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Experimental Renal Preservation
Vol. 103, June
THE JOURNAL OF UROLOGY
Copyright
©
Printed in U.S.A.
1970 by The Williams & Wilkins Co.
EXPERIMENTAL RENAL PRESERVATION DONALD C. MARTIN, GARY SMITH
AND
DONALD 0. FAREED
Frorn the Division of Urology, Department of Surgery, University of California, Los Angeles ancl the University of California Irvine, Irvine, California
Identification of leukocyte antigens by serologic methods provides a basis for matching donor-recipient pairs in organ transplantation. 1 Heterogeneous distribution of these antigens in thehumanpopulationrequires that many individuals be considered to form matched pairs. One institution cannot maintain a sufficient number of recipients on chronic hemodialysis to ensure a good match for all available organs. To overcome this deficiency many institutions must pool their potential recipients to provide sufficient numbers for good matching. 2 The limiting factor in the size of this pool is the length of time the kidney can be maintained outside of the body for transport to a specific recipient. Cold perfusion on an artificial circulation system has been shown to preserve the canine 3 •4 and human kidney 5 for 72 and 17 hours respectively. Humphries recently reported on successful preservation of the canine kidney for 5 days. 6 This system incurs renal injury manifest by transient
elevation of the blood urea nitrogen (BUN). The equipment is large and complex. Herrmann and Turcotte preserved the canine kidney for 18 and 30 hours with a single pass low flow perfusion. 7 We evaluated methods of short-term preservation to determine which would incur no renal damage during the interval of renal preservation. The quality of renal function rather than animal survival was of principal concern. MBTHODS
Accepted for publication July 25, 1969. Read at annual meeting of American Urological Association, San Francisco, California, May 1115, 1969. This study was supported by the University of California Kidney Fund. 1 Terasaki, P. I., Vredevoe, D. L., Mickey, M. R., Porter, K. A., Marchioro, T. 0., Faris, T. D. and Starzl, T. E.: Sero typing for homotransplantation VII, selection of kidney donors for thirtytwo recipients. Ann. N.Y. Acad. Sci., 129: 500, 1966. 2 Patel, R., Glassock, R. and Terasaki, P. I.: Serotyping for homotransplantation XIX, experience with an interhospital scheme of cadaverkidney sharing and tissue typing. J.A.M.A., 207: 1319, 1969. 3 Belzer, F. 0., Ashby, B. S. and Dunphy, J.E.: 24-hour and 72 hour-preservation of canine kidneys. Lancet, 2: 536, 1967. 4 Humphries, A. L., Jr., Russel, R., Stoddard, L. D. and Moretz, W. H.: Three-day kidney preservation: perfusion of kidneys with hypothermic, diluted blood or plasma. Surgery, 63: 646, 1968. 5 Belzer, F. 0., Ashby, B. S. and Gulyassy, P. F.: Successful seventeen-hour preservation and transplantation of human-cadaver kidney. New Engl. J. Med., 278: 608, 1968. 6 Humphries, A. L., Jr., Russell, R., Stoddard, L. D. and Moretz, W. H.: Successful five-day kidney preservation. Perfusion with hypothermic, diluted plasma. Invest. Ural., 5: 609, 1968.
Fifty-three dogs had renal autotransplantation with 8 hours of preservation outside of the body and immediate contralateral nephrectomy. Anesthesia was obtained by intravenous pentobarbital. All procedures were performed with careful aseptic technique. In the experimental group the left kidney was removed through a flank incision. Autotransplantation to the right iliAc vessels was accomplished through a midline or transrectal incision. Right nephrectomy was performed at implantation of the left kidney. The animals were allowed to awaken between operations. In the control group all of the operative procedure was carried out through a long midline incision. Heparin 2.5 mg. was injected into the renal artery immediately prior to nephrectomy of all test organs. The kidney was revascularized by anastomosis of the renal vein end-to-side with the iliac vein and the renal artery end-to-end with the external iliac artery. The ureter of the graft was implanted in the bladder. The dogs received 500 ml. 5 per cent dextrose in H normal saline intravenously during nephrectomy, again during implantation and subcutaneously for the initial 3 days postoperatively. This fluid was given to avoid dehydration with resultant false elevation of BUN and creatinine. On the day of operation 1.0 gm. ceph-
681
7 Herrmann, T. J. and Turcotte, J. G.: Preservation of canine kidneys by hypothermia and low flow perfusion with bloodless perfusate. Arch. Surg., 98: 121, 1969.
