Effects of Dehydration-freezing-thawing on the Preservation and Homotransplantability of Renal Tissue

THE JOURNAL OF UROLOGY

Vol. 82, No. 4, October 1959 Printed in U.S.A.

EFFECTS OF DEHYDRATION-FREEZING-THAWING ON THE PRESERVATION AND HOMOTRANSPLA-;\;"TABILITY OF RE-;\;"AL TISSUE JUAN C. BELTRAN

HERMAN T. BLUMENTHAL

AND

From the Institute of Experimental Pathology, the Jewish Hospital and the Homer G. Phillips Hospital, St. Louis, Nlo.

The modern era of transplantation investigation derives from the attempts of Ollier1 and also Thiersch, 2 towards the end of the last century, to transplant skin and bone. The host response to auto- and homografts of kidney tissue was studied in the guinea pig by Myer 3 as long ago as 1913, and by Loeb 4 in 1917. The latter investigator concluded that lymphocytic infiltration was a very prominent and constant feature found in homografts and was responsible in part for the destruction of the latter. On the other hand, lymphocytic infiltration into an autograft was insignificant and rarely observed. In general, Loeb observed that organization of a homograft of renal tissue was far more intense and advanced than in an autograft of comparable duration. Homografting of whole kidneys with establishment of a blood supply has been attempted in the human and other mammals, and has been uniformly unsuceessful. In the dog, for example, such renal homografts follow a relatively constant course, with survival and function for periods varying from 3 to 15 clays, at which time overt rejection occurs. On the other hand, autografted kidneys prepared in the same manner as homografts persist indefinitely, with excellent function. This manuscript was awarded second prize in the essay competition sponsored annually by the American Urological Association, Inc. Paper read at annual meeting of Association, Atlantic City, N.J., April 20-23, 1959. This investigation was supported by a grant from the Institute of Medical Education and Research, the City Hospitals of St. Louis, and the Harry Edison Fund of the Jewish Hospital. 1 Ollier, L. J.: Transplantation and regeneration of bone. C. R. Acad. d. Science, Paris, 126: 1252, 1898. 2 Thiersch, K.: Successful transplantation of white skin in Negroes and vice versa. Verh. d. deutsch. Gesellsch. f. Chir., 15: 17, 1886. 3 Myer, M. W.: Autoplastic and homeoplastic transplantations of kidney tissue. Archiv fiir Entwicklungsmechanik der Organism, 38: 1, 1913. 4 Loeb, L.: Further investigations on auto and homioplastic transplantation of kidney tissue. J. Med. Res., 37: 229, 1917. 424

In his extensive investigations on transplantation Locb 5 observed that the length of survival of a graft is directly related to the genetic relationship between host and donor: The more distant the relationship, the more intense the reaction to the graft and the shorter the survival of the latter. Thus syngynesio-transplants (host and donor are related) survive longer than homotransplants, and within the former group the longest survival is observed in unioval twins. On the basis of such observations Loeb derived the concept of the Individuality Differential, the latter being defined as all of those characteristics which distinguish an individual from all other individuals. With respect to transplantation, it means that the cells, tissues and body fluids of an individual have biochemical characteristics which distinguish him from all other individuals, except possibly in the case of the existence of an identical t,Yin. In Loeb's concept the cells, tissues and fluids of the graft interact with the tissue fluids of the host, and the immediate response in and about the graft is the result of this interaction. In part this interaction may be cytotoxic to the cells of the transplant, and in part it calls forth an infiltration of inflammatory cells and an ingrowth of fibroblasts and capillaries. I3lumenthal 6 has shown that there is also a concomitant systemic reaction characterized by an elevation in the blood lymphocytic count in the case of homotransplantation. In contrast with the concept that the immediate reaction to a homograft results from the interaction of individuality differentials is the idea that the rejection reaction is an immunologic phenomenon. Schi:ine 7 first formulated this theory on the basis of obserrntions on the fate of 5 Loeb, Leo: The Biological Basis of Individuality. Springfield, Ill.: C. C. Thomas, pub!., 1945. 6 Blumenthal, H. T.: Organismal differentials and leucocytes in circulating blood. Arch. Path., 27: 510, 1939; Ibid. 31: 295, 1941. 7 Schone, G.: Uber transplanta~ions immunitat. Munchen med. Wchnschr., 59: 451, 1912.

