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Comparison of the effect of 3- and 5-day hypothermic perfusion of dog kidneys on metabolism of tissue slices
CRYORIOI.OGY
21, 285-295 (1984)
Comparison
of the Effect of 3- and 5-Day Hypothermic Perfusion Dog Kidneys on Metabolism of Tissue Slices’
of
JAMES H. SOUTHARD, MASAHIRO KUNIYOSHI, MARY F. LUTZ, MARY AMETANI, AND FOLKERT 0. BELZER Madison,
Wi.sconsin 53792
Hypothermic perfusion effectively preserves the viability of kidneys for 3 days. Long-term preservation (5 days or greater) has not been consistently obtained. In this study, the differences between kidneys perfused for 3 and 5 days were compared by determining the “integrated-metabolic” capabilities of tissue slices incubated in vitro at 30°C. The “integrated-metabolic” parameters determined include (1) respiration rates, (2) cell volume regulation [total tissue water (TTW) and saccharide permeable space], (3) rate of reaccumulation of K’ and pumping of Na+, (4) maintenance of ATP concentrations, and (5) mitochondrial functions. Conditions that result in high and low concentrations of ATP following perfusion of kidneys for 5 days were also compared for effects on tissue slice metabolism. The results indicate that energy metabolism in tissue slices is well preserved under all conditions and times of perfusion of kidneys. This includes average respiration rates (315 -+ 50, 275 t 35, and 255 2 45 pmol OJhrig dry wt at 0. 3, and 5 days, respectively, mitochondrial function [respiratory control ratio (RCR) = 4.6, 4.0, and 4.1 for 0, 3, and 5 days, respectively], and steadystate concentration of ATP in slices after incubation (4.0 t 1.45, 3.9 2 1.28, and 3.3 t 0.81 pmol/g/ dry wt, for 0, 3, and 5 days, respectively). The primary differences between 3- and j-day perfused kidneys were the capability of the slices to regulate ccl1 volume and rcnccumulate K’. Slices from kidneys perfused for 3 days maintained the TTW at 3.X kg/kg dry wt, a value similar to that of control tissue slices. However, slices from 5-day perfused kidneys remained swollen (TTW = 4.6 kg/kg dry wt). Also, slices from the 5-day perfused kidney pumped K+ at less than one-half the rate found in slices from control or 3-day preserved kidneys. No significant differences were apparent in the permeability properties of the tissue slices from kidneys perfused for 3 and 5 days to radiolabeled saccharides. The defects in membrane-linked transpor-t functions, resulting fr-om long-term kidney perfusion, were reduced in kidneys containing a high concentration of ATP. The results suggest that one factor which may limit successful preservation of kidneys is the increased membrane permeability (to electrolytes) which is partially prevented by maintaining elevated concentrations of tissue ATP during perfusion.
Successful kidney preservation is limited to 3 days of continuous hypothermic perfusion and (in our hands) 5 days yields consistently nonviable kidneys. The fact that longer periods of preservation can be obtained is suggested by the results of a few studies demonstrating 5 (or more) days of Received September 13, 1983; accepted October 17, 1983. ’ This work was supported by NIH Grant AM18624 and presented at the 20th Annual Meeting of the Society of Cryobiology, August 29-September I, 1983, Cambridge, England.
preservation (6, 15, 17,32). However, these results have been difficult to reproduce and survival has not been reliable. Two problems exist in achieving successful long-term kidney preservation: one is the preservation of vascular (endothelial) integrity; the other is preservation of the functional integrity of cellular metabolism. We have compared the effects of time of preservation on the levels of adenine nucleotides (24, 26), mitochondrial properties (26, 27, 28), lipid peroxidation (2), free fatty acid levels (23, 2.5), and phospholipid composition (23) in the kidney. In one study (24)
285 001 l-2240184 $3.00 Cnpyrlghl ‘E 1984 by Academic Press, Inc. All rights of reproductmn in any form rexwed.
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we described a method to maintain high washout (33) at 0-4°C. In some cases kidconcentrations of ATP in kidneys perfused neys were washed out with Pegg’s washout for 5 days. The presence of an adenosine containing 40 mg/liter deoxycoformycin deaminase inhibitor (deoxycoformycin), (obtained as a gift from Dr. R. C. Jackson, and high concentrations of adenosine (10 Warner Lambert Co., Michigan) (24). The mM) and KH,PO, (25 mM) resulted in washout (250 ml/kidney) was delivered tissue concentrations of ATP three times from a height of 100 cm. Perfusion pressure greater than in kidneys perfused in the ab- was set initially at 60 mm Hg and stabilized sence of these additives. These data pro- for the duration of the experiment at 30-40 vide insight into the effects of hypothermic mm Hg. 0, (100%) was allowed to flow preservation on the kidney at the end of over the surface of the perfusate (2-3 liters/ perfusion, but do not indicate how well the hr); perfusate p0, = 300-400 mm Hg. kidney will function as an integrated metabolic unit following transplantation. These data also do not indicate if maintaining high Preparation and Incubation of Tissue Slices concentrations of tissue ATP contributes to the preservation of cell viability. At the end of the perfusion period, tissue In this study we compared the effects of slices were prepared from the kidney 3 and 5 days of continuous hypothermic cortex using a Stadie-Riggs microtome as perfusion of dog kidneys on the integrated described previously (7). The slices were metabolic functions of cortex tissue slices placed directly into the reaction medium (at incubated in a serum-like medium at nor- 0-4°C) for the time (20 min) necessary to mothermia (30°C). These studies were done complete the slicing. The normothermic to more fully understand the mechanism of (30°C) incubation was done in a Gilson difcryoinjury during perfusion and to observe ferential respirometer. Each flash conif maintaining high concentrations of tissue tained 5 ml of reaction medium. The reacATP suppressed the occurrence of cryoin- tion medium (KH) was similar to KrebsHenseleit solution and contained 120 mM jury. NaCl, 4.8 mM KCl, 1.16 mM CaCl,, 1.2 mM MgSO,, 16.7 mM NaH,P04, and 10 MATERIALS AND METHODS mM glucose. The pH was adjusted to 7.4 Perfusion of Dog Kidneys -+ 0.05 and the osmolarity was 292 + 5 Adult dogs of mixed breed (15-25 kg) mosmollliter. Three slices were placed in were used. Methods for perfusion preser- the reaction medium and incubated for 60 vation (6-10°C) have been described pre- to 120 min. 0, consumption was deterviously (3, 10). The perfusate contained hy- mined manometrically (29). For each experiment, four flasks were used. Two were redroxyethyl starch (HES), Na gluconate, KH,PO, (25 mM), and other common ad- moved at 60 min, and two at 120 min. Three ditives as described previously (11). In ad- slices were frozen in dry ice-acetone after dition, the perfusate also contained adeno- each time period and stored frozen for analsine (final concentration = 10 mM). Final ysis of ATP, and three slices were removed osmolality was 3 lo-320 mosmol/liter and for determination of total tissue water cation concentrations were Na+’ = 120- (TTW) and electrolytes (Na’ and K+). 130 meqlliter and K + = 25 meq/liter. The Tissue samples were also obtained from the basic perfusate has been shown to yield re- kidneys immediately after preservation, liable 3-day preservation (1 l), but inconsis- and after the cold incubation in KH, and tent survival following 5 days of perfusion. used for determination of ATP, TTW, and Kidneys were flushed out with Pegg’s electrolytes.