682
MARTIN, SMITH AND FAREED
alothin (keflin) was given intravenously. BUN and serum creatinine were obtained prior to the operation and daily for 3 days postoperatively. Blood chemistry values were obtained 3 times per week thereafter. An autopsy was done in all cases at death or sacrificed 2 to 3 weeks following transplantation. Renal tissue was studied with hematoxylin and eosin and with special stains for lipids. Group 1. Five dogs served as controls by autotransplantation of 1 kidney without preservation and with immediate contralateral nephrectomy. Perfusion: Forty-one canine kidneys were cooled and perfused by electrolyte solutions or homologous plasma. Group 2. Perfusion was used to wash blood cells out of the kidney and then to produce core cooling in 11 cases. The kidney was cleared of red blood cells with 100 ml. normal saline at 20 to 37C. To cool the organ 150 to 250 ml. plasma at 4C was perfused by gravity pressure (100 cm. H20). The renal artery and vein were ligated to maintain some perfusate in the vascular tree. The kidney was placed in a solution at 4C and maintained with ice chips for 8 hours. Plasma was pooled from several canine donors and prepared by passage through a 0.22 micron Millipore filter after cooling to 4C to remove lipids and fibrin. To each liter of plasma were added 250 mg. dextrose, 80 units of insulin, 200,000 units of penicillin, 100 mg. hydrocortisone and 4 meq. magnesium sulfate. The additives were similar to those used by Belzer.3 Thirty kidneys had a continuous intra-arterial perfusion with a pulsatile pump at a flow rate of 100 ml. per hour. During continuous perfusion the kidney and coils of tubing were immersed in a saline bath at 5C. Cooling was achieved by perfusion and surface contact (fig. 1). The renal artery was secured to a cannula with 1 ligature and venous effluent was allowed to enter the saline bath. The level of cold solution maintained a venous pressure of 3 to 5 cm. Three continuous perfusates were used. Group 3. Intracellular electrolyte composition (KCl-15 meg./L, Mg80 4-30 meg./L, NaHCO:r-10 meg./L, K2HP0.-70 meg./L, KH2P04-30 meg./L, pH-6.95). Group 4, Extracellular electrolyte composition (NaCl-117 meg./L, NaHC0 3-27 meg./ L, K2HP04-2 meg./L, Ca citrate-3 meg. /L , Mg acetate-3 meg./L, pH-7.35).
Thermometer
Plasma bottle
;-WJ. Electric stirrer
IV tuhing
·,.Jire mesh basket i':'9---...l Stainless stee 1
cooling coil
Styrofoam insulator
Fm. l. Diagram of system used to continuously perfuse canine kidneys. (Actual dimensions 22 x 15 x 24 inches.) Organ is in bath at 5C. Flow (100 ml. per hr.) is pulsatile but low pressure. Group 5. Homologous plasma with additives previously described. No perfusion was done. Group 6. Eleven kidneys were cooled by immersion in a bath at 5C. No perfusion was carried out. Preservation was obtained by surface cooling only. OBSERVATIONS
All animals survived except those with technical defects and those continuously perfused with extracellular electrolyte solution. Most kidneys resumed normal color and turgor immediately after release of the vascular clamps. In group 2 patchy areas of cortical cyanosis developed in many kidneys 5 to 10 minutes after revascularization. This was a transient condition as normal cortical color resumed in an additional 5 minutes. No permanent disease could be attributed to this observation. Most kidneys produced urine during anastomosis of the ureter to the bladder attesting to their healthy condition. Continuous infusion of extracellular electrolyte composition produced edema in the adventitia of the hilum and around the ureter. The mean of serum creatinine prior to and subsequent to autotransplantation is shown in figures 2 and 3. Impaired renal function, evident from a rise in the serum creatinine and BUN was mild and transient. In group 4, 9 of 10 kidneys continuously perfused with extracellular electolyte solution failed to support life. These dogs died of renal failure. BUN values paralleled the serum creatinine. Only the latter are reported since we believe the
683
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intracellular electrolyte composition. The glomeruli had minimal if any hydropic change. Group 4: There was a marked disturbance in cellular architecture of those kidneys perfused 8 hours with a solution of extracellular electrolyte composition. The tubular cells showed marked swelling, hydropic degeneration and necrosis. Some of the glomeruli showed similar changes. Group 5: The tubular cells were well following 8 hours of perfusion with homologous plasma. Lipid thrombi were not seen in renal vessels or in glomeruli.
