JOURNAL

OF SURGICAL

Functional V. Pharmacokinetics Perfused

27, 93-99 (1979)

RESEARCH

Preservation

of the Mammalian

of Dimethyl Sulfoxide at 37, 25, or 10°C followed

Kidney

(1.4 M) in Kidneys by Transplantation

(Rabbit and Dog) (Dog)‘s2

A. M. KAROW, JR., PH.D.,~ S. WIGGINS, B.S., G. 0. CARRIER, PH.D., R. BROWN, AND J.L. MATHENY, PH.D.~ Departments

of Pharmacology

and Surgery,

Medical

Submitted for publication

College of Georgia, September

Augusta,

Georgia 30902

15, 1978

Rabbit and dog kidneys were perfused for 30 min at 37°C with 1.4 M [“H]Me$O in a K+-Mg*+rich perfusate. Subsequently the kidneys were perfused for 30 min with MesSO-free perfusate. The rate of Me.$O uptake and washout as well as Me&SO distribution in the tissue were determined. It was found that equilibrium conditions were achieved within 30 min for both uptake and washout with T/M ratios approaching 1.0. The amount of MezSO in the cortex and medulla of rabbit kidneys was not significantly different. The same experiment was repeated with dog kidneys at 25 and 10°C. At these lower temperatures the rate of uptake and washout was significantly less, but the final concentration achieved within 30 min was the same as at 37°C. Dog kidneys flushed with a K+-Mg*+- rich solution, with or without 1.4 M dimethyl sulfoxide (Me,SO), were kept at lO”C, then reimplanted in the autologous host, and an immediate contralateral nephrectomy was performed. Of the dogs receiving kidneys treated with Me,SO-free solution, 86% survived; of the dogs receiving Me,SO-treated kidneys, 75% survived. Dog kidneys were perfused for 30 min with a K+-Mg2+-rich solution, with or without 1.4 M Me2S0, at 25 or 37°C. All kidneys were then perfused for 30 min with MetSO-free solution at the same temperature used for the first perfusion. All kidneys were then reimplanted in the autologous host and an immediate contralateral nephrectomy was performed. Of the dogs receiving kidneys perfused at 25°C with MezSO-free solution, 43% survived: of the dogs receiving kidneys perfused with Me*SO, 42% survived. Dog kidneys were also treated at 37°C in a manner similar to those at 25°C. Of the dogs receiving kidneys perfused at 37°C with Me$O-free solution, 80% survived; of the dogs receiving kidneys perfused with Me2S0, 67% survived. Other results indicate that perfusion with a closed circuit is superior to perfusion with an open circuit. Also, gradual administration and washout of Me,SO gives better renal survival than rapid changes in Me,SO concentration.

Dog kidney cryopreservation efforts must be reevaluated. Reports from several laboratories IS, 8, 9, 16, 17, 201 indicate that dog kidneys will occasionally survive freezing sufficiently well to serve as the sole source of renal function. The reasons for the disappointing results are difficult to deter-

mine, but certainly there is room for improvement. An analysis of these reports clearly demonstrates two points: Dimethyl sulfoxide (Me,SO) in concentrations of 1 to 2 M is a useful cryoprotectant for dog kidneys and electromagnetic radiation is suitable for thawing these kidneys. This paper focuses on Me2S0. The problem of initial perfusion in organ cryopreservation is substantially different from that of perfusion preservation. The purpose of perfusion preservation is mainly to maintain a temperature that will minimize metabolic injury. Much collective experience from many laboratories proves that

’ A portion of this report, that dealing with rabbit kidneys, was presented at the 12th annual meeting (1975) of the Society for Cryobiology [ 193. 2 The research described in this report was supported by USPHS-NIH Grants AM-17816 and GM-08472, 3 To whom reprint requests should be addressed. 4 Present address: Department of Oral Biology, School of Dentistry, University of Kentucky, Lexington, Ky. 40506. 93

0022-4804/79/080093-07$01.00/O Copyright All rights

6 1979 by Academic Press, Inc. of reproduction in any form reserved.

