7 8.3 2.1 4.5 5.4 f 2.5
20 1.6 3.4 16.0 1.2 1.6 2.0 f 0.9
1.8 1.5 2.2 Expired Sacrificed 1.2 1.7 f 0.4
Group II-Sachs I solution 5.3 4.1 4.4 3.0 7.9 2.9 3.9 ?r 1.4
7.3 3.6 4.6 2.9 Expired 1.7 4.0 * 2.1
5.5 1.5 4.7 2.0 1.8 3.1 * 1.9
Group III-7% mannitol 1 2 3 4 5 6
4.7 3.0 8.4 6.2 8.1 8.6
9.6 2.2 Expired Expired Expired Expired
Expired 1.9
aMean and standard deviations of survivors.
DISCUSSION Sachs I solution rather than Sachs II solution was chosen to carry out preservations because of the simplicity of preparation. A commercially available bottle of kidney preservation solution can be removed from the refrigerator, mannitol which is readily available can be added, and the solution is ready for use. The 24 hr preservations with Sachs I and pulsatile preservation were both successful. There were too few animals to allow conclusions comparing pulsatile preservation with Sachs I preservation based on the mean creatinine values at the various intervals in the two groups. Judging from the poor flow which occurred immediately following revascularization in the 7% mannitol group of kidneys, there appeared to be vascular injury. This was confirmed by biopsies. The large dark kidneys, with clot in both artery and vein
STERLING
ET AL.: RENAL PRESERVATION
105
FIG. 1. Glomerulus showing focal necrotizing glomerulonephritis (H & E, x 6000).
that were seenat autopsy, may have beenthe result of the low flow state with renal vein thrombosis and then retrograde clotting throughout the vasculature. We are unable to explain the success of one kidney preserved with 7% mannitol. A poor flushout so that the method of preservation was predominately by surface cooling could ex-
plain it. Toledo-Pereyra and his colle:agues [12] attempted to evaluate the effecl t of a flush-out with hypertonic mannitol (40 mM) in protecting canine kidneys with 120to 40 min of warm ischaemia precedinlg the flush and 24 hr of pulsatile preservation. They concluded that the hypertonic mannitol was detrimental.
106
JOURNAL OF SURGICAL
RESEARCH, VOL. 20, NO. 2, FEBRUARY
We conclude that flush-out with Sachs I solution was as successful as pulsatile perfusion for 24 hr preservation of canine kidneys. A 7% mannitol solution was not successful. It presumably causedirreversible vascular injury. ACKNOWLEDGMENTS This research was supported by Project Number 8 131-01, Birmingham VA Hospital, Birmingham, Alabama. We thank Mrs. Patricia Soule for her assistance in carrying out the preservations.
REFERENCES 1. Belzer, F. O., Current concepts of renal preservation. N. Engl. J. Med. 291:402-404, 1974. 2. Belzer, F. O., Ashby, B. S., and Dunphy, J. E., 24 Hour and 72 hour preservation of canine kidneys. Lancer 1:536-539, 1967. 3. Clark, E. A., Opelz, G., Mickey, M. R., and Terasaki, P. I., Lancer 1:361-364,1973. 4. Clark, E. A., Terasaki, P. I., Opelz, G., and Mickey, M. R., Cadaver-kidney transplant failures at one month. N. Engl. J. Med. 291:1099-l 102, 1974. 5. Collins, G. M., Bravo-Shugarman, M., and Terasaki, P. I. Kidney preservation for trans-
1976
portation. Initial perfusion and 30 hours ice storage. Lancet2:1219-1222, 1969. 6. Cross, E. W., Whittier, F. C., Cuppage, F., Crouch, T., Manuel, E., and Grantham, J., Hyperacute rejection of renal allografts following pulsatile perfusion with a perfusate containing specific antibody. J. Transplantation.
11~626-629, 1974.
7. Filo, R. S., Dickson, L. G., Suba, E. A., and Sell, K. W. Immunologic injury induced by Ex-Viva perfusion of canine renal allografts. Surgery 76:88100,1974. 8. Hartley, L. C. J., Collins, G. M., and Clunig, G. J. A., Kidney preservation for transportation function of 29 human cadaver-kidneys preserved with an intracellular perfusate. N. Engl. J. Med. 285: 1049-1052, 1971. 9. McCabe, R., Applebaum, H., Lorieo, D., and Stevens, L., Failure of canine kidney preservation using Sachs II solution. Lancer 1:517, 1975. 10. Sachs, S. A., Petritsch, P. H., and Kaufman, J. J., Canine kidney preservation using a new perfusate. Lancer 2:10241028, 1973. 11. Sterling, W. A., Pierce, J. C., Lee, H. M., Hume, D. M., Hutcher, N. E., and Mendez-Picon, G., Renal preservation by hypothermic storage plus pulsatile perfusion. Surg. Gynec. Obstet. 135:589592. 12. Toledo-Pereyra, L. H., Kunau, R. F., and Najarian, J. S., Effect of hypertonic mannitol on ischemic kidneys. J. Surg. Res. 17:35&358, 1974.