RENAL PRESERVATION
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FIG. 2. A, renal function in control (group 1) and washout perfused (group 2) kidneys. B, renal function in continuously perfused organs. Intracellular electrolyte composition (group 3) produced minimal histological changes. Extracellular electrolyte composition (group 4) produced marked histological changes and progressive azotemia.
BUN is more susceptible to alteration by nonrenal causes. PATHOLOGY
Gross. The transplanted kidneys examined at sacrifice of animals 2 to 3 weeks postoperatively were of normal size, color and consistency. The capsule was thin. Normal separation of cortex and medulla was seen on section of the kidneys. Small wedge-shaped cortical infarcts were seen in some of the perfused kidneys but never in the non-perfused kidneys. There were no gross abnormalities in kidneys of group 4 which failed to support life. J1ficroscopic. Groups 1 and 6: No histological changes were seen by light microscopy in controls nor in those kidneys preserved for 8 hours by surface cooling alone. Group 2: Cortical infarcts were seen in several kidneys when perfusion was used to remove blood cells and produce core cooling. No other abnormalities were seen. Group 3: There was minimal swelling of renal tubular cells with hydropic degeneration in kidneys continuously perfused with 2, solution of
Preservation of the kidney for 8 hours allows time for serological testing of leukocyte antigens and transportation to a remote recipient. If antigen typing were obtained ante mortem in cadaveric donors, this period of preservation would allow for transcontinental air transport of the kidney. Low flow continuous perfusion might provide nutrients and remove wastes from an organ with marked depression of metabolism due to cooling. Continuous perfusion with electrolyte solutions of either intracellular or extracellular composition without osmotic balance, i.e. lacking plasma A ~
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Frn. 4. A, temperatures determined by thermisters at various depths in canine kidney. Surface cooling. B, temperatures determined by thermisters at various depths in canine kidney. Cooling by intra-arterial infusion of Ringer's solution at 5C. Flow rate is as rapid as possible with infusion bottle 100 cm. above kidney. Solution warms to 7C in passage through tubing. proteins, resulted in physical damage to renal cells characterized by swelling and hydropic degeneration. The extracellular composition was more deleterious than intracellular. Continuous perfusion with homologous plasma maintained osmotic stability and no histological changes were seen. Simple perfusion methods in this study offered no advantage over surface cooling. Occlusion of intrarenal vessels during perfusion resulted in small cortical infarcts in some cases. Renal function following 8 hours storage of the kidney outside the body without perfusion was comparable to simple autotransplantation and contralateral nephrectomy. The anesthesia, surgical procedure, unilateral nephrectomy and 20 minutes of ischemia required for implantation combined to cause a rise in the postoperative serum creatinine in controls (group 1). Eight hours of storage with immersion cooling did not
produce increased elevation of serum creatinine indicating excellent preservation. Fisher and associates observed minimal histological changes in canine kidneys after 12 hours of cold storage. 8 More marked anatomical and functional changes were observed after 15 hours. Renal preservation with surface cooling for 8 hours appears to be excellent. Longer storage may require the perfusion system described by Humphries and Belzer but preservation in excess of 8 hours would rarely be necessary. We have used a perfusion of lactated Ringer's solution at 4C to. produce rapid cooling of human kidneys in cadaver transplantation. The rate of surface and perfusion cooling in the canine kidney is shown in figure 4. Three thermister probes were placed on the surface, 1.0 cm. into the cortex and 1.7 cm. into the renal parenchyma. The latter was at the corticomedullary junction. Cooling