94

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temperatures of 6 to 10°C achieve this objective for kidneys. Furthermore, the best solution for pump perfusion-preservation has traditionally been one similar in composition to plasma. Previous efforts by most investigators studying renal cryopreservation, including those cited above, have attempted to use perfusion preservation techniques during the initial perfusion. But the purpose of perfusion prior to cryopreservation is mainly to administer a cryoprotectant drug such as dimethyl sulfoxide. Drug diffusion into tissues will theoretically be facilitated by warm temperatures, but the risk of metabolic injury to tissues increases at warm temperatures. Me,SO increases this risk of metabolic injury because it accelerates the loss of cellular K+ in hypothermic kidneys [15]. We learned from other investigators [3, 151 that this Me,SO effect can be reduced by a K+-Mg2+-rich perfusate. We [6, II] performed previous in vitro experiments on rabbit kidneys which affirmed the superiority of a K+-Mg2+-rich perfusate in comparison to a plasma-like perfusate at temperatures warmer than 6 to 10°C. In other in vitro experiments we [12, 141 investigated the toxicity of various Me,SO concentrations perfused for various periods of time in the normothermic rabbit kidney. We learned that rabbit kidneys perfused for 60 min at 37°C with 1.4 M (10 mUdI) Me,SO in a K+-Mg2+-rich perfusate yielded physiological and ultrastructural changes quite similar to control (Me,SO-free) kidneys, but higher Me,SO concentrations (2.1 and 2.8 M) produced appreciable toxic effects. The purpose of this report is to substantiate that perfusion at warm temperatures does improve renal distribution of Me$O and that dog kidneys treated in this way, when transplanted, can serve as the sole source of renal support.

VOL. 27, NO. 2, AUGUST

1979

nonrecirculating perfusion system. Details of the surgical procedure and the nonrecirculating perfusion apparatus have been previously described [6]. Each liter of perfusate (solution A) contained NaCl, 96.2 mM; KCl, 40.3 mM; NaCHO,, 11.9 mM; CaCl,, 1.7 mM; MgS04, 12.5 mM; dextrose, 11.1 mM; heparin, 2000 units per liter; and isoxsuprine HCl (Vasodilan, Mead Johnson), 100 mg. Solution A has an osmolarity of 310 ? 10 mOsm. Solution A containing Me,SO (1.4 M) was made by substituting Me,SO on a volume basis for 10% of solvent water. The flow rate was maintained between 1.O and 1.3 ml/g/min; the pressure fluctuated between 80 and 90 mm Hg. Throughout the experiments the temperature was maintained at 37”C, the oxygen tension was approximately 550 mm Hg, and the pH was 7.35 to 7.38. Following a lo-min control perfusion, kidneys were perfused with 1.41 M [3H]Me,S0 (0.88 $X/ml). The total volume of venous and of ureteral effluents were collected at 2-min intervals for 30 min. The kidneys were then perfused with MezSO-free perfusate for 30 min and similar 2-min samples were collected. A portion (0.1 ml) of each perfusate sample was mixed with Dimilume 30 (Packard) scintillation “cocktail” and counted with a Packard Tri-Carb spectrometer. In other experiments kidneys were perfused for either 5, 10, 15, or 30 min with [3H]Me,S0. Tissue samples (2 100mg) were immediately taken from cortex and medulla. Tissue samples were also taken from kidneys following the 30-min washout perfusion. Tissue samples were blotted for 90 set on Whatman No. 1 filter paper and weighed to 20.1 mg. The tissue was solubilized with 1 mL Soluene 350 (Packard) overnight at 40°C. The solubilized tissue was then added to 9 ml Dimilume 30 (Packard) scintillation “cocktail” and counted. MATERIALS AND METHODS Me,SO distribution in dog kidneys. Adult Me,SO distribution in rabbit kidneys. The male mongrel dogs (15 to 20 kg) were anesleft kidney was removed from New Zealand thetized by intravenous injection of sodium albino rabbits (2 to 3 kg) and connected to a pentobarbital (20 mg/kg). The left kidney

KAROW ET AL,: PRESERVATION

3 fi 0

OF MAMMALIAN

3tkht+S(r IN URINE 8 VENOUS EFFLUENT FROM PERFUSED RABBIT KIWEYS loo-

E ::

60-

2

60-

% t

40-

g

20.