METABOLISM
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Determination of Saccharide Permeable Spaces The slices were placed in an incubation medium containing radiolabeled saccharides for determination of the permeability of the tissue to compounds of varying molecular weights (22). Six slices were placed into 10 ml of reaction medium and incubated for 60 and 120 min at 30°C in a Dubnoff metabolic shaking incubator under an atmosphere of 100% 0,. The reaction medium was basically Krebs-Henseleit buffer containing 0.1 mM inulin (MW = SSOO),1 mM sucrose (MW = 342), and I mM mannitol (MW = 180). The reaction medium used for the normothermic incubation also contained either [‘HI inulin, 0.25-0.35 $Zi/ ml; [‘4C]sucrose, 0.25-0.35 pCi/ml; or [‘4C]mannitol, 0.25-0.35 $i/ml. At the end of the incubation period, three slices from each flask were used to determine TTW. Three slices were also used for determination of radioactivity by the procedure of Rosenberg et al. (22). Briefly, slices were rinsed two times in cold reaction medium, blotted (two times to a side) on Whatmann No. 1 filter paper, weighed, and placed into 2 ml of H20. The slices were boiled for 5 min to completely equilibrate tissue water with the added H,O (16, 21). After centrifugation aliquots were placed into Aquasol (New England Nuclear), for counting in a Packard liquid scintillation spectrometer. The method for calculating the volume of tissue occupied by a given radiolabeled marker is based upon the level of radioactivity in the reaction medium, the level in the tissue slice, and the wet weight of the tissue slice as described by Rosenberg et al. (22). Analytical Methods Total tissue water and tissue concentrations of Nat and K+ were determined as previously described (14). Frozen tissue was quickly weighed, homogenized in HClO,, and extracted for adenine nucleo-
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tides (24, 26). Adenine nucleotide concentrations were determined by high-performance liquid chromatography (28, 34). Mitochondrial Isolation and Analysis Following preservation a portion of the kidney cortex was minced in cold sucrose for isolation of mitochondria (13). Mitochondrial protein (PC) was adjusted to 20 mgiml (Biuret) and the mitochondria were used for the determination of respiratory activity, respiratory control ratios, and ADP:O ratios as described previously (8, 25). The respiratory substrate was pyruvate plus malate. A minimum of six kidneys were used for each preservation period studied (perfusion for 3 days, 5 days, and 5 days + deoxycoformycin washout) and six kidneys were used as fresh controls. Kidneys were perfused in pairs and one was removed at 3 days and one at 5 days. Comparisons between 3- and 5-day perfused kidneys were studied using a nonpaired Student t test. RESULTS
Effect of Preservation Time on Swelling of Incubated Tissue Slices Hypothermic perfusion of kidneys leads to an increase in total tissue water due primarily to an expansion of the extracellular space (ECS) (25). At the end of perfusion, total tissue water increased from 3.5 kg/kg dry wt (control) to 4.0 kg/kg dry wt (3 day) and 4.25 kg/kg dry wt (5 day) (Fig. 1). Prior to incubating slices at 3O”C, there was an additional 30% increase in TTW due to the anoxia associated with the slicing technique and the incubation of slices in cold reaction medium (20 min). The restoration of normothermic conditions by incubating the slices at 30°C in a well-oxygenated serumlike medium (KH) resulted in a decrease in TTW to almost normal levels in slices from fresh tissue or from kidneys perfused for 3 days (Fig. 1). Slices obtained from 5-day perfused kidneys, however, remained,
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SOUTHARD ET AL Potassium
5 Day
5Day
3 Day C0ntV3
FIG. 1. Total tissue water (TTW) in kidney slices from kidneys preserved for 3 and 5 days. Dog kidneys were perfused as described under Materials and Methods. Following the indicated time of perfusion tissue slices were prepared from the cortex and incubated for 20 min in cold KH. Total tissue water was determined as described under Materials and Methods, on tissue immediately after preservation, after 20 min of cold-anoxic incubation, and after 60 and 120min of incubation at 30°C. Values reported are averages + SEM. The SEM at the end of preservation averages 0.35 kg/kg dry wt. The difference between TTW in 3- and 5-day perfused kidneys is statistically significant (P < 0.05) at each time interval tested.
swollen and there was only a slight tendency to decrease cellular swelling during the normothermic incubation period. Effect of Preservation Time on Ion Pumping of Incubated Tissue Slices
During preservation the concentration of tissue K+ was reduced from about 278 meql kg dry wt (control) to 254 meq/kg dry wt (3-day) and 193 meq/kg dry wt (5day). The loss of K+ was accompanied by a gain in Na+ (Fig. 2). When returned to the normothermic incubation medium, control tissue slices reaccumulated K+ within 60 min and maintained a high concentration of K+ for up to 120 min. Sodium was pumped out of the cell concomitant with the uptake of K+. Slices from the 3-day preserved kidney also rapidly accumulated K+ at a rate approximately equal to that of control slices and also showed a decrease in the
1 I 1 t End Of 0 Ptarervotlon
1 60
I I20
Time (min) FIG. 2. Tissue Na+ and K+ in kidney slices from kidney preserved for 3 and 5 days. Methods are identical to those described in the legend to Fig. 1. Na+ and K* were determined as described under Materials and Methods. Values are averages with SEM in parentheses. K+ is expressed per kg dry wt and Na+ per kg wet wt.
concentration of tissue Na+’ The slices from the 5-day preserved kidney were much less able to reaccumulate K+. During the first 60 min of incubation the concentration of K+ increased by only 40 meq/kg dry wt compared to an increase of 90-100 meq/kg dry wt in slices from control or 3-day perfused kidneys. Furthermore, during the second 60-min incubation of slices from the 5-day perfused kidney there was a loss of K+ and gain of Na+, indicating a decrease in the capability of slices from 5-day perfused kidneys to maintain ionic equilibrium. Effects of Perfusion Time on Energy Metabolism
Control tissue slices respire at a rate of 3 15 ? 75 bmol 0, consumed/hr, g dry wt. Slices from kidneys preserved for 3 and 5 days respire at rates 15% (267 2 50 p,mol
METABOLISM
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O,/hr, g dry wt) and 24% (240 _t 65 pmol O,/hr, g dry wt) less than control tissue, respectively. The differences between respiration rates of slices from 3- and 5-day preserved kidneys (9%) was not statistically significant. The rate of respiration in tissue slices from kidneys pretreated with deoxycoformycin and perfused for 3 and 5 days was not significantly different from that in kidneys perfused without this inhibitor. Mitochondria isolated from kidney cortex tissue preserved under these conditions are functionally similar to control mitochondria. The rate of ADP-stimulated respiration with pyruvate as substrate was decreased by 13% after 3 days of preservation; however, no decrease was observed in mitochondria isolated from 5-day perfused kidneys. There was a slight decrease in the respiratory control ratios (RCR) from about 4.6 to 4.0-4. I at 3 and 5 days of perfusion, respectively. The loss of RCR, however, was not reflected in a decrease in the coupling of oxidation to phosphorylation as indicated by a constant ADP:O ratio (2.11 2 0.1) in mitochondria isolated from control, 3-day, and 5-day perfused kidneys. Deoxycoformycin washout had no effect on mitochondrial activity. Slices prepared from control tissue lost ATP during the 20 min anoxic-cold incubation (Fig. 3). Normothermic incubation of these slices stimulated the resynthesis of ATP, but the final concentration was only about 50% of the initial control tissue concentration. This appeared attributable to a decrease in the total adenine nucleotide content of the slices from lo- 12 kmollg dry wt to 4-6 pmol/g dry wt (sum of AMP, ADP, and ATP determined by HPLC) during anoxic-cold incubation. Thus, the loss of ATP precursors limits, by about 50%, the maximum amount of ATP resynthesized following normothermic incubation. The ATP content in cortex tissue from 3-day preserved kidneys was greater than control concentrations due to perfusion
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1
Time
(min)
FIG. 3. ATP levels in tissue slices from kidneys perfused for 3 and 5 days. Methods are identical to those described in the legend to Fig. 1 and under Materials and Methods. Values are averages i- SEM (in parentheses). There is a statistically significant difference (P < 0.05) between ATP levels in tissue slices from 3-day (120 min incubation) and j-day (120 min incubation) perfused kidneys.
with adenosine and PO, (24). During the anoxic-cold incubation, these slices lost a smaller proportion of the ATP than control slices (Fig. 3). Normothermic incubation results in a rapid loss of ATP during the first 60 min. However, a steady-state concentration of ATP was maintained near control levels during the 120-min incubation of tissue slices. The initial loss of ATP may be due to the time required to activate oxidative phosphorylation and the rapid utilization of ATP to restore ionic equilibrium (Na+/K+ATPase) and cell volume during the initial period of normothermia. However, after 60 min the rate of synthesis and utilization of ATP was stabilized and concentrations of ATP equal to that in control tissue were obtained. The concentration of ATP in cortex tissue from 5-day perfused kidneys was less than that of the control. This is due to the fact that after 5 days of perfusion the adenosine concentration in the perfusate was reduced from the initial value of 10 mM to 1.O mM or less (24). During the anoxic-cold incubation a further decrease in the ATP concentration occurred. Normothermic incubation of these slices resulted in main-
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taining a steady-state concentration of ATP slightly lower than in slices from 3-day perfused kidneys (Fig. 3). Slices from the 5-day perfused kidneys are capable, however, of maintaining a rate of ATP synthesis approximately equal to the rate of utilization as indicated by the constancy of the ATP concentrations at 60 and 120 min of normothermic incubation. Thus, even though the initial concentration of ATP was lower in the kidney following 5 days of perfusion than after 3 days of perfusion, slices from kidneys preserved for the longer time period appeared capable of carrying out respiration, mitochondrial function, and ATP synthesis similar to slices from the 3day perfused kidney. Effect of Maintaining High Tissue ATP Levels on Functions of Tissue Slices after 5 Days of Preservation The breakdown of adenosine during perfusion of kidneys is suppressed by deoxycoformycin (24). Thus, at the end of preservation the concentration of cortex tissue ATP was approximately three times higher (in kidneys washed out with deoxycoformycin) than in kidneys perfused in the absence of deoxycorformycin (Fig. 4). During the anoxic-cold incubation associated with slice preparation there was a loss of ATP which continued upon rewarming the slices. This result was identical to results obtained with slices from kidneys preserved for 3 days. A steady-state concentration of ATP was reached at 60 min of incubation and maintained up to 120 min. The steady-state concentration of ATP (approximately 7.5 pmol/g dry wt) was over twofold greater than obtained in slices prepared from S-day preserved kidneys without deoxycoformycin. These data suggest that following 5 days of perfusion the cortex cells are fully capable of maintaining normal concentrations of ATP in the cell if precursors of ATP synthesis are not rate limiting.