to 15C at a depth of 1.7 cm. requires only 5 minutes longer for surface cooling. Although the human kidney is larger than the canine kidney it cools from a larger surface area. Results of this experimental study suggest that there is no functional damage to canine kidneys maintained outside the body for 8 hours at 5C. Once the organ is preserved by cooling it would appear there is no urgency to revascularize. Careful selection and preparation of the recipient would be possible. In our clinical experience temporary oliguria or anuria with tubular necrosis will frequently develop in cadaveric kidneys. This renal damage has been attributed to the agonal events of death and the euthermic ischemia prior to nephrectomy and cooling. The injury may be increased by 8 hours of hypothermic preservation. We plan further laboratory studies to test this theory. Dogs have survived on a kidney preserved by cooling for 24 hours. 9 • 10 Renal function is initially 8 Fisher, E. R., Copeland, C. and Fisher, B.: Correlation of ultrastructure and function following hypothermic preservation of canine kidneys. Lab. Invest., 17: 99, 1967. 9 Hendry, W. F., Struthers, N. W., Duguid, W. P. and Hopkinson, W. I.: Twenty-four-hour storage of kidneys. Lancet, 1: 1221, 1968. 10 Ackerman, J. R., Hopkinson, W. I., Murphy, D. M. G. and Kenyon, J. R.: Successful 24-48 hour canine kidney preservation. In: Advance in Transplantation. Proceedings of the First International Congress of the Transplant Society. Edited by J. Dausset, J. Hamburger and G. Mathe. Baltimore: The Williams and Wilkins Co., p. 743, 1968.
EXPERIMENTAL RENAL PRESERVATION
poor and mortality is frequent. Most successes are achieved when the contralateral kidney is left in situ to support the animal while the experimental kidney recovers. We have attempted to achieve perfect preservation for a short interval. This seems possible up to 12 hours with cold immersion alone based on our observations and those reported by Fisher. 8 Beyond 12 hours of storage a perfusion system described by Belzer and Humphries would appear necessary to maintain the kidney in a healthy state. Renal storage in excess of 12 hours is associated with temporary impaired renal function. Fibrin and lipids precipitate out of whole plasma upon cooling. It is important to remove them by filtration prior to use in organ perfusion to avoid vascular occlusions. SUMMARY
The canine renal autograft has been used to determine the optimum technique for 8-hour preservation. The quality of renal function rather than animal survival has been of principal concern. Fifty-three dogs had renal autotransplantation with 8 hours of preservation with the kidney outside the body and immediate contralateral nephrectomy. Five dogs served as controls by autotransplantation of one kidney without preservation and with immediate contralateral
685
nephrectomy. Renal function was monitored by daily serum creatinine and BUN. Forty-one kidneys were cooled and preserved by perfusion with cold electrolyte solutions or homologous plasma. In 11 cases the perfusion was used to cool the kidney only. The remaining 30 kidneys had continuous intra-arterial infusion of electrolyte solution or homologous plasma at a flow rate of 100 ml. per hour. Eleven kidneys were cooled by immersion in a solution at 5C. No perfusion was carried out. Preservation was achieved by surface cooling only. All animals survived 8-hour cold preservation except those with technical failure and those continuously perfused with a solution of extracellular electrolyte composition. The maximum rise in serum creatinine of the perfused group was to 3.0 mg. per cent (mean value). The maximum rise in serum creatinine in the surface cooled and control groups was to 2.5 mg. per cent (mean value). Small cortical infarcts were seen in some of the perfused kidneys but never in the nonperfused. Surface cooling of the canine kidney to 5C can provide excellent preservation for 8 hours. Perfusion offers no advantages for 8-hour storage. Drs. Richard Hall, Ian Grey and Joe D'Assis assisted with the operation. Dr. Freburn James reviewed the histopathology.