PRESERVATION A>fD HONIOTUAXSPLAXTABILFrY OF REKAL TISSUB

tumor and skin homotransplants in animals previously immunizecl with bomologous organs. Under these conditions he observed a more rapid reaction of the host against the homograft and a shorter survival of the latter, Subsequently, Lehmann and Tammann 8 further emphasized the idea that the immunization of the host was the causative factor responsible for the rejection of a homograft. ::VIorc, recently, ~VIedawar 9 • 10 presented further evidence to support this concept, observing that when homografts of skin are performed on a host previously homografted with skin from the sanw donor, the survival time of the second set of grafts is shorter than the average survival time of the first set. He has also observed that if skin homografting is r:arried out in the anterior chambrr of the eye, there is an apparent inability of the host to develop antibodies; if a second skin graft from the same donor is then transplanted elsewhere in the recipient, rejection of both grafts ocr:urs as a consequence of the formation of antibodies to the second graft. 13illingham11 believes that the antigens responsi-ble for this immune response arc derived from the nuclei of the grafted cells and are clcsoxyribonuclcoprotrins. On the other hand, Fleisber12 has Qbserved that the immunization of an animal by homologous kidney does not in any definite way modify the course of the typical reaction to a kidney homograft. 01.lthough a great deal of further study is required before a complete understanding of the rejection phenomenon to a lmmograft is accomplished, it would appear that there may be two stages to the reaction: 1) an immediate or early response of the host, based on the interaction of individuality differentials, and 2) a delayed reaction which may reprrsent an immune response. As Loeb' has pointed out, there is a reaction to the homograft almost immediately after it has been placed, and it would appear unlikely that 8 Lehmann, vV. and Tanummn, H.: Immunization function of reticuloendothelial. system. Z.f. Imm. Forch., 45: 493, HJ26. 9 Medawar, P. B. · The behavious and fate of skin homografts in rabbits. J. Anat., 78: 176, 1944. 10 .l\-Iedawar, P. B.: Immunity to homologous grafted skin. III. The faLe of skin homografts transplanted to the brain, to subcutaneous tissue, and to the anterior chamber of the eye. Brit. J. Exp, Path., 29: 1948. 11 Billingham, E., Brent, L. and }Vfedawar, P. B.: The antigenic stinrnlns in transplantation immunity. Nature, 178: 514, 1956. 12 Fleisher, ]VI. S.: Immunit.v and tissue transplantation. J. Med. Res., 39: 1, Hl18

an immune resporrne could hr cle\'(']opC'd rapidly ..Judging from both the local and the systemic reaction, this first phase attains a maximum intensity in the case of homntransplantation in rodents at about the se,·enth It is also possible that a later immune n•;.,ponse develops, although the evidence for such iR largely based on either successive homogrnfts or presensitization of the host. And in auy C'YPnt, this would constitute the immunization of tbe host to the individuality differential of the clouor. Nevertheless, the case for an immune response to a single homograft has not been clearly c:stab-lishecl. There has been no demonstration of cir-· culating antibodies utilizing conventional sen)c logic techniques, and while it is true that a second set of homografts is rejected more rapidly than first set under some circumstances, the has also been reported, namdy that a longer survival or acquired tolerance may also occur when tumort:i or ovarian tissuc 11 is homografted. The foregoing considerations are particularly important as regards available directed at overcoming the rl'jcction reaction to a homograft. :\Iost current n•scarch ha~ the first response and has been directed at overcoming the development of the immurw rc:,u:tion by attempts to depress the prodm:tion of irnmtme substances by the host. In this regard d('Sensitiz:J,· tion of the host, 15 antihistamines, 16 tomy,17 nitrogen mustard,1 7 cortisone, 17 · " and total body irracliation19 have been utilized. The use of such drastic mechanisms is often lrthal in itself, or destroys those mechanisms of tlic l10st which normally eombat infections. By the present study is direetecl at attempting to 13 Snell, G. D.: The immunogenetics of tumor transplantation. Cancer Res., 12: 5°13, Hl52, 14 Parkes, A. S.: Attenuation of host reaction to ovarian homografts. Nature, 178:_1228, 1956. 15 Baetzner, W. and Beck. S.: Ulwr Homoiotransplantation. Zcntralbl. f. 55: Hl22l 16 Foster, D. G. and Hanrahan, M.: vations on skin homogrnfts after 60 day, of benzamine therapy. Bull. Johns Hopkin,