OF SURGICAL
RESEARCH
Sachs
20,
103-106 (1976)
I Solution
and 7% Mannitol
for Renal W. A. STERLING,
Solution
Preservation
M.D.,’ B. DATNOW, M.D. AND A. G. DIETHELM,
M.D.
Departments of Surgery and Surgical Pathology Birmingham VA Hospital and University of Alabama Medical Center, Birmingham, Alabama 35294 Submitted August 5, 1975
Presently there are two chief methods of preserving cadaver kidneys prior to transplantation. The most common method in the United States today is by machine preservation as first described by Belzer and his colleagues in 1967 [2]. Another method is by flush-out of kidneys with hypothermic solutions of extracellular or intracellular electrolyte composition. Most recent experience with flush-out and cold storage is with so-called Collins [5] or Sachs [lo] solutions, which have intracellular electrolyte compositions. Currently, there is uncertainty regarding the optimal method to preserve cadaver kidneys. Reports of immediate kidney failure, both in dogs [7] and man [6] due to cytotoxic antibody in the perfusate during pulsatile preservation as well as human kidney failures at one month and one year presumably related to pulsatile preservation [3, 41 have appeared. Canine kidneys [5, lo] and human kidneys [8] have been successfully preserved by cold storage following flush-out with hypothermic solutions of intracellular electrolyte composition; however, a recent report describes failure of this method to preserve canine kidneys for 24 hr [9]. Although most of our experience in human renal preservation is with pulsatile perfusion, which immediately follows core cooling with an extra-cellular solution or by the so-called combined method, namely, 1 to 9 hr of core cooling ‘Present address: William A. Sterling, M.D., Department of Surgery, University of New Mexico, School of Medicine, and Department of Surgery, Veterans Administration Hospital Albuquerque, New Mexico 87108.
and cold storage followed by pulsatile perfusion [ll], we have preserved canine and human kidneys successfully using Sachs I solution. In addition, to test the assumption that the beneficial effects of intracellular wash-out solutions are due to prevention of cellular swelling by high concentrations of substances that are poorly permeable and osmotically active, rather than to the high potassium content [l], 24 hr canine kidney preservations were attempted using a solution of distilled water to which mannitol was added to approximate the osmolality of Sachs I solution. The present report describes results of 24 hr Sachs I preservations of canine kidneys compared with 24 hour pulsatile preservation as well as the failure of flush-out with 7% mannitol to preserve canine kidneys for 24 hr. MATERIALS AND METHODS Cryoprecipitated pooled dog plasma was prepared and machine preservations were carried out as described previously [ 111.The Sachs I solution was prepared by adding 50 g of 25% mannitol to commercially prepared kidney preservation solution after removing approximately 100 cm3 from the one liter bottle in order to accommodate the volume of mannitol. The mannitol solution was prepared by adding 25% mannitol to distilled water to reach an osmolality of approximately 400 mM. This was a 7% solution. Eighteen mongrel dogs of both sexes weighing 15 kg to 20 kg were divided into three groups. Anesthesia was by intravenous nembutal. During surgery, dogs received
103 Copyright 01976 by Academic Press, Inc. All rights of reproduction in any form reserved.
104
JOURNAL OF SURGICAL
RESEARCH, VOL. 20, NO. 2, FEBRUARY
1000 to 1500 cm3 of 5% dextrose in normal saline. There was no attempt to use the “notouch” technique of nephrectomy. Five minutes before nephrectomy, 12.5 g of mannitol was given intravenously. Group I consisted of six dogs. Kidneys from these dogs were flushed with a balanced pH extracellular solution then placed directly on the preservation machine. Group II consisted of six dogs whose kidneys were removed and flushed with 100 to 150 ml of Sachs I solution, then immersed in the solution and stored at 5°C. Group III consisted of six dogs whose kidneys were flushed with 100 to 150 ml of the 7% mannitol solution then immersed in this solution and stored at 5°C. Following 24 hr of preservation, dogs in each group were again anesthetized and given 1000 to 1500 cm3 of 5% dextrose in saline while kidneys were reimplanted. Mannitol(12.5 g) was administered intravenously while the ureteroneocytostomy was being done. Following contralateral nephrectomy, dogs were placed in their cages and given food and water ad lib. Blood samples for serum creatinine levels were drawn at one day, one week and two weeks. RESULTS Table I gives serum creatinine values for the three intervals in each group. One dog in Group I and one dog in Group II died of uremia. In addition, one dog in Group I was sacrificed after one week due to infection of the wound and dehiscence. Five dogs in Group III died of uremia. All other animals lived. Grossly all of the kidneys in Group III, except in dog #2 appeared mottled and cyanotic at the time of reimplantation, and failed to make urine. Biopsies of these kidneys at one hour after reimplantation (Fig. 1) showed necrotizing glomerulonephritis with marked congestion as well as hydropic degeneration and necrosis of the tubules with intraluminal deposits of eosinophilic material. At autopsy, the kidneys were very large with both venous and arterial occlusion.