THE JOURNAL OF UROLOGY
Copyright
©
Printed in U.S.A.
1970 by The Williams & Wilkins Co.
EXPERIMENTAL RENAL PRESERVATION DONALD C. MARTIN, GARY SMITH
AND
DONALD 0. FAREED
Frorn the Division of Urology, Department of Surgery, University of California, Los Angeles ancl the University of California Irvine, Irvine, California
Identification of leukocyte antigens by serologic methods provides a basis for matching donor-recipient pairs in organ transplantation. 1 Heterogeneous distribution of these antigens in thehumanpopulationrequires that many individuals be considered to form matched pairs. One institution cannot maintain a sufficient number of recipients on chronic hemodialysis to ensure a good match for all available organs. To overcome this deficiency many institutions must pool their potential recipients to provide sufficient numbers for good matching. 2 The limiting factor in the size of this pool is the length of time the kidney can be maintained outside of the body for transport to a specific recipient. Cold perfusion on an artificial circulation system has been shown to preserve the canine 3 •4 and human kidney 5 for 72 and 17 hours respectively. Humphries recently reported on successful preservation of the canine kidney for 5 days. 6 This system incurs renal injury manifest by transient
elevation of the blood urea nitrogen (BUN). The equipment is large and complex. Herrmann and Turcotte preserved the canine kidney for 18 and 30 hours with a single pass low flow perfusion. 7 We evaluated methods of short-term preservation to determine which would incur no renal damage during the interval of renal preservation. The quality of renal function rather than animal survival was of principal concern. MBTHODS
Accepted for publication July 25, 1969. Read at annual meeting of American Urological Association, San Francisco, California, May 1115, 1969. This study was supported by the University of California Kidney Fund. 1 Terasaki, P. I., Vredevoe, D. L., Mickey, M. R., Porter, K. A., Marchioro, T. 0., Faris, T. D. and Starzl, T. E.: Sero typing for homotransplantation VII, selection of kidney donors for thirtytwo recipients. Ann. N.Y. Acad. Sci., 129: 500, 1966. 2 Patel, R., Glassock, R. and Terasaki, P. I.: Serotyping for homotransplantation XIX, experience with an interhospital scheme of cadaverkidney sharing and tissue typing. J.A.M.A., 207: 1319, 1969. 3 Belzer, F. 0., Ashby, B. S. and Dunphy, J.E.: 24-hour and 72 hour-preservation of canine kidneys. Lancet, 2: 536, 1967. 4 Humphries, A. L., Jr., Russel, R., Stoddard, L. D. and Moretz, W. H.: Three-day kidney preservation: perfusion of kidneys with hypothermic, diluted blood or plasma. Surgery, 63: 646, 1968. 5 Belzer, F. 0., Ashby, B. S. and Gulyassy, P. F.: Successful seventeen-hour preservation and transplantation of human-cadaver kidney. New Engl. J. Med., 278: 608, 1968. 6 Humphries, A. L., Jr., Russell, R., Stoddard, L. D. and Moretz, W. H.: Successful five-day kidney preservation. Perfusion with hypothermic, diluted plasma. Invest. Ural., 5: 609, 1968.
Fifty-three dogs had renal autotransplantation with 8 hours of preservation outside of the body and immediate contralateral nephrectomy. Anesthesia was obtained by intravenous pentobarbital. All procedures were performed with careful aseptic technique. In the experimental group the left kidney was removed through a flank incision. Autotransplantation to the right iliAc vessels was accomplished through a midline or transrectal incision. Right nephrectomy was performed at implantation of the left kidney. The animals were allowed to awaken between operations. In the control group all of the operative procedure was carried out through a long midline incision. Heparin 2.5 mg. was injected into the renal artery immediately prior to nephrectomy of all test organs. The kidney was revascularized by anastomosis of the renal vein end-to-side with the iliac vein and the renal artery end-to-end with the external iliac artery. The ureter of the graft was implanted in the bladder. The dogs received 500 ml. 5 per cent dextrose in H normal saline intravenously during nephrectomy, again during implantation and subcutaneously for the initial 3 days postoperatively. This fluid was given to avoid dehydration with resultant false elevation of BUN and creatinine. On the day of operation 1.0 gm. ceph-
681
7 Herrmann, T. J. and Turcotte, J. G.: Preservation of canine kidneys by hypothermia and low flow perfusion with bloodless perfusate. Arch. Surg., 98: 121, 1969.