82: 501, 1948. 17 Baker, R., Gordon, R., Huffer, ,J. ancl G. H.: Experimental renal transplantation: Effect of nitrogen mustard, cortisone :mcl splun · ectomy. Arch. Surg., 65: 702, 1952. 18 Persky, L. and Stanley, ,l.: Effect of c\CTH and cortisone on homogeneous kidney Proc. Soc. Exp. Biol. and l\led., 77: (l6, 19 Trentin, J. J.: The immunological ba,is for induced tolerance to skin homografts in irrndiated mice receiving bone marrow trnn,fu,si011,-;. Tra1rnp Bull., 4: 7,5, 1957.

426

JUAN C. BELTRAN AND HERMAN T. BLUMENTHAL

alter the individuality differential of the transplant so that it will not call forth the immediate response of the host. If this can be accomplished, it is also likely that the graft will be altered in its capability of calling forth the later immune reaction. This would appear to be a more promising approach than one aimed at altering response mechanisms of the host. In recent years dehydration-freezing-thawing of grafts has come into common use, but for the most part simply as a means of preserving and storing tissues for subsequent transplantation. Such procedures have not been generally considered to have any influence on the survival of the graft, but recent studies by JVIorgan20 with tumors, Parkes 21 with ovary, Andresen and associates22 with musculofascial tissues, and Joshi and Blumenthal23 with adrenal and aorta, have shown that under certain conditions of freezingdehydration-thawing the immediate or early reaction of the host to the graft is suppressed. In addition, in those of the foregoing reports in which the tissue used possessed a substantial regenerative capacity, the injury of the grafted tissues resulting from the mechanics of transplantation or from the delay in establishing a blood supply could be overcome by a subsequent proliferation of the surviving cells. There appear to be three problems with respect to the application of the foregoing techniques to the transplantation of renal tissue; these derive from the complex structure of the kidney. In the first place, since different tissues appear to have different sensitivities as regards dehydrationfreezing-thawing (Taylor and Gerstner24), the question arises as to whether or not conditions 20 Morgan, J. F., Guerin, L. F. and Morton, H. J.: The effect of low temperature and storage on the viability and mouse strain specificity of ascitic tumor cells. Cancer Res., 16: 907, 1956. 21 Parkes, A. S. and Smith, A. U.: Regeneration of rat ovarian tissue grnfted after exposure to low temperatures. Proc. Roy. Soc. Bull., 140: 455,

1952. 22 Andresen, R. H., JVIonroe, C. W., Hass, G. M. and Madden, D. A.: Tissue reactions to autologous and homologous musculofascial transplants. A.M.A. Arch. Path., 27: 272, 1956. 23 Joshi, R. A. and Blumenthal, H. T.: Alterations of homotransplantability of rat adrenal and aorta by dehydration-freezing. Lab. Invest.,

7: 19, 1958.

24 Taylor, A. C. and Gerstner, R.: Tissue survival after exposure to low temperatures and the effectiveness of protective treatments. I. Evaluation by growth in tissue culture. J. Cell and Comp. Physiol., 46: 477, 1955.

which permit the survival of glomeruli would also permit the survival of the tubular and collecting systems. Secondly, since in the mechanics of transplantation there is inevitably a period of anoxia until a blood supply is established, there is also the problem of the relative sensitivities of the various components of the nephron to lack of oxygen. Thirdly, since some degree of injury with respect to the foregoing considerations is unavoidable, the relative abilities of the various components of the nephron to regenerate is also of importance. The present experiments are, therefore, designed pri1narily to evaluate these factors, since if they can be overcome, it may become possible to successfully homograft kidney. MATERIAL AND METHOD

White rats of the Wistar strain were used throughout these experiments. Transplantation consisted of the insertion of a slice of kidney into a subcutaneous pocket in the abdominal wall. This procedure was carried out under ether anesthesia and with strict aseptic precautions. A total of 160 autotransplants and 300 homotransplants were made. In all instances in which the graft was frozen prior to transplantation, slices of kidney tissue so frozen were immediately fixed for study of the effects of the treatment prior to transplantation. All tissues were fixed in 10 per cent formalin, dehydrated, embedded in paraffin, sectioned at 6 mu. and stained with hematoxylin and eosin. The experiments in this study fall into three categories: 1) control auto- and homografts; 2) modified auto- and homografts in which the tissue to be transplanted was subjected to various conditions of dehydration-freezing-thawing, as described below, and 3) tissue preservation studies in which the effects of various conditions of freezing-thawing were used and the per cent of preserved tissue estimated. In the control group donor kidneys were removed, sliced and immediately transplanted. This group afforded a basis for comparison with experimental transplants as well as a means of studying the differences between the auto- and homotransplantation reactions. It also provided a means of evaluating the effects of the mechanics of transplantation and anoxia on the components of the nephron as well as the regenerating ability of the latter. There were 32 autotransplants and 64 homotransplants, each divided into four groups