ET AL
Maintaining high concentrations of ATP in the kidney for 5 days suppressed tissue edema that occurred during preservation (TTW = 3.9 vs 4.3 kg H,O/kg dry wt). The capacity of slices prepared from 5-day perfused kidneys to pump H,O out of the tissue, however, appeared similar (? deoxycoformycin) and was not affected by the presence of high concentrations of ATP. The only difference was that the steadystate level of TTW was about 20% less in the slices prepared from the kidney containing the high concentration of ATP. Following 60-120 min of incubation, the total tissue water in slices averaged 4.85 I+_0.75 kg/kg dry wt (low ATP, -deoxycoformycin) versus 3.90 t 0.50 kg/kg dry wt (high ATP, + deoxycoformycin). A comparison of the effects of 5-day perfusion ( + deoxycoformycin washout) on the reaccumulation of K+ and changes in tissue Naf in incubated slices is shown in Fig. 5. Although the rate of K+ uptake was similar in both cases during the initial 60min incubation, slices from kidneys containing a high concentration of ATP continued to reaccumulate K + and did not gain Na+ . Slices from kidneys with low concentrations of ATP (- deoxycoformycin) lost K+ and gained Naf during the second 60min incubation period. Effects of Perfusion on Saccharide Permeable Spaces The permeability of kidney slices from control, 3-day perfused, and 5-day perfused (2 deoxycoformycin) kidneys to radiolabeled saccharides of different molecular weight was measured to determine the effect of preservation on the passive permeability of tissue slices. The inulin permeable space (IPS) averaged 45-59% under all conditions of perfusion and was similar to the IPS obtained in control slices. The mannitol permeable space (MPS) also did not change as a result of the perfusion conditions and averaged 50-65%. The sucrose permeable space (SPS) increased from 47
METABOLISM
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PERFUSED KIDNEYS
I3 21
6 4-
2-
291
5 Dayf Deoxycoformyctn
(2 21
(0 77)
(O.ftl) yn
(0 701
5 DOY Deoxycoformycm
start of 30-c Incubotlon I
1
60
t b End of PreSerV0tlOn
Time
120
(min)
FIG. 4. Effect of deoxycoformycin washout on ATP synthesis in kidney slices from kidneys perfused for 5 days. Dog kidneys were initially washed out with deoxycoformycin as described under Materials and Methods. Slices were incubated and ATP was determined as indicated in the legends to Figs. 1 and 3. Values are averages + SEM (in parentheses).
t 3% (control) to 55 ? 9% (3-day per- SPS as a result of perfusion for 5 days fused) (statistically significant at P < 0.05, (t deoxycoformycin) from 54 t 7 to 44 & Student t test). Extending the preservation 6% (statistically significant at P < 0.05). time to 5 days had no further effect on the SPS. There was an apparent decrease in the DISCUSSION 300
-
Potossum
The viability of hypothermically perfused (27,50oy+ Deoxycofo’myc’n kidneys is dependent upon the capability of the organ after transplantation to rapidly restore normal cellular metabolism and repair preservation-induced damage. To effec‘351 5 cmySodum d-0 Deoxycoformycln tively initiate tissue repair, the transplanted E 150$o~------~?-‘--organ must not be subjected to normofq, 3I , e 100df thermic reperfusion damage. This could DO” + (m------a 06, 5Deorycoformycr t G-0) .s occur as a result of inefficient resynthesis w 50 of energy-rich compounds, increased cel:;z%Y c lular edema, loss of intracellular electroI , tEnd lytes, and the fatal sequellae of these events 120 0‘0’ 60 Prelcr”o11o” including decreased renal perfusion. Time Imin) In this study we attempted to simulate Fig. 5. Tissue Na+ and K+ in kidney slices from kidneys preserved (2 deoxycoformycin) for 5 days. the reperfusion conditions by using slices Methods are identical to those described in the legend from the preserved kidneys to study the into Fig. 1. Values are averages ? SEM (in parentheses) tegrated metabolic response during nor-
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mothermic (30°C) incubation in a serumlike, well-oxygenated medium. A comparison of the integrated metabolic response of tissue slices from kidneys preserved for 3 and for 5 days indicates the following similarities:
ET AL
cant difference between the concentration of ATP in slices incubated for 120 min from 3- and 5-day perfused kidneys, the actual intracellular concentration of ATP in tissue slices following 120 min incubation averaged 2.15 mM in the control, 1.8 mM in 3-day perfused, 1.35 mM in j-day perfused (1) 0, consumption of tissue slices is (- deoxycoformycin), and 3.77 mM in similar after 3 or 5 days of preservation and S-day perfused (f deoxycoformycin). is not greatly different from control tissue These values were obtained by dividing the slices. tissue concentration of ATP (p.mol/g dry (2) Mitochondrial activity is similar after wt) by the intracellular volume of water both periods of preservation and nearly (mg/g dry wt). The intracellular volume of identical to control values. water remained relatively constant at 51(3) The capability of slices to synthesize 56% of the total tissue water as determined and maintain a steady-state concentration by measuring the [3H]inulin permeable of ATP is similar after both periods of presspace. The value of the apparent concenervation. tration of ATP at half-maximal transport ac(4) The distribution of radiolabeled sactivity of the Na+/K+ ATPase in kidney tischarides in tissue slice water was similar for sues averages about 0.7 mM (12). Thus, kidneys preserved for 3 and 5 days. using the Michaelis relationship of enzyme The primary differences in the integrated rate to substrate concentration, an intrametabolic response are: cellular concentration of 1.35 mM ATP (120 hr-deoxycoformycin) would yield a V,,, of (I) Kidneys perfused for 3 days are about 75%. At ATP concentrations of 1.8 better able to regulate tissue edema than mM (3-day) the V,,, is about 81%, and at kidneys perfused for 5 days as indicated by ATP concentrations of 2.15 mM (control) the level of TTW and electrolytes at the end 0-E vmaxis about 86%. These values are, at of perfusion. best, only an approximate indication of the (2) Kidneys perfused for 3 days yield concentration of intracellular ATP available slices that are able to more effectively reacfor the Na+/K+ ATPase since the calculacumulate and maintain intracellular K+ and tion assumes an even distribution of ATP pump Na+ than slices from kidneys perthroughout the intracellular water. Howfused for 5 days. ever, on the basis of a comparison between the intracellular concentration of ATP in Under our conditions of hypothermic perfusion perservation, kidneys perfused tissue slices from kidneys perfused for 3 for 5 days appear less capable of carrying and 5 days and the similarities of estimated out important membrane-linked reactions V,,, (6% difference at 3 and 5 days), the (Na+/K+ pumping, volume control) than differences in ATP concentrations do not 3-day perfused kidneys. The cause of this appear to explain the differences observed difference is not completely clear, but does in volume regulation or ion pump activities. not appear due to a major difference in en- Other indications that ATP is not rate limergy metabolism. Slices from kidneys pre- iting to Na+/K+ ATPase acitivty in slices served for both time periods respire equally from kidneys preserved for 5 days come well, mitochondrial functions appear sim- from the results of Wheeler and Whittam ilar, and maintenance of a steady-state con- (31) and Pettersson (19). They show that centration of ATP in slices appear similar. maximal ATPase activity is obtained at conAlthough there was a statistically signifi- centrations of 0.5 mM ATP. The concentra-
METABOLISM
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tion of ATP in slices from 5-day preserved kidneys is maintained at approximately three times this concentration. The similar concentrations of ATP in slices from 3- and 5-day preserved kidneys cannot be taken to indicate metabolic competence of slices. The most that can be inferred is that under these conditions the metabolic machinery is capable of maintaining essentially similar concentrations of ATP. Another explanation for the loss of cell volume regulation and ion pump activity could be the loss of activity of the Naf/Kf ATPase. The effects of hypothermic storage of kidneys on the activity of the Na+/K+ ATPase has not been fully characterized. In kidneys cold-stored in an extracellular-type solution for up to 48 hr without continuous perfusion there was a loss of enzyme activity (30). However, the capability of slices to accumulate K+ and extrude Na+ was unimpaired. Cold storage of kidneys in an intracellular-type solution, however, did not affect the activity of the Na+/K+ ATPase (30). The effects of long-term hypothermic perfusion of kidneys with cryoprecipitated plasma on whole cell Na+/K+ ATPase activity are not clear. In three kidneys perfused for 168 hr there was a large loss of enzyme activity, whereas in two kidneys the enzyme activity was well preserved (20). The results reported here indicate that the IPS remains constant in tissue slices obtained from dog kidneys perfused for up to 5 days. Thus, hypothermic perfusion of dog kidney appears less damaging to the cell membranes than ischemia which results in an increase in the IPS in tissue slices (9). Mannitol rapidly equilibrates with over 60% of the fluid in tissue slices and the MPS is also unaffected by perfusion for up to 5 days. The SPS increases slightly (8%) after 3 days of perfusion and remains at this level for up to 5 days (7%) of perfusion (relative to control levels). Although this increase is small, it may reflect an increase in tissue
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water that is in equilibrium with sucrose and an increase in membrane permeability. However, the fact that 3-day perfused kidneys are viable whereas kidneys perfused for 5 days are nonviable suggests that the increase in the SPS is not related to viability or the differences in permeability observed in this study. The fact that there is no dramatic increase in permeability of tissue slices from 3- and 5-day perfused kidneys to saccharides suggests that the loss of K+ transport capabilities by the slices from the 5-day perfused kidney is not due to the formation of “pores” in the cell membrane. Thus, the cause of the loss of cell volume control and ion pumping activity in kidneys preserved for 5 days may be due to the inability of the tissue to utilize available ATP, a loss of enzyme activity (Na+/K+ ATPase), or an increase in the permeability properties of the cell membrane to electrolytes. A question often raised in organ preservation research concerns the role of the concentration of cellular ATP in organ viability (4, 5, 18). We have shown in this study that maintaining a high concentration of ATP for up to 5 days of perservation (by deoxycoformycin) does affect some aspects of tissue slice metabolism that may be important for preserving the viability of the tissue. In the presence of high concentrations of ATP, the tissue is less swollen at the end of preservation, and slices maintain a more normal level of TTW and cellular electrolytes during incubation at normothermia. Also, slices from kidneys containing a high concentration of ATP were able to maintain ATP concentrations equal to normal tissue concentrations during normothermic incubation. From these results, the maintenance of high concentrations of ATP during long-term preservation appears to preserve the metabolic integrity of the tissue. The true test of effectiveness of preservation is survival following transplantation.