682
MARTIN, SMITH AND FAREED
alothin (keflin) was given intravenously. BUN and serum creatinine were obtained prior to the operation and daily for 3 days postoperatively. Blood chemistry values were obtained 3 times per week thereafter. An autopsy was done in all cases at death or sacrificed 2 to 3 weeks following transplantation. Renal tissue was studied with hematoxylin and eosin and with special stains for lipids. Group 1. Five dogs served as controls by autotransplantation of 1 kidney without preservation and with immediate contralateral nephrectomy. Perfusion: Forty-one canine kidneys were cooled and perfused by electrolyte solutions or homologous plasma. Group 2. Perfusion was used to wash blood cells out of the kidney and then to produce core cooling in 11 cases. The kidney was cleared of red blood cells with 100 ml. normal saline at 20 to 37C. To cool the organ 150 to 250 ml. plasma at 4C was perfused by gravity pressure (100 cm. H20). The renal artery and vein were ligated to maintain some perfusate in the vascular tree. The kidney was placed in a solution at 4C and maintained with ice chips for 8 hours. Plasma was pooled from several canine donors and prepared by passage through a 0.22 micron Millipore filter after cooling to 4C to remove lipids and fibrin. To each liter of plasma were added 250 mg. dextrose, 80 units of insulin, 200,000 units of penicillin, 100 mg. hydrocortisone and 4 meq. magnesium sulfate. The additives were similar to those used by Belzer.3 Thirty kidneys had a continuous intra-arterial perfusion with a pulsatile pump at a flow rate of 100 ml. per hour. During continuous perfusion the kidney and coils of tubing were immersed in a saline bath at 5C. Cooling was achieved by perfusion and surface contact (fig. 1). The renal artery was secured to a cannula with 1 ligature and venous effluent was allowed to enter the saline bath. The level of cold solution maintained a venous pressure of 3 to 5 cm. Three continuous perfusates were used. Group 3. Intracellular electrolyte composition (KCl-15 meg./L, Mg80 4-30 meg./L, NaHCO:r-10 meg./L, K2HP0.-70 meg./L, KH2P04-30 meg./L, pH-6.95). Group 4, Extracellular electrolyte composition (NaCl-117 meg./L, NaHC0 3-27 meg./ L, K2HP04-2 meg./L, Ca citrate-3 meg. /L , Mg acetate-3 meg./L, pH-7.35).
Thermometer
Plasma bottle
;-WJ. Electric stirrer
IV tuhing
·,.Jire mesh basket i':'9---...l Stainless stee 1
cooling coil
Styrofoam insulator
Fm. l. Diagram of system used to continuously perfuse canine kidneys. (Actual dimensions 22 x 15 x 24 inches.) Organ is in bath at 5C. Flow (100 ml. per hr.) is pulsatile but low pressure. Group 5. Homologous plasma with additives previously described. No perfusion was done. Group 6. Eleven kidneys were cooled by immersion in a bath at 5C. No perfusion was carried out. Preservation was obtained by surface cooling only. OBSERVATIONS
All animals survived except those with technical defects and those continuously perfused with extracellular electrolyte solution. Most kidneys resumed normal color and turgor immediately after release of the vascular clamps. In group 2 patchy areas of cortical cyanosis developed in many kidneys 5 to 10 minutes after revascularization. This was a transient condition as normal cortical color resumed in an additional 5 minutes. No permanent disease could be attributed to this observation. Most kidneys produced urine during anastomosis of the ureter to the bladder attesting to their healthy condition. Continuous infusion of extracellular electrolyte composition produced edema in the adventitia of the hilum and around the ureter. The mean of serum creatinine prior to and subsequent to autotransplantation is shown in figures 2 and 3. Impaired renal function, evident from a rise in the serum creatinine and BUN was mild and transient. In group 4, 9 of 10 kidneys continuously perfused with extracellular electolyte solution failed to support life. These dogs died of renal failure. BUN values paralleled the serum creatinine. Only the latter are reported since we believe the
683
EXPERIJ\IENTAL RENAL PRESERVATION
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intracellular electrolyte composition. The glomeruli had minimal if any hydropic change. Group 4: There was a marked disturbance in cellular architecture of those kidneys perfused 8 hours with a solution of extracellular electrolyte composition. The tubular cells showed marked swelling, hydropic degeneration and necrosis. Some of the glomeruli showed similar changes. Group 5: The tubular cells were well following 8 hours of perfusion with homologous plasma. Lipid thrombi were not seen in renal vessels or in glomeruli.