-VENOUS -URINE

EFFLUENT

f” ‘0

B

IO

40

20

UPTAKE 8 EFFLUX 2 $ 0

95

KIDNEY

OF k-fd.~O

50

IN RABBIT

60

I 120

KIDNEY

100 MEDULLA o CORTEX

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P a 2

60

p L rf

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TI&IN~ FIG. 1. (Top) Time course of change in concentration of tritiated dimethyl sulfoxide ([3H]Me2SO) in urine and venous effluents from rabbit kidneys perfused at 37°C with 1.4 M MelSO in a K+-Mg2+rich perfusate. After 30 min the kidneys were perfused with MelSO-free perfusate. (Bottom) Time course of change in rabbit kidney concentration of Me,SO when kidneys are perfused as described for the top graph. Each point represents the mean of samples from four or more rabbit kidneys. Error bars indicate ? 1.0 SE.

was exposed by a ventral midline abdominal incision. The kidney, freed of fat and connective tissue, was flushed with solution A and then placed in a perfusion machine (MOX , Waters Instruments). Each experiment was performed at one of three temperatures: 37, 25, or 10°C. Arterial pressure was held constant at 70 mm Hg. Perfusate flow rate was on the order of 1 ml/g/min. Kidneys were perfused with 1.41 M ,[3H]Me,S0 (1 .O @Zi/rnl) in solution A; the MezSO concentration was not gradually increased during the perfusion. From each left kidney, perfusate samples and a needle biopsy were obtained at 0, 2, 5,10,20, and 30 min during perfusion. Each right kidney was perfused for 30 min in a manner identical to the left kidneys except that tissue samples were not taken. Each right kidney was then perfused for 30 min

with MeeSO-free solution A. Perfusate samples and a needle biopsy were obtained at 0, 2, 5, 10, 20, and 30 min from each right kidney undergoing washout perfusion. The perfusate effluent and renal tissue from all kidneys were prepared for scintillation counting in a manner identical to that described for rabbit kidneys. Tissuelmedium ratios (TIM) were calculated and reported as percentage of maximum Me2S0 concentration. Transplantation

of Me2S0 dog kidneys.

Adult male mongrel dogs (15 to 20 kg) were anesthetized with thiamylal and the left kidney was removed as described above. Additional anesthetic was administered as required throughout the operative procedure. Intravenous lactated Ringer’s solution, 500 ml, maintained dog hydration and provided venous access during surgery.