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We are unable to test the effect of maintaining high concentrations of ATP by deoxycoformycin on viability since deoxycoformycin induces in combination with 10 mM adenosine some form of toxicity in kidneys preserved for periods of only l-3 days. Furthermore, although maintaining high concentrations of ATP during 5 days of perfusion yields tissue slices that appear to function better (TTW and ion pump activity) than slices from kidney preserved for 5 days without high concentrations of ATP, these slices do not function as well as slices from 3-day preserved kidneys. Thus, although it may be important to maintain high concentrations of ATP is tissue during longterm preservation, other changes in cellular metabolism (i.e., membrane dysfunction) may limit viability. Successful long-term preservation will require the development of methods to therapeutically correct multiple sites of tissue damage. REFERENCES 1. Belzer, E O., Hoffmann, R. M., and Southard, J. H. Kidney preservation. Surg. C/in. North Amer. 58, 261 (1978). 2. Belzer, F. O., Hoffmann, R. M., and Southard, J. H. Aerobic and anaerobic preservation of kidneys. In “Organ Preservation: Basic and Applied Aspects” (D. E. Pegg, I. A. Jacobsen, and N. A. Halasz, Eds.), pp. 253-260. MTP Press, Lancaster, England, 1982. 3. Belzer, F. O., Hoffmann, R. M., and Southard, J. H. A new perfusate for kidney preservation. Transplantation
33, 322-323 (1982).
4. Buhl, M. R., and Jensen, M. H. Metabolic inhibitors. In “Organ Preservation for Transplantation,” (A. M. Karow, Jr., and D. E. Pegg, Eds.), 2nd ed., pp. 497-515. Dekker, New York, 1981. 5. Calman, K. C. The prediction of organ viability. 1. An hypothesis. Cryobiology 11, l-6 (1974). 6. Cohen, G. L., Ballardie, F. W., Mainwaring, A., and Johnson, R. W. G. Lysosomal enzyme release during successful S-, 7-, and 8-day canine kidney storage. In “Organ Preservation: Basic and Applied Aspects” (D. E. Pegg, I. A. Jacobsen, and N. A. Halasz, Eds.), pp. 249-252. MTP Press, Lancaster, England, 1982. 7. Cohen, J. J., Chesney, R. W., Brand, P. H., Neville, H. F., and Blanchard, C. F. cY-Ketoglutarate metabolism and K’ uptake by dog
kidney slices. Amer. J. Physiol. 217, 161-169 (1969). 8. Estabrook, R. W. Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. In “Methods in Enzymology (R. W. Estabrook and M. E. Pullman, Eds.), Vol. 10, pp. 41-47. Academic Press, New York, 1967. 9. Ganote, C. E., Jennings, R. B., Hill, M. L., and Grochowski, E. C. Experimental myocardial ischemic injury. II. Effect of in viva ischemia on dog heart slice function in vitro. J. Mol. Cell. Cardiol. 8, 189-204 (1976). 10. Hoffmann, R. M., and Southard, J. H. Preservation technology and kidney transplantation. In “Renal Transplantation: A Multidisciplinary Approach” (S. N. Chatterjee, Ed.), pp. 59-88. Raven Press, New York, 1980. 11. Hoffmann, R. M., Southard, .I. H., Lutz, M. F., Mackety, A., and Belzer. E 0. Synthetic perfusate for kidney preservation: Its use in 72 hour preservation of dog kidneys. Arch. Surg. 118, 919-921 (1983). 12. Jacobsen, M. P., Rodriguez, H. J., Hogan, W. C., and Klahr, S. Mechanism of activation of renal Na+-K+-ATPase in the rat: Effects of reduction of renal mass. Amer. J. Physiol. 239, F281-F288 (1980). 13. Johnson, D., and Lardy, H. Isolation of liver or kidney mitochondria. In “Methods in Enzymology (R. W. Estabrook and M. E. Pullman, Eds.), Vol. IO, pp. 94-96. Academic Press, New York, 1967. 14. Little, J. R. Determination of water and electrolytes in tissue slices. Anal. Biochem. 7, 87-95 (1964). 15. Liu, W. P., Humphries, A. L., Russell, R., Stoddard, L. D., Garcia, L. A. and Serkes, K. D. Three- and seven-day preservation of dog kidneys with human plasma protein fraction IV. Surg. Forum 24, 316-318 (1973). 16. Manchester, K. L., and Young, F. G. The effect of insulin in vitro on the accumulation of amino acids by isolated rat diaphragm. Biochem. J. 75, 487-495 (1960). 17. Ozaki, A., Fukao, K., Sano, M., Okamura, T., and Iwasaki, Y. Five-day preservation of canine kidneys using a preservation machine. In “Organ Preservation: Basic and Applied Aspects” (D. E. Pegg, I. A. Jacobsen, and N. A. Halasz, Eds.), pp. 245-248. MTP Press, Lancaster, England, 1982. 18. Pegg, D. E. The biology of cell survival in vivo. In “Organ Preservation for Transplantation” (A. M. Karow, Jr., and D. E. Pegg, Ed.), 2nd ed., pp. 31-52. Dekker, New York, 1981.
METABOLISM
OF HYPOTHERMICALLY
19. Pettersson, S. Kidney viability during hypothermic preservation. Scund. J. Ural. Nephrol. Suppl. 23, l-24 (1974). 20. Pettersson, S., Claes, G., and Schersten, T. Correlation between sodium-, potassium-stimulated ATPase activity and renal function after tranplantation of canine kidneys. Ew. Surg. Res. 6, 18-25 (1974). 21. Resnick, O., and Hechter, 0. Studies on the permeability of galactose in muscle cells of the isolated rat diaphragm. J. Biol. Chrm. 224, 941954 (1957). 22. Rosenberg, L. E., Downing, S. J., and Segal, S. Extracellular space estimation in rat kidney slices using 14C-saccharides and phlorizin. Amer. J. Physiol. 202, 800-804 (1962). 23. Southard, J. H., Ametani, M. S.. Lutz, M. F., and Belzer, E 0. Effects of hypothermic perfusion of kidneys on tissue and mitochondrial phospholipids. Cryobiology 21, 20-24 (1984). 24. Southard, J. H., Lutz, M. F., Ametani, M. S., and Belzer, F. 0. Stimulation of ATP synthesis in hypothermically perfused dog kidneys by adenosine and PO,. Cryobiology 21, 13-19 (1984). 25. Southard, J. H., Senzig, K. A., and Belzer, F. 0. Effect of hypothermia on canine kidney mitochondria. C~obiology 17, 148- 153 (1980). 26. Southard, J. II., Senzig, K. A., Hoffmann, R. M., and Belzer, F. 0. Energy metabolism in kidneys stored by simple hypothermia. Transplant. Proc. 9, 1535 (1977). 27. Southard, J. H., Senzig, K. A., Hoffmann, R. M., and Belzer, F. 0. Toxicity of oxygen to mito-
28.