RENAL PRESERVATION
B 6
DISCUSSION
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FIG. 2. A, renal function in control (group 1) and washout perfused (group 2) kidneys. B, renal function in continuously perfused organs. Intracellular electrolyte composition (group 3) produced minimal histological changes. Extracellular electrolyte composition (group 4) produced marked histological changes and progressive azotemia.
BUN is more susceptible to alteration by nonrenal causes. PATHOLOGY
Gross. The transplanted kidneys examined at sacrifice of animals 2 to 3 weeks postoperatively were of normal size, color and consistency. The capsule was thin. Normal separation of cortex and medulla was seen on section of the kidneys. Small wedge-shaped cortical infarcts were seen in some of the perfused kidneys but never in the non-perfused kidneys. There were no gross abnormalities in kidneys of group 4 which failed to support life. J1ficroscopic. Groups 1 and 6: No histological changes were seen by light microscopy in controls nor in those kidneys preserved for 8 hours by surface cooling alone. Group 2: Cortical infarcts were seen in several kidneys when perfusion was used to remove blood cells and produce core cooling. No other abnormalities were seen. Group 3: There was minimal swelling of renal tubular cells with hydropic degeneration in kidneys continuously perfused with 2, solution of
Preservation of the kidney for 8 hours allows time for serological testing of leukocyte antigens and transportation to a remote recipient. If antigen typing were obtained ante mortem in cadaveric donors, this period of preservation would allow for transcontinental air transport of the kidney. Low flow continuous perfusion might provide nutrients and remove wastes from an organ with marked depression of metabolism due to cooling. Continuous perfusion with electrolyte solutions of either intracellular or extracellular composition without osmotic balance, i.e. lacking plasma A ~
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40
Frn. 4. A, temperatures determined by thermisters at various depths in canine kidney. Surface cooling. B, temperatures determined by thermisters at various depths in canine kidney. Cooling by intra-arterial infusion of Ringer's solution at 5C. Flow rate is as rapid as possible with infusion bottle 100 cm. above kidney. Solution warms to 7C in passage through tubing. proteins, resulted in physical damage to renal cells characterized by swelling and hydropic degeneration. The extracellular composition was more deleterious than intracellular. Continuous perfusion with homologous plasma maintained osmotic stability and no histological changes were seen. Simple perfusion methods in this study offered no advantage over surface cooling. Occlusion of intrarenal vessels during perfusion resulted in small cortical infarcts in some cases. Renal function following 8 hours storage of the kidney outside the body without perfusion was comparable to simple autotransplantation and contralateral nephrectomy. The anesthesia, surgical procedure, unilateral nephrectomy and 20 minutes of ischemia required for implantation combined to cause a rise in the postoperative serum creatinine in controls (group 1). Eight hours of storage with immersion cooling did not
produce increased elevation of serum creatinine indicating excellent preservation. Fisher and associates observed minimal histological changes in canine kidneys after 12 hours of cold storage. 8 More marked anatomical and functional changes were observed after 15 hours. Renal preservation with surface cooling for 8 hours appears to be excellent. Longer storage may require the perfusion system described by Humphries and Belzer but preservation in excess of 8 hours would rarely be necessary. We have used a perfusion of lactated Ringer's solution at 4C to. produce rapid cooling of human kidneys in cadaver transplantation. The rate of surface and perfusion cooling in the canine kidney is shown in figure 4. Three thermister probes were placed on the surface, 1.0 cm. into the cortex and 1.7 cm. into the renal parenchyma. The latter was at the corticomedullary junction. Cooling to 15C