96

JOURNAL OF SURGICAL RESEARCH: VOL. 27, NO. 2, AUGUST 1979

in oxygen, resulting in a final pH of 7.40 2 0.05 and an oxygen tension of approxi25°C mately 250-275 mm Hg. IOT After an hour’s preservation each kidney was reimplanted autologously, connected to the right iliac artery and vein using the Teflon cuff-ligature method of end-to-end anastomosis. The ureter was reimplanted by munions through the bladder. Heparin, 250 units/kg, was administered intravenously TIMEhIN) immediately prior to releasing the renal vasFIG. 2. Time course of change in tissue concentration cular clamps. With the resumption of renal of tritiated dimethyl sulfoxide ([3H]MeZSO) in dog kidblood flow, a solution (1 .Oliter) of dextrose neys perfused at 37, 25, or 10°C with 1.4 M MelSO in a K+-Mg*+-rich perfusate. After 30 min the kidneys (25 g) and mannitol (12.5 g) was adminiswere perfused with Me.$O-free perfusate. Each point tered intravenously. An immediate contrarepresents the mean of samples from four or more dog lateral nephrectomy was performed. kidneys. The TliZ at 37°C is significant (P < 0.05) from Postoperative management included penthat at 25 and 10°C. icillin, one million units daily for 7 days, and warfarin sodium (Coumadin), 5 mg Some kidneys were flushed (not continu- orally every other day for 30 days. Serum ously perfused) at a pressure of 20 to 30 creatinine was measured periodically. Glomm Hg with cold (10°C) solution A. Other merular filtration rate (GFR) was deterkidneys were continuously perfused with mined periodically by creatinine clearance. solution A at either 25 or 37°C. In each in- Dogs which survived 14 days were termed in our experience, anephric stance, hydroxyethyl starch (McGaw), 30 “survivors”; g per liter, was added to solution A which dogs live no longer than 5 days and attained was then filtered (0.22 pm, Millipore) im- a mean creatinine of 16 mg/dl at death. Stamediately prior to perfusion, Two different tistical analysis was performed on data from “matched” kidneys. perfusion systems were used. At 25” kidneys were perfused on an apparatus assembledby connecting with Silastic RESULTS tubing the following components in seMe$O Distribution quence: a peristaltic “finger” pump (Model The results with rabbit kidneys are sum120, Harvard Apparatus), a membrane oxygenator (Travenol), a heat exchanger (Trav- marized in Fig. 1. Rabbit kidneys perfused enol), a bubble trap, and a kidney chamber at 37°C with 1.4 M Me,SO in solution A (Travenol). Flow and pressure were meas- achieve uniform tissue distribution throughured at the bubble trap. This apparatus was out the cortex and medulla within 30 min; used as both an open, nonrecirculating unit, T1,2is 6 min, There is no significant differand as a closed circuit. In the open circuit ence between the cortex and medullary conmode a perfusate reservoir “fed” the pump centrations at the end of 30 min. MezSO and the perfusate from the kidney was dis- is washed from rabbit kidneys by perfusion carded. In the closed circuit mode the drain at 37°C with solution A; T1,* is 7 min. This washout is 90% complete within 30 min; perfrom the kidney chamber fed the pump. At 37°C kidneys were perfused on a com- fusion for an additional hour has minimal mercially manufactured renal preservation effect in reducing the residual tissue concentration. Me,SO concentration in urine system (MOX-100, Waters Instruments). In all perfusion experiments the arterial and venous effluents followed the same time pressure was held constant at 70 mm Hg. course as the concentration in tissue. The results with dog kidneys are sumThe perfusate was oxygenated with 3% COz UPTAKE 8 EFFLUX OF 3H-Me>0

IN DOG KIDNEY -

IO

20

30

40

50

374c

60

KAROW ET AL.: PRESERVATION

OF MAMMALIAN

97

KIDNEY

TABLE 1 EFFECT OFRATEOF MepSO ADMINISTRATION AND WASHOUT" Post-transplant Perfusion dynamics Group (rate) Rapid Slow

n

Flow rate (ml/g/min)

Pressure (mm Hg)

Weight gain (%)

Survivors (n)

Serum creatinine (mddl)

Resistance (pressure/flow)

6 9

0.14 2 0.06 0.91 + 0.03

65 2 4 49 * 3

42 f 7 33 f 6

2 8

2.2 r 0.8 4.2 f 0.6

91 -c 10 54 f 4

DAll kidneys perfused on open circuit for 60 min at 2X, then autologously reimplanted. All values are mean f SE. See text for description of method of Me,SO administration and washout.