PRESERVATION AND HOMOTRANSPLANTABILITY OF RENAL TISSUE TABLE

427

l. Effects of various conditions of dehydration-freezing-thawing on renal tissue preservation

Experiment

Tissue

Dehydration

Freezing

Thawing

Preservation

---- --------1-------1--------1--------1----

I.

II.

Slices of kidney in Petri dishes

30% glycerol

Slices of kidney in I 30% glycerol Petri dishes

0°C. in refrigerator

Room temperature

0-0.5%

-90°C. in deep freeze

Room temperature

0-0.5%

------------------------------------------------

III.

----

Slices of kidney in perforated metallic tissue capsules

None

-190°C. by fast immersion in liquid nitrogen

Room temperature

0.5%

---------- -------- ----------- ---------- -----

IV.

Slices of kidney in pyrex tubes

30% glycerol

Same

Room temperature

10-20%

V.

Slices of kidney in pyrex tubes

30% glycerol

Same

In boiling water until glycerol liquefied

30-70%

VJ.

Slices of kidney in pyrex tubes

30% glycerol

Same

In boiling water, then placed m water bath for 30 min. at 37°C.

30-50%

VII.

Slices of kidney in pyrex tubes

30% glycerol

Same

In boiling water until glycerol began to liquefy

30-40%

VIII.

Slices of kidney in pyrex tubes

Same

At 37°C., then at room temperature

10-20%

IX.

Whole kidney in pyrex tubes

Same

In boiling water until glycerol liquefied

70-80%

30% glycerol

30% glycerol

of equal number and sacrificed on the fourth, eighth, sixteenth and twenty-first days after transplantation. In the group of modified transplants, the latter were frozen at 0, -90 and -190°C. prior to transplantation. The first was accomplished by placing the transplants in a refrigerator, the second by exposure in a deep freeze, and the third by immersion in liquid nitrogen. In all instances exposure to low temperatures was for 24 hours. In the experiments with O and -90°C. the tissue slices to be grafted were first immersed in 30 per cent glycerol in Petri dishes and kept at room temperature for 30 minutes before being placed under refrigeration, while at the lowest temperature the slices were placed in perforated

metallic capsules, without immersion in glycerol, and immediately frozen. There were 32 autotransplants and 64 homotransplants in each temperature series; again they were divided into groups of equal numbers and sacrificed at 4, 8, 16 and 21 days after transplantation. In the study on tissue preservation with variations in the freezing-thawing technique both slices and whole kidney were used (table 1). Eight variations were used in this regard; they were as follows: 1) Glycerol immersion, freezing at 0°C., thawing at room temperature for about 30 minutes. 2) Glycerol immersion, freezing at -90°C.,

428

JUAN C. BELTRAN AND HERMAN T. BLUMENTHAL

thawing at room temperature for about 30 minutes. 3) No glycerin immersion, freezing at -190°C., tha,ving at room temperature for about 30 minutes. 4) Immersion in glycerin, freezing at -190°C., thawing at room temperature for about 30 minutes. 5) Glycerin immersion, freezing at -190°C., thawing rapidly in boiling water until glycerin was completely liquefied. 6) Glycerin immersion, freezing at -190°C., thawing rapidly in boiling ,rnter until glycerin was completely liquefied, and then in 37°C. water bath for 30 minutes. 7) Glycerin immersion, freezing at -190°C., thawing in boiling water until glycerin was partially liquefied, and then at room temperature for 30 minutes to avoid the possibility of overheating. 8) Glycerin immersion, freezing at -190°C., thawing in 37 C. water bath for 30 minutes. In all instances the tissues were fixed immediately after the thawing procedure was completed, sectioned and an estimate of per cent of preserved tissue made. Tissue slices subjected to the third and fourth techniques were auto- and homografted. In addition, grafts of tissue slices obtained from whole kidneys subjected to technique 5 were also auto- and homografted, since they showed the greatest per cent of preserved tissue. RESULTS