29.
30.
31.
32.
33.
34.
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chondrial respiratory activity in hypothermitally perfused canine kidneys. Trunspluntation 29, 459-461 (1980). Southard, J. H., van der Laan, N. Ch., Lutz, M. F., Pavlock, G. S., Belzer. J. P., and Belzer, E 0. Comparison of the effects of temperature on kidney cortex mitochondria from rabbit. dog, pig and human: Arrhenius plots of ADPstimulated respiration. Cryobiology 20, 395-400 (1983). Umbreit, W. W., Burris, R. H., and Stauffer, J. E “Mamometric Techniques,” pp. 79-93. Burgess, Minneapolis, Minn., 1957. Varkarakis. M. J., Papahadjopoulos, D., and Murphy, G. P. Renal adenosine triphosphatase in hypothermically stored canine kidneys. Cryobiology 12, 219-223 (1975). Wheeler, K. P., and Whittam, R. Some properties of a kidney adenosine triphosphatase relevant to active cation transport. Biochem. J. 85, 495507 (1962). Woods, J. E. Successful three- and seven-day preservation of canine kidneys. Arch. Surg. 102, 614-618 (1971). Wusteman, M. C., Jacobsen, I. A., and Pegg, D. E. A new solution for initial perfusion of transplant kidney. Scund. .I. Ural. Nephrol. 12, 281-286 (1978). Zakaria, M., and Brown, P. R. High performance liquid column chromatography of nucleotides, nucleosides and bases. J. Chromutog. 226, 267-290 (1981).
21, 285-295 (1984)
Comparison
of the Effect of 3- and 5-Day Hypothermic Perfusion Dog Kidneys on Metabolism of Tissue Slices’
of
JAMES H. SOUTHARD, MASAHIRO KUNIYOSHI, MARY F. LUTZ, MARY AMETANI, AND FOLKERT 0. BELZER Madison,
Wi.sconsin 53792
Hypothermic perfusion effectively preserves the viability of kidneys for 3 days. Long-term preservation (5 days or greater) has not been consistently obtained. In this study, the differences between kidneys perfused for 3 and 5 days were compared by determining the “integrated-metabolic” capabilities of tissue slices incubated in vitro at 30°C. The “integrated-metabolic” parameters determined include (1) respiration rates, (2) cell volume regulation [total tissue water (TTW) and saccharide permeable space], (3) rate of reaccumulation of K’ and pumping of Na+, (4) maintenance of ATP concentrations, and (5) mitochondrial functions. Conditions that result in high and low concentrations of ATP following perfusion of kidneys for 5 days were also compared for effects on tissue slice metabolism. The results indicate that energy metabolism in tissue slices is well preserved under all conditions and times of perfusion of kidneys. This includes average respiration rates (315 -+ 50, 275 t 35, and 255 2 45 pmol OJhrig dry wt at 0. 3, and 5 days, respectively, mitochondrial function [respiratory control ratio (RCR) = 4.6, 4.0, and 4.1 for 0, 3, and 5 days, respectively], and steadystate concentration of ATP in slices after incubation (4.0 t 1.45, 3.9 2 1.28, and 3.3 t 0.81 pmol/g/ dry wt, for 0, 3, and 5 days, respectively). The primary differences between 3- and j-day perfused kidneys were the capability of the slices to regulate ccl1 volume and rcnccumulate K’. Slices from kidneys perfused for 3 days maintained the TTW at 3.X kg/kg dry wt, a value similar to that of control tissue slices. However, slices from 5-day perfused kidneys remained swollen (TTW = 4.6 kg/kg dry wt). Also, slices from the 5-day perfused kidney pumped K+ at less than one-half the rate found in slices from control or 3-day preserved kidneys. No significant differences were apparent in the permeability properties of the tissue slices from kidneys perfused for 3 and 5 days to radiolabeled saccharides. The defects in membrane-linked transpor-t functions, resulting fr-om long-term kidney perfusion, were reduced in kidneys containing a high concentration of ATP. The results suggest that one factor which may limit successful preservation of kidneys is the increased membrane permeability (to electrolytes) which is partially prevented by maintaining elevated concentrations of tissue ATP during perfusion.
Successful kidney preservation is limited to 3 days of continuous hypothermic perfusion and (in our hands) 5 days yields consistently nonviable kidneys. The fact that longer periods of preservation can be obtained is suggested by the results of a few studies demonstrating 5 (or more) days of Received September 13, 1983; accepted October 17, 1983. ’ This work was supported by NIH Grant AM18624 and presented at the 20th Annual Meeting of the Society of Cryobiology, August 29-September I, 1983, Cambridge, England.
preservation (6, 15, 17,32). However, these results have been difficult to reproduce and survival has not been reliable. Two problems exist in achieving successful long-term kidney preservation: one is the preservation of vascular (endothelial) integrity; the other is preservation of the functional integrity of cellular metabolism. We have compared the effects of time of preservation on the levels of adenine nucleotides (24, 26), mitochondrial properties (26, 27, 28), lipid peroxidation (2), free fatty acid levels (23, 2.5), and phospholipid composition (23) in the kidney. In one study (24)
285 001 l-2240184 $3.00 Cnpyrlghl ‘E 1984 by Academic Press, Inc. All rights of reproductmn in any form rexwed.
286
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ET AL
we described a method to maintain high washout (33) at 0-4°C. In some cases kidconcentrations of ATP in kidneys perfused neys were washed out with Pegg’s washout for 5 days. The presence of an adenosine containing 40 mg/liter deoxycoformycin deaminase inhibitor (deoxycoformycin), (obtained as a gift from Dr. R. C. Jackson, and high concentrations of adenosine (10 Warner Lambert Co., Michigan) (24). The mM) and KH,PO, (25 mM) resulted in washout (250 ml/kidney) was delivered tissue concentrations of ATP three times from a height of 100 cm. Perfusion pressure greater than in kidneys perfused in the ab- was set initially at 60 mm Hg and stabilized sence of these additives. These data pro- for the duration of the experiment at 30-40 vide insight into the effects of hypothermic mm Hg. 0, (100%) was allowed to flow preservation on the kidney at the end of over the surface of the perfusate (2-3 liters/ perfusion, but do not indicate how well the hr); perfusate p0, = 300-400 mm Hg. kidney will function as an integrated metabolic unit following transplantation. These data also do not indicate if maintaining high Preparation and Incubation of Tissue Slices concentrations of tissue ATP contributes to the preservation of cell viability. At the end of the perfusion period, tissue In this study we compared the effects of slices were prepared from the kidney 3 and 5 days of continuous hypothermic cortex using a Stadie-Riggs microtome as perfusion of dog kidneys on the integrated described previously (7). The slices were metabolic functions of cortex tissue slices placed directly into the reaction medium (at incubated in a serum-like medium at nor- 0-4°C) for the time (20 min) necessary to mothermia (30°C). These studies were done complete the slicing. The normothermic to more fully understand the mechanism of (30°C) incubation was done in a Gilson difcryoinjury during perfusion and to observe ferential respirometer. Each flash conif maintaining high concentrations of tissue tained 5 ml of reaction medium. The reacATP suppressed the occurrence of cryoin- tion medium (KH) was similar to KrebsHenseleit solution and contained 120 mM jury. NaCl, 4.8 mM KCl, 1.16 mM CaCl,, 1.2 mM MgSO,, 16.7 mM NaH,P04, and 10 MATERIALS AND METHODS mM glucose. The pH was adjusted to 7.4 Perfusion of Dog Kidneys -+ 0.05 and the osmolarity was 292 + 5 Adult dogs of mixed breed (15-25 kg) mosmollliter. Three slices were placed in were used. Methods for perfusion preser- the reaction medium and incubated for 60 vation (6-10°C) have been described pre- to 120 min. 0, consumption was deterviously (3, 10). The perfusate contained hy- mined manometrically (29). For each experiment, four flasks were used. Two were redroxyethyl starch (HES), Na gluconate, KH,PO, (25 mM), and other common ad- moved at 60 min, and two at 120 min. Three ditives as described previously (11). In ad- slices were frozen in dry ice-acetone after dition, the perfusate also contained adeno- each time period and stored frozen for analsine (final concentration = 10 mM). Final ysis of ATP, and three slices were removed osmolality was 3 lo-320 mosmol/liter and for determination of total tissue water cation concentrations were Na+’ = 120- (TTW) and electrolytes (Na’ and K+). 130 meqlliter and K + = 25 meq/liter. The Tissue samples were also obtained from the basic perfusate has been shown to yield re- kidneys immediately after preservation, liable 3-day preservation (1 l), but inconsis- and after the cold incubation in KH, and tent survival following 5 days of perfusion. used for determination of ATP, TTW, and Kidneys were flushed out with Pegg’s electrolytes.