marized in Fig. 2. Dog kidneys perfused at 37°C with 1.4 M Me,SO in solution A achieve uniform tissue distribution within 30 min; T1,2is 0.5 min. At colder temperatures, 25 and lO”C, the rate of tissue saturation is significantly less than at 37°C but is complete within 30 min. All measurements were made on tissue samples containing approximately equal amounts of medullary and cortical material. Me$O is washed from dog kidneys by perfusion at 37°C with solution A; Tllz is 0.5 min. Washout at colder temperatures (25 and 1O’C) is slower than at 37°C; T,,, is about 6 min. Transplantation or 37°C

after Me,SO at 10, 25,

One kidney taken from each of seven dogs and flushed (not continuously perfused) with solution A at 10°C was maintained at this temperature for 1 hr and then reimplanted. Six dogs (86%) survived. The creatinine postoperatively, measured on Days 1, 4, 7, 10, 14, and 21, had a mean peak value of 1.75 + 0.01 mg/dl and never exceeded 2.5 mg/dl. One kidney taken from each of four dogs and flushed with solution A containing 1.4 M Me,SO was maintained at 10°C for 1 hr and then reimplanted. Three dogs (75%) survived. The creatinine postoperatively, measured on Days 1, 4, 7, 10, 14, and 21, had a mean peak value of 5.2 + 1.8 mg/dl. One kidney taken from each of seven dogs and continuously perfused with solution A at 25°C was maintained at this temperature

for 1 hr and then reimplanted. Three dogs (43%) survived. Serum creatinine of the survivors had a mean peak value of 4.5 2 1.2 mg/dl and creatinine clearance (GFR) was 1.0 + 0.2 mL/min. One kidney taken from each of 12 dogs and perfused with solution A containing 1.4 M Me,SO was maintained at 25°C for 1 hr and then reimplanted. Five dogs (42%) survived. Serum creatinine of the survivors had a mean peak value of 4.0 + 0.7 mg/dl and creatinine clearance (GFR) was 1.0 +- 0.3 mL/min . One kidney taken from each of five dogs and perfused with solution A at 37°C was maintained at 37°C for 1 hr and then reimplanted. Four dogs (80%) survived. Serum creatinine of the survivors had a mean peak of 3.5 mg/dl and creatinine clearance (GFR) was 1.3 mYmin. One kidney taken from each of three dogs and perfused with solution A containing 1.4 M MezSO was maintained at 37°C for 1 hr and then reimplanted. Two dogs (67%) survived. Serum creatinine of the survivors had a mean peak value of 3.3 mg/dl and creatinine clearance (GFR) was 0.90 ml/min. Rate of MezSO Administration Washout

and

To evaluate whether rapid or slow administration of Me,SO affected post-transplant renal survival, six dog kidneys placed on the open circuit at 25°C were perfused for 30 min with 1.4 M Me,SO in solution A, and then perfused with Me,SO-free solution A

98

JOURNAL TABLE

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2

EFFECT OF OPEN AND CLOSED CIRCUITS ON POST-TRANSPLANT SURVIVAL”

Pressure (mm Hg)

Resistance (pressure/ ROW)

Group

n

FIN (mL/gfmin)

Closed Closed + Me,SO Open Open + Me,SO

6

1.6 f 0.1

65 + 2

36 t 1.7

3 5

1.8 + 0.1 1.0 ? 0.1

63 -c 1 71 k 7

41 k 1.6 66 t 2

15

0.8 + 0.1

71 2 6

57 k 3

a All kidneys perfused for 60 min at 2X, then autologously reimplanted. All values are mean 2 SE. See text for desctiption of method of Me,SO administration and washout.

for 30 min. Nine other kidneys placed on the open circuit at 25°C were perfused with gradually increasing concentration of Me,SO so that after 15 min the final concentration was 1.4 M. This second group was perfused with 1.4 M Me,SO for 30 min and then the Me&SO concentration was reduced to zero over a 15min period. All kidneys were transplanted. The results are summarized in Table 1. Effect of Open and Closed Circuits on Post-Transplant Survival Some kidneys were perfused at 25°C on an open circuit; others were perfused on a closed circuit. The results, presented in Table 2, show that closed-circuit kidneys have lower resistance regardless of whether the perfusate contains Me&SO. DISCUSSION