at a depth of 1.7 cm. requires only 5 minutes longer for surface cooling. Although the human kidney is larger than the canine kidney it cools from a larger surface area. Results of this experimental study suggest that there is no functional damage to canine kidneys maintained outside the body for 8 hours at 5C. Once the organ is preserved by cooling it would appear there is no urgency to revascularize. Careful selection and preparation of the recipient would be possible. In our clinical experience temporary oliguria or anuria with tubular necrosis will frequently develop in cadaveric kidneys. This renal damage has been attributed to the agonal events of death and the euthermic ischemia prior to nephrectomy and cooling. The injury may be increased by 8 hours of hypothermic preservation. We plan further laboratory studies to test this theory. Dogs have survived on a kidney preserved by cooling for 24 hours. 9 • 10 Renal function is initially 8 Fisher, E. R., Copeland, C. and Fisher, B.: Correlation of ultrastructure and function following hypothermic preservation of canine kidneys. Lab. Invest., 17: 99, 1967. 9 Hendry, W. F., Struthers, N. W., Duguid, W. P. and Hopkinson, W. I.: Twenty-four-hour storage of kidneys. Lancet, 1: 1221, 1968. 10 Ackerman, J. R., Hopkinson, W. I., Murphy, D. M. G. and Kenyon, J. R.: Successful 24-48 hour canine kidney preservation. In: Advance in Transplantation. Proceedings of the First International Congress of the Transplant Society. Edited by J. Dausset, J. Hamburger and G. Mathe. Baltimore: The Williams and Wilkins Co., p. 743, 1968.
EXPERIMENTAL RENAL PRESERVATION
poor and mortality is frequent. Most successes are achieved when the contralateral kidney is left in situ to support the animal while the experimental kidney recovers. We have attempted to achieve perfect preservation for a short interval. This seems possible up to 12 hours with cold immersion alone based on our observations and those reported by Fisher. 8 Beyond 12 hours of storage a perfusion system described by Belzer and Humphries would appear necessary to maintain the kidney in a healthy state. Renal storage in excess of 12 hours is associated with temporary impaired renal function. Fibrin and lipids precipitate out of whole plasma upon cooling. It is important to remove them by filtration prior to use in organ perfusion to avoid vascular occlusions. SUMMARY
The canine renal autograft has been used to determine the optimum technique for 8-hour preservation. The quality of renal function rather than animal survival has been of principal concern. Fifty-three dogs had renal autotransplantation with 8 hours of preservation with the kidney outside the body and immediate contralateral nephrectomy. Five dogs served as controls by autotransplantation of one kidney without preservation and with immediate contralateral
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nephrectomy. Renal function was monitored by daily serum creatinine and BUN. Forty-one kidneys were cooled and preserved by perfusion with cold electrolyte solutions or homologous plasma. In 11 cases the perfusion was used to cool the kidney only. The remaining 30 kidneys had continuous intra-arterial infusion of electrolyte solution or homologous plasma at a flow rate of 100 ml. per hour. Eleven kidneys were cooled by immersion in a solution at 5C. No perfusion was carried out. Preservation was achieved by surface cooling only. All animals survived 8-hour cold preservation except those with technical failure and those continuously perfused with a solution of extracellular electrolyte composition. The maximum rise in serum creatinine of the perfused group was to 3.0 mg. per cent (mean value). The maximum rise in serum creatinine in the surface cooled and control groups was to 2.5 mg. per cent (mean value). Small cortical infarcts were seen in some of the perfused kidneys but never in the nonperfused. Surface cooling of the canine kidney to 5C can provide excellent preservation for 8 hours. Perfusion offers no advantages for 8-hour storage. Drs. Richard Hall, Ian Grey and Joe D'Assis assisted with the operation. Dr. Freburn James reviewed the histopathology.