Control series. At 4 days after auto- and homotransplantation, both groups show central areas of necrosis and a peripheral area with variable amounts of surviving tubules and glomeruli. In general there is more surviving tissue in autothan in bomografts. :l\Iitotic figures are seen in the epithelium of some tubules and of Bowman's capsule; these are present more frequently in auto- than in bomografts. The transplants are surrounded by fibroblasts and capillaries, which penetrate into the outer rim of the transplant. The fibroblastic reaction is somewhat more intense in homo- than in autografts. In the homografts lymphocytic infiltration into the periphery of the transplant is also seen. By the eighth day there is beginning organization of the necrotic zone in both groups. The peripheral zone of surviving kidney tissue appears larger in relation to the necrotic zone than after four days and

mitoses are more numerous; these changes are more marked in auto- than in homografts. There is more fibroblastic ingrowth than after four days and capillary penetration is also deeper. Lymphocytic infiltration is more marked in homografts than after four days and occasional lymphocytes are sometimes also seen in autotransplants. Beginning endothelial proliferation of arteries of the transplant is seen in homografts, but this change is not seen in autotransplants. By the sixteenth clay the bomografts show advanced organization of necrotic areas and marked lymphocytic infiltration; lymphocytes penetrate into tubules and glomeruli and destroy many of the structures which have apparently survived up to about this time. Endothelial proliferation of arteries of the homotransplants is also marked (fig. 1 B, C and D). By contrast, the autotransplants sh01v more surviving kidney tissue (fig. 1, 11) and mitotic activity is still present although less marked than at 4 and 8 days. The arteries of autotransplants still show no endothelial proliferation and lymphocytic infiltration is absent in most grafts, ,vhile occasional ones show a few scattered lymphocytes. By the twenty-first day homografts show a very few surviving tubules and glomeruli, and those which apparently survive often show partial destruction by the ingrowth of interstitial connective tissue. There is almost complete organization of necrotic tissue and areas of calcification are present in this zone. Lymphocytic infiltration is marked and endothelial proliferation has advanced to the point of partially or completely occluding arteries. On the other hand, autotransplants show many ,vell preserved tubules and glomeruli. In general, collecting tubules show a better state of preservation than convoluted ones. There is less organization than in homografts and less interstitial connective tissue is seen in areas of preserved tissue. Lymphocytic infiltration is absent in all autografts. Jfodified transplants. In grafts frozen at 0°C. there was little to distinguish between auto- and homotransplants by the fourth day. Both groups showed large areas of necrosis, with only small peripheral areas of sun·iving tubules and glomeruli. There was an absence of lymphocytic infiltration in both groups and fibroblastic proliferation was minimal. By the eighth day there was little or no surviving tissue in both groups; fibroblastic reaction was 1101,· marked,

PRESERVATION AND HOMOTRANSPLANTABILITY OF RENAL TISSUE

429

Fm. 1. A, 16 day subcutaneous autotransplant of unfrozen kidney slice. Hematoxylin-eosin. Mag. approx. 125X. Section shows fibrous tissue in which there are scattered small tubules. Lymphocytic infiltration is absent. B, 16 day subcutaneous homotransplant of unfrozen kidney slice. Hematoxylin-eosin. Mag. approx. 250X. Note central vessel with advanced endarteritis and tiny eccentrically placed lumen towards left side of artery wall. C, same as B. Hematoxylin-eosin. Mag. approx. 500X. Several tubules (arrows) show various stages of degeneration. D, same as B and C. Hematoxylin-eosin. Mag. approx. 250X. Arrow indicates degenerating tubule. Almost all of area is occupied by intense lymphocytic infiltration. penetrating along with capillaries into the necrotic areas. Homografts showed marked lymphocytic infiltration, but autotransplants showed little or no infiltration by these cells. By the sixteenth day preserved tubules or glomeruli were rare in either group and fibroblastic organization was well advanced in both groups (fig. 2, A). Again the homografts showed marked lymphocytic infiltration, while in the autotransplants inflammatory cells were essentially absent. By the twenty-first day organization was complete and no preserved elements were found in either group. The only distinguishing feature between auto- and homotransplants was the marked lymphocytic infiltration in the latter.