Our results demonstrate that dog kidneys survive perfusion of 1.4 M Me,SO at normothermia. Perfusion at normothermia results in significantly faster distribution of Me,SO in renal tissues than perfusion at 25 or 10°C. This is an important contribution to solving the problem of large organ cryopreservation because it provides a means by which good tissue distribution of cryoprotectant Me&SO can be attained [19, 211 without inflicting lethal damage to dog kidneys. Small, Feduska, and Filo [21] reported the perfusion of dog kidneys with 1.4 M MezSO at 8

VOL. 27, NO. 2, AUGUST

1979

to 10°C. The rate and quality of Me,SO distribution was substantially less than that achieved by us. They reported that after 80 min of perfusion, the cortex attained a T/M ratio of about 90% whereas the medulla attained about 60%. After a 40-min washout the cortex and medulla had a residual tissue concentration of about 9%. When these kidneys were transplanted and an immediate contralateral nephrectomy was performed, the dogs survived with good renal function. A K+-Mg2+-rich solution is a useful vehicle with which to administer and to wash Me,SO from dog kidneys. In 1950, Cross and Taggart [4] first recognized the potential usefulness of K+-Mg2+-rich solution for renal preservation. Keeler and associates [ 151demonstrated that such a solution perfused for 3 hr at 0°C into dog kidneys yielded viable kidneys upon reimplantation with delayed contralateral nephrectomy, whereas Tyrode’s solution did not yield viable kidneys; our [12, 141results with normothermic perfusion of rabbit kidneys confirmed this difference. Our observation that a closed perfusion circuit provides better renal preservation is of some importance. We would have predicted that an open, nonrecirculating circuit would have been superior in that metabolites and other cellular products would be removed from further renal exposure. The reason for the closed circuit superiority is not obvious; perhaps the closed circuit “conditions” the perfusate much in the same fashion that cells in tissue culture condition their own media, improving its growth-promoting qualities. The observation that rapid Me,SO administration has certain deleterious effects (increased weight gain, increased resistance) ultimately manifested in a low survival rate can be explained by osmotic pressure gradients. Me2S0, even at 25”C, exerts considerable osmotic force because of its low diffusability relative to water. Perfusion of large quantities of Me,SO into kidneys over a short period of time results in cellular dehydration and water accumulation in the extracellular spaces. This tissue water compressed the vascular bed and thereby in-

KAROW

ET AL.:

PRESERVATION

creased vascular resistance. Similar osmotic phenomena has been observed by investigators of glycerol as a cryoprotectant

[l, 21. For optimal administration of Me,SO used as a renal cryoprotectant, we recommend a brief (2- to 5-min) perfusion with Me,SO at 37°C. During this normothermic perfusion Me&SOconcentration should gradually be raised from zero to 1.4 M in the perfusate. After this initial perfusion, the temperature of the kidney should be reduced to 10°C over a period of 20 min. The importance of slow cooling has been previously reported [7]. For optimal removal of Me,SO from the kidney, we recommend that the concentration of MezSO be gradually reduced to zero over a 20- to 30-min period. Throughout this time the temperature should be maintained at 25°C. ACKNOWLEDGMENT We are grateful to McGaw Laboratories for the gift of hydroxyethyl starch.

REFERENCES 1. Brada, D. R., and Schloerb, P. R. Dynamics of

glycerol addition to the kidney. Surg. Gynecof. Obster. 121: 1004, 1965. 2. Cady, B., Barner, H. B., Rivers, R. J., Jr., Haynes, L. L., and Watkins, E., Jr. Glycerolization of the canine kidney. I. Fluid exchanges. Cryobiology 3: 76, 1%6. 3. Carruthers, R. K., Clark, P. B., Anderson, C. K., and Parsons, F. M. Tubular function demonstrated in rat kidneys after storage at -79°C. Brir. J. Ural. 41: 186, 1969.