1976
TABLE 1 Results Following Preservation by Pulsatile Flow, Flush with SachsI and Flush with 7% Mannitol Dog
SERUM CREATININE IN mg/lOOml 1 Day 1 Week 2 Weeks Group I-Pulsatile preservation 4.7 8.0 7.7 8.3 2.1 4.5 5.4 f 2.5
20 1.6 3.4 16.0 1.2 1.6 2.0 f 0.9
1.8 1.5 2.2 Expired Sacrificed 1.2 1.7 f 0.4
Group II-Sachs I solution 5.3 4.1 4.4 3.0 7.9 2.9 3.9 ?r 1.4
7.3 3.6 4.6 2.9 Expired 1.7 4.0 * 2.1
5.5 1.5 4.7 2.0 1.8 3.1 * 1.9
Group III-7% mannitol 1 2 3 4 5 6
4.7 3.0 8.4 6.2 8.1 8.6
9.6 2.2 Expired Expired Expired Expired
Expired 1.9
aMean and standard deviations of survivors.
DISCUSSION Sachs I solution rather than Sachs II solution was chosen to carry out preservations because of the simplicity of preparation. A commercially available bottle of kidney preservation solution can be removed from the refrigerator, mannitol which is readily available can be added, and the solution is ready for use. The 24 hr preservations with Sachs I and pulsatile preservation were both successful. There were too few animals to allow conclusions comparing pulsatile preservation with Sachs I preservation based on the mean creatinine values at the various intervals in the two groups. Judging from the poor flow which occurred immediately following revascularization in the 7% mannitol group of kidneys, there appeared to be vascular injury. This was confirmed by biopsies. The large dark kidneys, with clot in both artery and vein
STERLING
ET AL.: RENAL PRESERVATION
105
FIG. 1. Glomerulus showing focal necrotizing glomerulonephritis (H & E, x 6000).
that were seenat autopsy, may have beenthe result of the low flow state with renal vein thrombosis and then retrograde clotting throughout the vasculature. We are unable to explain the success of one kidney preserved with 7% mannitol. A poor flushout so that the method of preservation was predominately by surface cooling could ex-
plain it. Toledo-Pereyra and his colle:agues [12] attempted to evaluate the effecl t of a flush-out with hypertonic mannitol (40 mM) in protecting canine kidneys with 120to 40 min of warm ischaemia precedinlg the flush and 24 hr of pulsatile preservation. They concluded that the hypertonic mannitol was detrimental.
106
JOURNAL OF SURGICAL
RESEARCH, VOL. 20, NO. 2, FEBRUARY
We conclude that flush-out with Sachs I solution was as successful as pulsatile perfusion for 24 hr preservation of canine kidneys. A 7% mannitol solution was not successful. It presumably causedirreversible vascular injury. ACKNOWLEDGMENTS This research was supported by Project Number 8 131-01, Birmingham VA Hospital, Birmingham, Alabama. We thank Mrs. Patricia Soule for her assistance in carrying out the preservations.
REFERENCES 1. Belzer, F. O., Current concepts of renal preservation. N. Engl. J. Med. 291:402-404, 1974. 2. Belzer, F. O., Ashby, B. S., and Dunphy, J. E., 24 Hour and 72 hour preservation of canine kidneys. Lancer 1:536-539, 1967. 3. Clark, E. A., Opelz, G., Mickey, M. R., and Terasaki, P. I., Lancer 1:361-364,1973. 4. Clark, E. A., Terasaki, P. I., Opelz, G., and Mickey, M. R., Cadaver-kidney transplant failures at one month. N. Engl. J. Med. 291:1099-l 102, 1974. 5. Collins, G. M., Bravo-Shugarman, M., and Terasaki, P. I. Kidney preservation for trans-
1976
portation. Initial perfusion and 30 hours ice storage. Lancet2:1219-1222, 1969. 6. Cross, E. W., Whittier, F. C., Cuppage, F., Crouch, T., Manuel, E., and Grantham, J., Hyperacute rejection of renal allografts following pulsatile perfusion with a perfusate containing specific antibody. J. Transplantation.
11~626-629, 1974.
7. Filo, R. S., Dickson, L. G., Suba, E. A., and Sell, K. W. Immunologic injury induced by Ex-Viva perfusion of canine renal allografts. Surgery 76:88100,1974. 8. Hartley, L. C. J., Collins, G. M., and Clunig, G. J. A., Kidney preservation for transportation function of 29 human cadaver-kidneys preserved with an intracellular perfusate. N. Engl. J. Med. 285: 1049-1052, 1971. 9. McCabe, R., Applebaum, H., Lorieo, D., and Stevens, L., Failure of canine kidney preservation using Sachs II solution. Lancer 1:517, 1975. 10. Sachs, S. A., Petritsch, P. H., and Kaufman, J. J., Canine kidney preservation using a new perfusate. Lancer 2:10241028, 1973. 11. Sterling, W. A., Pierce, J. C., Lee, H. M., Hume, D. M., Hutcher, N. E., and Mendez-Picon, G., Renal preservation by hypothermic storage plus pulsatile perfusion. Surg. Gynec. Obstet. 135:589592. 12. Toledo-Pereyra, L. H., Kunau, R. F., and Najarian, J. S., Effect of hypertonic mannitol on ischemic kidneys. J. Surg. Res. 17:35&358, 1974.