METABOLISM
OF HYPOTHERMICALLY
Determination of Saccharide Permeable Spaces The slices were placed in an incubation medium containing radiolabeled saccharides for determination of the permeability of the tissue to compounds of varying molecular weights (22). Six slices were placed into 10 ml of reaction medium and incubated for 60 and 120 min at 30°C in a Dubnoff metabolic shaking incubator under an atmosphere of 100% 0,. The reaction medium was basically Krebs-Henseleit buffer containing 0.1 mM inulin (MW = SSOO),1 mM sucrose (MW = 342), and I mM mannitol (MW = 180). The reaction medium used for the normothermic incubation also contained either [‘HI inulin, 0.25-0.35 $Zi/ ml; [‘4C]sucrose, 0.25-0.35 pCi/ml; or [‘4C]mannitol, 0.25-0.35 $i/ml. At the end of the incubation period, three slices from each flask were used to determine TTW. Three slices were also used for determination of radioactivity by the procedure of Rosenberg et al. (22). Briefly, slices were rinsed two times in cold reaction medium, blotted (two times to a side) on Whatmann No. 1 filter paper, weighed, and placed into 2 ml of H20. The slices were boiled for 5 min to completely equilibrate tissue water with the added H,O (16, 21). After centrifugation aliquots were placed into Aquasol (New England Nuclear), for counting in a Packard liquid scintillation spectrometer. The method for calculating the volume of tissue occupied by a given radiolabeled marker is based upon the level of radioactivity in the reaction medium, the level in the tissue slice, and the wet weight of the tissue slice as described by Rosenberg et al. (22). Analytical Methods Total tissue water and tissue concentrations of Nat and K+ were determined as previously described (14). Frozen tissue was quickly weighed, homogenized in HClO,, and extracted for adenine nucleo-
PERFUSED
KIDNEYS
287
tides (24, 26). Adenine nucleotide concentrations were determined by high-performance liquid chromatography (28, 34). Mitochondrial Isolation and Analysis Following preservation a portion of the kidney cortex was minced in cold sucrose for isolation of mitochondria (13). Mitochondrial protein (PC) was adjusted to 20 mgiml (Biuret) and the mitochondria were used for the determination of respiratory activity, respiratory control ratios, and ADP:O ratios as described previously (8, 25). The respiratory substrate was pyruvate plus malate. A minimum of six kidneys were used for each preservation period studied (perfusion for 3 days, 5 days, and 5 days + deoxycoformycin washout) and six kidneys were used as fresh controls. Kidneys were perfused in pairs and one was removed at 3 days and one at 5 days. Comparisons between 3- and 5-day perfused kidneys were studied using a nonpaired Student t test. RESULTS
Effect of Preservation Time on Swelling of Incubated Tissue Slices Hypothermic perfusion of kidneys leads to an increase in total tissue water due primarily to an expansion of the extracellular space (ECS) (25). At the end of perfusion, total tissue water increased from 3.5 kg/kg dry wt (control) to 4.0 kg/kg dry wt (3 day) and 4.25 kg/kg dry wt (5 day) (Fig. 1). Prior to incubating slices at 3O”C, there was an additional 30% increase in TTW due to the anoxia associated with the slicing technique and the incubation of slices in cold reaction medium (20 min). The restoration of normothermic conditions by incubating the slices at 30°C in a well-oxygenated serumlike medium (KH) resulted in a decrease in TTW to almost normal levels in slices from fresh tissue or from kidneys perfused for 3 days (Fig. 1). Slices obtained from 5-day perfused kidneys, however, remained,
288
SOUTHARD ET AL Potassium
5 Day
5Day
3 Day C0ntV3
FIG. 1. Total tissue water (TTW) in kidney slices from kidneys preserved for 3 and 5 days. Dog kidneys were perfused as described under Materials and Methods. Following the indicated time of perfusion tissue slices were prepared from the cortex and incubated for 20 min in cold KH. Total tissue water was determined as described under Materials and Methods, on tissue immediately after preservation, after 20 min of cold-anoxic incubation, and after 60 and 120min of incubation at 30°C. Values reported are averages + SEM. The SEM at the end of preservation averages 0.35 kg/kg dry wt. The difference between TTW in 3- and 5-day perfused kidneys is statistically significant (P < 0.05) at each time interval tested.
swollen and there was only a slight tendency to decrease cellular swelling during the normothermic incubation period. Effect of Preservation Time on Ion Pumping of Incubated Tissue Slices
During preservation the concentration of tissue K+ was reduced from about 278 meql kg dry wt (control) to 254 meq/kg dry wt (3-day) and 193 meq/kg dry wt (5day). The loss of K+ was accompanied by a gain in Na+ (Fig. 2). When returned to the normothermic incubation medium, control tissue slices reaccumulated K+ within 60 min and maintained a high concentration of K+ for up to 120 min. Sodium was pumped out of the cell concomitant with the uptake of K+. Slices from the 3-day preserved kidney also rapidly accumulated K+ at a rate approximately equal to that of control slices and also showed a decrease in the
1 I 1 t End Of 0 Ptarervotlon
1 60
I I20
Time (min) FIG. 2. Tissue Na+ and K+ in kidney slices from kidney preserved for 3 and 5 days. Methods are identical to those described in the legend to Fig. 1. Na+ and K* were determined as described under Materials and Methods. Values are averages with SEM in parentheses. K+ is expressed per kg dry wt and Na+ per kg wet wt.
concentration of tissue Na+’ The slices from the 5-day preserved kidney were much less able to reaccumulate K+. During the first 60 min of incubation the concentration of K+ increased by only 40 meq/kg dry wt compared to an increase of 90-100 meq/kg dry wt in slices from control or 3-day perfused kidneys. Furthermore, during the second 60-min incubation of slices from the 5-day perfused kidney there was a loss of K+ and gain of Na+, indicating a decrease in the capability of slices from 5-day perfused kidneys to maintain ionic equilibrium. Effects of Perfusion Time on Energy Metabolism
Control tissue slices respire at a rate of 3 15 ? 75 bmol 0, consumed/hr, g dry wt. Slices from kidneys preserved for 3 and 5 days respire at rates 15% (267 2 50 p,mol
METABOLISM
OF HYPOTHERMICALLY
O,/hr, g dry wt) and 24% (240 _t 65 pmol O,/hr, g dry wt) less than control tissue, respectively. The differences between respiration rates of slices from 3- and 5-day preserved kidneys (9%) was not statistically significant. The rate of respiration in tissue slices from kidneys pretreated with deoxycoformycin and perfused for 3 and 5 days was not significantly different from that in kidneys perfused without this inhibitor. Mitochondria isolated from kidney cortex tissue preserved under these conditions are functionally similar to control mitochondria. The rate of ADP-stimulated respiration with pyruvate as substrate was decreased by 13% after 3 days of preservation; however, no decrease was observed in mitochondria isolated from 5-day perfused kidneys. There was a slight decrease in the respiratory control ratios (RCR) from about 4.6 to 4.0-4. I at 3 and 5 days of perfusion, respectively. The loss of RCR, however, was not reflected in a decrease in the coupling of oxidation to phosphorylation as indicated by a constant ADP:O ratio (2.11 2 0.1) in mitochondria isolated from control, 3-day, and 5-day perfused kidneys. Deoxycoformycin washout had no effect on mitochondrial activity. Slices prepared from control tissue lost ATP during the 20 min anoxic-cold incubation (Fig. 3). Normothermic incubation of these slices stimulated the resynthesis of ATP, but the final concentration was only about 50% of the initial control tissue concentration. This appeared attributable to a decrease in the total adenine nucleotide content of the slices from lo- 12 kmollg dry wt to 4-6 pmol/g dry wt (sum of AMP, ADP, and ATP determined by HPLC) during anoxic-cold incubation. Thus, the loss of ATP precursors limits, by about 50%, the maximum amount of ATP resynthesized following normothermic incubation. The ATP content in cortex tissue from 3-day preserved kidneys was greater than control concentrations due to perfusion
PERFUSED KIDNEYS I2
289
1
Time
(min)
FIG. 3. ATP levels in tissue slices from kidneys perfused for 3 and 5 days. Methods are identical to those described in the legend to Fig. 1 and under Materials and Methods. Values are averages i- SEM (in parentheses). There is a statistically significant difference (P < 0.05) between ATP levels in tissue slices from 3-day (120 min incubation) and j-day (120 min incubation) perfused kidneys.