4. Cross, R. J., and Taggart, J. V. Renal tubular transport: Accumulation of p-aminohippurate by rabbit kidney slices. Amer. J. Physiol. 161: 181, 1950. 5. Deitzman, R. H., Rebelo, A. E., Graham, E. F., Crabo, B. G., and Lillehei, R. C. Long-term functional success following freezing of canine kidneys. Surgery 74: 181, 1973. 6. Fonteles, M. C., Jeske, A. H., and Karow, A. M., Jr. Functional preservation of the mammalian kidney. I. Normothermia, low flow perfusion. J. Surg. Res. 14: 7, 1973.

7. Fonteles, M. C., and Karow, A. M., Jr. Vascular alpha adrenotropic responses of the isolated rabbit

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KIDNEY

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kidney at 1s”C. Arch. Int. Pharmacodyn. Ther. 227: 195, 1977. 8. Guttman, F. M., Lizin, J., Robitaille, P., Blanchard, H., and Turgion-Knaak, C. Survival of canine kidneys after treatment with dimethyl sulfoxide, freezing at -80°C and thawing by microwave illumination. Cryobiology 14: 559, 1977. 9. Halasz, N. A., Rosenfield, H. A., Orloff, M. J., and Seifert, L. N. Whole organ preservation. II. Freezing studies. Surgery 61: 417, 1967. 10. Henderson, I. W. D., Bickis, I. J., and Edwards, P. Some observations about the dimethyl sulfoxide permeation in tissues of dog kidneys during perfusion. Cryobiology 3: 373, 1967. 11. Jeske, A. H., Fonteles, M. C., and Karow, A. M., Jr. Functional preservation of the mammalian kidney. II. Ultrastructure with low-flow perfusion at normothermia. J. Surg. Res. 15: 4, 1973. 12. Jeske, A. H., Fonteles, M. C., and Karow, A. M., Jr. Functional preservation of the mammalian kidney. III. Ultrastructural effects of perfusion with dimethylsulfoxide (DMSO). Cryobiology 11: 170, 1974.

13. Karow, A. M., Jr., Holst, H. I., and Ecker, H. A. Organ cryopreservation: Renal and cardiac experience. In A. M. Karow, Jr., G. Abouna, and A. L. Humphries, Jr., (Eds.), Organ Preservation for Transplantation. Boston: Little, Brown, 1974. p. 274. 14. Karow, A. M., Jr., and Jeske, A. H. Functional preservation of the mammalian kidney. IV. Functional effects of perfusion with dimethyl sulfoxide (DMSO) at normothermia. Cryobiology 13: 448, 1976. 15. Keeler, R., Swinney, J., Taylor, R. M. R., and Uldah, M. B. The problem of renal preservation. Brit. J. Ural. 38: 653, 1966.

16. Kubota, S., and Lillehei, R. C. Some problems associated with kidneys frozen to -50°C or below. Low Temp. Med. 2: 95, 1976.

17. Lehr, H. B. Progress in long-term organ freezing. Transplant. Proc. 3: 1565, 1971. 18. Malinin, G. I. Cytotoxic effect of dimethyl sulfoxide on the ultrastructure of cultured rhesus kidney cells. Cryobiology 10: 22, 1973. 19. Matheny, J. L., and Karow, A. M., Jr. Kinetics of dimethyl sulfoxide uptake and washout from the rabbit kidney. Cryobiology 12: 576, 1975. 20. Mundth, E. D., Defalco, A. J., and Jacobson, Y. G. Functional survival of kidneys subjected to extracorporeal freezing and transplantation. Cryobiology 2: 62, 1965. 21. Small, A., Feduska, N. J., and Filo, R. S. Function of autotransplanted kidneys after hypothermic perfusion with dimethylsulfoxide. Cryobiology 14: 23, 1977.