While in transplants frozen at 0°C. auto- and homotransplants could be distinguished on the basis of lymphocytic infiltration, this distinction disappeared when transplants were frozen at -90°C. In the latter there was almost no surviving renal tissue in either group by the fourth day; fibroblastic reaction was minimal and lymphocytic reaction absent. During succeeding periods there was a progressive ingrowth of fibroblasts and capillaries (fig. 2, B), until organization was almost complete by the twenty-first day. Surviving glomeruli and tubules were rarely seen, and in almost all instances these were collecting tubules (fig. 2, C, D). Lympho-

430

JUAN C. BELTRAN AND HERMAN T. BLUMENTHAL

FIG. 2 A, 16 day subcutaneous homotransplant of kidney slice immersed in glycerin, frozen at 0°C. for 24 hours and thawed at room temperature. Hematoxylin-eosin. Mag. approx. 250X. Fibrosis with lymphocytic infiltration is present in a band along left margin, from which fibrous extensions penetrate parenchyma. Atrophying tubule is present in center. B, 16 day subcutaneous homotransplant of kidney slice immersed in glycerin, frozen at -90°C. for 24 hours and thawed at room temperature. Mag. approx. 125X. Fibrosis with less lymphocytic infiltration than in preceding figure is present along upper edge, from which fibrous extensions penetrate transplant. Organization is less than in A. C, same as B. Hematoxylin-eosin. Mag. approx. 500X. Center is occupied by glomerulus. Note extensive proliferation of epithelium of Bowman's membrane. D, 21 day subcutaneous homotransplant of kidney slice immersed in glycerin, frozen at -90°C. for 24 hours and thawed at room temperature. Hematoxylin-eosin. JVIag. approx. 125X. Note well-preserved proliferating transitional epithelium of a dilated calyx. cytic infiltration of significant degree was not seen in either group even as late as 21 clays after transplantation. The results with auto- and homotransplants frozen at -190°C. without immersion in glycerin were essentially the same as with grafts frozen at -90°C., except that fibroblastic proliferation was considerably less intense. Thus, by the twenty-first clay considerable calcified necrotic tissue remained, there was no surviving renal tissue, and in both groups there was an absence of lymphocytic infiltration (fig. 3, A).

Variations in techniques of dehydration-freezingthawing. The results of studies dealing with the preservation of tissue following various methods of dehydration-freezing-thawing are shown in table 1. It can be seen that by far the best preservation was obtained when whole kidney was immersed in 30 per cent glycerol, frozen at -190°C. and thawed by placing the tube containing the kidney in boiling water until the glycerol was completely liquefied (fig. 3, B and C). In general, when the estimated percentage was below 30 per cent, the principal preserved

PRESERVATIO.\' A:>;D HOMOTRA"iSPLA\'TABILITY OF RENAL 'fISSUE

Fm. 3. A, 16 day subcutaneous homotransplant of kidney slice not immersed in glycerin, frozen 111 -190°0. for 24 hours and thawed at room temperatme. Hematoxylin-eosin. Mag. approx. 250X. Organization of graft by fibrous tissue is about complete and lymphocytes are loosely scattered through area. B, section of untransplanted whole kidney immersed in glycerin, frozen at -190°0. for 24 hours, and thawed by immersion of tube in boiling water until glycerin liquefied. Hematoxylin-eosin. Mag. approx. 125X. Left margin shows degenerated tubules, but center and right show well preserved glomeruli and tubules. C, same as B. Hematoxyl.in-eosin. l\fag. 250X. Note excellent preservation of glomeruli and tubules. D, section of untransplanted kidney slice immersed in glycerin, frozen at -190°0. for 24 homs, and thawed by immersion of tube in boiling water until glycerin liquefied. Hematoxylin-eosin. Mag. approx 125X. Degeneration of parench:vma is more extensive than in B and C. element was the collecting tubule. When the pen:entage preserved was between 30 and 50 per cent, preserved glomeruli were seen in addition. In those instances in which preservation was over 50 per cent more collecting tubules and glomeruli were SPen and, in addition, there were islands of apparently pn'servecl convoluted tubules (fig. 3, D). Renal arteries appeared fairly well preserved with all conditions of dehydration-freezing-thawing. Since preservation appeared best when whole kidney was immersed in 30 pn CC'nt glycerol,

frozen at -190°C. and thawed in boiling water until glycerol was liquefied, tissue slices from such specimens were auto- and homotransplanted and were compared with slices of kidney hydrated in 30 per cent glycerol, frozen at -190°C. and thawed at room tempera.ture. Despite better preservation of the frozen whole kidney, thawed rapidly prior to transplantation, auto- and homotra1rnplants in both these groups gave the same results (fig. 4, :1 and . By 21 there was complete destruction of renal ,Yith minimal fibrosis and no significant lyrnphocytic infiltration.