with adenosine and PO, (24). During the anoxic-cold incubation, these slices lost a smaller proportion of the ATP than control slices (Fig. 3). Normothermic incubation results in a rapid loss of ATP during the first 60 min. However, a steady-state concentration of ATP was maintained near control levels during the 120-min incubation of tissue slices. The initial loss of ATP may be due to the time required to activate oxidative phosphorylation and the rapid utilization of ATP to restore ionic equilibrium (Na+/K+ATPase) and cell volume during the initial period of normothermia. However, after 60 min the rate of synthesis and utilization of ATP was stabilized and concentrations of ATP equal to that in control tissue were obtained. The concentration of ATP in cortex tissue from 5-day perfused kidneys was less than that of the control. This is due to the fact that after 5 days of perfusion the adenosine concentration in the perfusate was reduced from the initial value of 10 mM to 1.O mM or less (24). During the anoxic-cold incubation a further decrease in the ATP concentration occurred. Normothermic incubation of these slices resulted in main-
290
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taining a steady-state concentration of ATP slightly lower than in slices from 3-day perfused kidneys (Fig. 3). Slices from the 5-day perfused kidneys are capable, however, of maintaining a rate of ATP synthesis approximately equal to the rate of utilization as indicated by the constancy of the ATP concentrations at 60 and 120 min of normothermic incubation. Thus, even though the initial concentration of ATP was lower in the kidney following 5 days of perfusion than after 3 days of perfusion, slices from kidneys preserved for the longer time period appeared capable of carrying out respiration, mitochondrial function, and ATP synthesis similar to slices from the 3day perfused kidney. Effect of Maintaining High Tissue ATP Levels on Functions of Tissue Slices after 5 Days of Preservation The breakdown of adenosine during perfusion of kidneys is suppressed by deoxycoformycin (24). Thus, at the end of preservation the concentration of cortex tissue ATP was approximately three times higher (in kidneys washed out with deoxycoformycin) than in kidneys perfused in the absence of deoxycorformycin (Fig. 4). During the anoxic-cold incubation associated with slice preparation there was a loss of ATP which continued upon rewarming the slices. This result was identical to results obtained with slices from kidneys preserved for 3 days. A steady-state concentration of ATP was reached at 60 min of incubation and maintained up to 120 min. The steady-state concentration of ATP (approximately 7.5 pmol/g dry wt) was over twofold greater than obtained in slices prepared from S-day preserved kidneys without deoxycoformycin. These data suggest that following 5 days of perfusion the cortex cells are fully capable of maintaining normal concentrations of ATP in the cell if precursors of ATP synthesis are not rate limiting.
ET AL
Maintaining high concentrations of ATP in the kidney for 5 days suppressed tissue edema that occurred during preservation (TTW = 3.9 vs 4.3 kg H,O/kg dry wt). The capacity of slices prepared from 5-day perfused kidneys to pump H,O out of the tissue, however, appeared similar (? deoxycoformycin) and was not affected by the presence of high concentrations of ATP. The only difference was that the steadystate level of TTW was about 20% less in the slices prepared from the kidney containing the high concentration of ATP. Following 60-120 min of incubation, the total tissue water in slices averaged 4.85 I+_0.75 kg/kg dry wt (low ATP, -deoxycoformycin) versus 3.90 t 0.50 kg/kg dry wt (high ATP, + deoxycoformycin). A comparison of the effects of 5-day perfusion ( + deoxycoformycin washout) on the reaccumulation of K+ and changes in tissue Naf in incubated slices is shown in Fig. 5. Although the rate of K+ uptake was similar in both cases during the initial 60min incubation, slices from kidneys containing a high concentration of ATP continued to reaccumulate K + and did not gain Na+ . Slices from kidneys with low concentrations of ATP (- deoxycoformycin) lost K+ and gained Naf during the second 60min incubation period. Effects of Perfusion on Saccharide Permeable Spaces The permeability of kidney slices from control, 3-day perfused, and 5-day perfused (2 deoxycoformycin) kidneys to radiolabeled saccharides of different molecular weight was measured to determine the effect of preservation on the passive permeability of tissue slices. The inulin permeable space (IPS) averaged 45-59% under all conditions of perfusion and was similar to the IPS obtained in control slices. The mannitol permeable space (MPS) also did not change as a result of the perfusion conditions and averaged 50-65%. The sucrose permeable space (SPS) increased from 47
METABOLISM
OF HYPOTHERMICALLY
PERFUSED KIDNEYS
I3 21
6 4-
2-
291
5 Dayf Deoxycoformyctn
(2 21
(0 77)
(O.ftl) yn
(0 701
5 DOY Deoxycoformycm
start of 30-c Incubotlon I
1
60
t b End of PreSerV0tlOn
Time
120
(min)
FIG. 4. Effect of deoxycoformycin washout on ATP synthesis in kidney slices from kidneys perfused for 5 days. Dog kidneys were initially washed out with deoxycoformycin as described under Materials and Methods. Slices were incubated and ATP was determined as indicated in the legends to Figs. 1 and 3. Values are averages + SEM (in parentheses).
t 3% (control) to 55 ? 9% (3-day per- SPS as a result of perfusion for 5 days fused) (statistically significant at P < 0.05, (t deoxycoformycin) from 54 t 7 to 44 & Student t test). Extending the preservation 6% (statistically significant at P < 0.05). time to 5 days had no further effect on the SPS. There was an apparent decrease in the DISCUSSION 300
-
Potossum
The viability of hypothermically perfused (27,50oy+ Deoxycofo’myc’n kidneys is dependent upon the capability of the organ after transplantation to rapidly restore normal cellular metabolism and repair preservation-induced damage. To effec‘351 5 cmySodum d-0 Deoxycoformycln tively initiate tissue repair, the transplanted E 150$o~------~?-‘--organ must not be subjected to normofq, 3I , e 100df thermic reperfusion damage. This could DO” + (m------a 06, 5Deorycoformycr t G-0) .s occur as a result of inefficient resynthesis w 50 of energy-rich compounds, increased cel:;z%Y c lular edema, loss of intracellular electroI , tEnd lytes, and the fatal sequellae of these events 120 0‘0’ 60 Prelcr”o11o” including decreased renal perfusion. Time Imin) In this study we attempted to simulate Fig. 5. Tissue Na+ and K+ in kidney slices from kidneys preserved (2 deoxycoformycin) for 5 days. the reperfusion conditions by using slices Methods are identical to those described in the legend from the preserved kidneys to study the into Fig. 1. Values are averages ? SEM (in parentheses) tegrated metabolic response during nor-
292
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mothermic (30°C) incubation in a serumlike, well-oxygenated medium. A comparison of the integrated metabolic response of tissue slices from kidneys preserved for 3 and for 5 days indicates the following similarities:
ET AL
cant difference between the concentration of ATP in slices incubated for 120 min from 3- and 5-day perfused kidneys, the actual intracellular concentration of ATP in tissue slices following 120 min incubation averaged 2.15 mM in the control, 1.8 mM in 3-day perfused, 1.35 mM in j-day perfused (1) 0, consumption of tissue slices is (- deoxycoformycin), and 3.77 mM in similar after 3 or 5 days of preservation and S-day perfused (f deoxycoformycin). is not greatly different from control tissue These values were obtained by dividing the slices. tissue concentration of ATP (p.mol/g dry (2) Mitochondrial activity is similar after wt) by the intracellular volume of water both periods of preservation and nearly (mg/g dry wt). The intracellular volume of identical to control values. water remained relatively constant at 51(3) The capability of slices to synthesize 56% of the total tissue water as determined and maintain a steady-state concentration by measuring the [3H]inulin permeable of ATP is similar after both periods of presspace. The value of the apparent concenervation. tration of ATP at half-maximal transport ac(4) The distribution of radiolabeled sactivity of the Na+/K+ ATPase in kidney tischarides in tissue slice water was similar for sues averages about 0.7 mM (12). Thus, kidneys preserved for 3 and 5 days. using the Michaelis relationship of enzyme The primary differences in the integrated rate to substrate concentration, an intrametabolic response are: cellular concentration of 1.35 mM ATP (120 hr-deoxycoformycin) would yield a V,,, of (I) Kidneys perfused for 3 days are about 75%. At ATP concentrations of 1.8 better able to regulate tissue edema than mM (3-day) the V,,, is about 81%, and at kidneys perfused for 5 days as indicated by ATP concentrations of 2.15 mM (control) the level of TTW and electrolytes at the end 0-E vmaxis about 86%. These values are, at of perfusion. best, only an approximate indication of the (2) Kidneys perfused for 3 days yield concentration of intracellular ATP available slices that are able to more effectively reacfor the Na+/K+ ATPase since the calculacumulate and maintain intracellular K+ and tion assumes an even distribution of ATP pump Na+ than slices from kidneys perthroughout the intracellular water. Howfused for 5 days. ever, on the basis of a comparison between the intracellular concentration of ATP in Under our conditions of hypothermic perfusion perservation, kidneys perfused tissue slices from kidneys perfused for 3 for 5 days appear less capable of carrying and 5 days and the similarities of estimated out important membrane-linked reactions V,,, (6% difference at 3 and 5 days), the (Na+/K+ pumping, volume control) than differences in ATP concentrations do not 3-day perfused kidneys. The cause of this appear to explain the differences observed difference is not completely clear, but does in volume regulation or ion pump activities. not appear due to a major difference in en- Other indications that ATP is not rate limergy metabolism. Slices from kidneys pre- iting to Na+/K+ ATPase acitivty in slices served for both time periods respire equally from kidneys preserved for 5 days come well, mitochondrial functions appear sim- from the results of Wheeler and Whittam ilar, and maintenance of a steady-state con- (31) and Pettersson (19). They show that centration of ATP in slices appear similar. maximal ATPase activity is obtained at conAlthough there was a statistically signifi- centrations of 0.5 mM ATP. The concentra-
METABOLISM
OF HYPOTHERMICALLY
tion of ATP in slices from 5-day preserved kidneys is maintained at approximately three times this concentration. The similar concentrations of ATP in slices from 3- and 5-day preserved kidneys cannot be taken to indicate metabolic competence of slices. The most that can be inferred is that under these conditions the metabolic machinery is capable of maintaining essentially similar concentrations of ATP. Another explanation for the loss of cell volume regulation and ion pump activity could be the loss of activity of the Naf/Kf ATPase. The effects of hypothermic storage of kidneys on the activity of the Na+/K+ ATPase has not been fully characterized. In kidneys cold-stored in an extracellular-type solution for up to 48 hr without continuous perfusion there was a loss of enzyme activity (30). However, the capability of slices to accumulate K+ and extrude Na+ was unimpaired. Cold storage of kidneys in an intracellular-type solution, however, did not affect the activity of the Na+/K+ ATPase (30). The effects of long-term hypothermic perfusion of kidneys with cryoprecipitated plasma on whole cell Na+/K+ ATPase activity are not clear. In three kidneys perfused for 168 hr there was a large loss of enzyme activity, whereas in two kidneys the enzyme activity was well preserved (20). The results reported here indicate that the IPS remains constant in tissue slices obtained from dog kidneys perfused for up to 5 days. Thus, hypothermic perfusion of dog kidney appears less damaging to the cell membranes than ischemia which results in an increase in the IPS in tissue slices (9). Mannitol rapidly equilibrates with over 60% of the fluid in tissue slices and the MPS is also unaffected by perfusion for up to 5 days. The SPS increases slightly (8%) after 3 days of perfusion and remains at this level for up to 5 days (7%) of perfusion (relative to control levels). Although this increase is small, it may reflect an increase in tissue
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water that is in equilibrium with sucrose and an increase in membrane permeability. However, the fact that 3-day perfused kidneys are viable whereas kidneys perfused for 5 days are nonviable suggests that the increase in the SPS is not related to viability or the differences in permeability observed in this study. The fact that there is no dramatic increase in permeability of tissue slices from 3- and 5-day perfused kidneys to saccharides suggests that the loss of K+ transport capabilities by the slices from the 5-day perfused kidney is not due to the formation of “pores” in the cell membrane. Thus, the cause of the loss of cell volume control and ion pumping activity in kidneys preserved for 5 days may be due to the inability of the tissue to utilize available ATP, a loss of enzyme activity (Na+/K+ ATPase), or an increase in the permeability properties of the cell membrane to electrolytes. A question often raised in organ preservation research concerns the role of the concentration of cellular ATP in organ viability (4, 5, 18). We have shown in this study that maintaining a high concentration of ATP for up to 5 days of perservation (by deoxycoformycin) does affect some aspects of tissue slice metabolism that may be important for preserving the viability of the tissue. In the presence of high concentrations of ATP, the tissue is less swollen at the end of preservation, and slices maintain a more normal level of TTW and cellular electrolytes during incubation at normothermia. Also, slices from kidneys containing a high concentration of ATP were able to maintain ATP concentrations equal to normal tissue concentrations during normothermic incubation. From these results, the maintenance of high concentrations of ATP during long-term preservation appears to preserve the metabolic integrity of the tissue. The true test of effectiveness of preservation is survival following transplantation.
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We are unable to test the effect of maintaining high concentrations of ATP by deoxycoformycin on viability since deoxycoformycin induces in combination with 10 mM adenosine some form of toxicity in kidneys preserved for periods of only l-3 days. Furthermore, although maintaining high concentrations of ATP during 5 days of perfusion yields tissue slices that appear to function better (TTW and ion pump activity) than slices from kidney preserved for 5 days without high concentrations of ATP, these slices do not function as well as slices from 3-day preserved kidneys. Thus, although it may be important to maintain high concentrations of ATP is tissue during longterm preservation, other changes in cellular metabolism (i.e., membrane dysfunction) may limit viability. Successful long-term preservation will require the development of methods to therapeutically correct multiple sites of tissue damage. REFERENCES 1. Belzer, E O., Hoffmann, R. M., and Southard, J. H. Kidney preservation. Surg. C/in. North Amer. 58, 261 (1978). 2. Belzer, F. O., Hoffmann, R. M., and Southard, J. H. Aerobic and anaerobic preservation of kidneys. In “Organ Preservation: Basic and Applied Aspects” (D. E. Pegg, I. A. Jacobsen, and N. A. Halasz, Eds.), pp. 253-260. MTP Press, Lancaster, England, 1982. 3. Belzer, F. O., Hoffmann, R. M., and Southard, J. H. A new perfusate for kidney preservation. Transplantation
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4. Buhl, M. R., and Jensen, M. H. Metabolic inhibitors. In “Organ Preservation for Transplantation,” (A. M. Karow, Jr., and D. E. Pegg, Eds.), 2nd ed., pp. 497-515. Dekker, New York, 1981. 5. Calman, K. C. The prediction of organ viability. 1. An hypothesis. Cryobiology 11, l-6 (1974). 6. Cohen, G. L., Ballardie, F. W., Mainwaring, A., and Johnson, R. W. G. Lysosomal enzyme release during successful S-, 7-, and 8-day canine kidney storage. In “Organ Preservation: Basic and Applied Aspects” (D. E. Pegg, I. A. Jacobsen, and N. A. Halasz, Eds.), pp. 249-252. MTP Press, Lancaster, England, 1982. 7. Cohen, J. J., Chesney, R. W., Brand, P. H., Neville, H. F., and Blanchard, C. F. cY-Ketoglutarate metabolism and K’ uptake by dog
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19. Pettersson, S. Kidney viability during hypothermic preservation. Scund. J. Ural. Nephrol. Suppl. 23, l-24 (1974). 20. Pettersson, S., Claes, G., and Schersten, T. Correlation between sodium-, potassium-stimulated ATPase activity and renal function after tranplantation of canine kidneys. Ew. Surg. Res. 6, 18-25 (1974). 21. Resnick, O., and Hechter, 0. Studies on the permeability of galactose in muscle cells of the isolated rat diaphragm. J. Biol. Chrm. 224, 941954 (1957). 22. Rosenberg, L. E., Downing, S. J., and Segal, S. Extracellular space estimation in rat kidney slices using 14C-saccharides and phlorizin. Amer. J. Physiol. 202, 800-804 (1962). 23. Southard, J. H., Ametani, M. S.. Lutz, M. F., and Belzer, E 0. Effects of hypothermic perfusion of kidneys on tissue and mitochondrial phospholipids. Cryobiology 21, 20-24 (1984). 24. Southard, J. H., Lutz, M. F., Ametani, M. S., and Belzer, F. 0. Stimulation of ATP synthesis in hypothermically perfused dog kidneys by adenosine and PO,. Cryobiology 21, 13-19 (1984). 25. Southard, J. H., Senzig, K. A., and Belzer, F. 0. Effect of hypothermia on canine kidney mitochondria. C~obiology 17, 148- 153 (1980). 26. Southard, J. II., Senzig, K. A., Hoffmann, R. M., and Belzer, F. 0. Energy metabolism in kidneys stored by simple hypothermia. Transplant. Proc. 9, 1535 (1977). 27. Southard, J. H., Senzig, K. A., Hoffmann, R. M., and Belzer, F. 0. Toxicity of oxygen to mito-
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