432

JUAN C. BELTRAN

.i._:-m HERMAN T. BLUMENTHAL

Fm. 4. A, 16 day subcutaneous homotransplant of kidney slice immersed in glycerin, frozen at -190°C., for 24 hours, and thawed at room temperature. Hematoxylin-eosin. Mag. approx. 250X. Thin rim of fibrous tissue is present at upper left. Parenchyma shows infarction-necrosis, but fibrosis is minimal and there is no lymphocytic infiltration. B, 16 day subcutaneous homotransplant of slice of whole kidney previously immersed in glycerin,. frozen at -190°C. for 24 hours, and thawed by immersion of tube in boiling water until glycerin liquefied. Hematoxylin-eosin. Mag. approx. 125X. Thin fibrous rim along left margin probably represents, renal capsule. Lymphocytes are rare. Parenchyma shows infarction-necrosis with calcification at lower right, but there is no evidence of fibrosis or lymphocytic infiltration. DISCUSSION

The control experiments with autotransplants show that the kidney has a significant degree of regenerative capacity following injury due either to the mechanics of transplantation and/or to anoxia resulting from the delay in establishing a blood supply. Fresh, unfrozen homografts also exhibit some degree of regenerative ability, although it does not appear to be as great as in autotransplants. The difference between the two is, in all likelihood, due to the incompatibility state existing betvrnen host and donor, which Loeb 5 would attribute to the development of homotoxins resulting from the interaction of the individuality differentials, and others to the immunologic state developing following such transplantation. It would appear that the relative resistance to injury and regenerative capacities of the various components of the nephron is the same as their sensitivity to injury by freezing and thawing as discussed below, namely, that the greatest resistance to injury and capacity for regeneration resides in the collecting tubules, the next greatest in the glomerular capsule and the least in the convoluted tubules. In addition, the endothelium of blood vessels of the kidney in homotransplants appears not to be injured to a significant degree, but rather appears to be stimulated to proliferation to the point where

it may completely close off some arteries. This, too parallels the reaction to freezing, in that. again endothelium 1s little injured and retains a marked ability to proliferate following homotransplantation. Dehydration-freezing of renal tissue at 0°C. does not appear to influence the host-graft interaction following homotransplantation. The lymphocytic infiltration and organization of the homologous tissue are just as marked as. with unfrozen homotransplants. On the other hand, when kidney slices arc subjected to dehydration-freezing at -90°C. or -190°C., or arc frozen at the latter temperature without. prior dehydration, the host-graft interaction following homotransplantation appears to be markedly diminished. The lymphocytic infiltration seen with fresh homografts is largely absent and fibroblastic organization and ingrowth of capillaries markedly diminished. The regenerative capacity of the vascular endothelium is increased. The effects of dehydration-freezing-thawing on cells have been evaluated in several ways, i.e., by tissue culture (Taylor and Gerstner 24; Swim and associates 25 ; by microscopic exami25 Swim, H. E., Haff, R. F. and Parker, R. F.: Some practical aspects of storing mammalian cells in the dry ice chest. Cancer Res., 18: 711, 1958.

PRESERVATION AND HOJVIOTRANSPL,L\'TABILITY OF RENAL TISSl:E

nation (Smith and Smiks 2 6; and by autotransplantation (Klinke 27; Briggs and .Jund 28; Blumenthal and 1Yalsh"l; Billingham and ~\Iedawar 80 "~s Taylor and Gerntner 24 have pointed out, autotransplantation has proved to be a better technique than tissur culture, since; it evickntly provided a more complete medium for growth, although in some instancc·s cultures of deh:nlratccl-frozn1-thawed cells have bern suec:essfuL Utilizing such tissne cultun!s, Swim and associates" have shown that a variet)' of normal and cancer celb diffor in thC'ir sc:nsitivity to injur~' by ckhydration-fn·ezing-thawing, but it appears not to have been shown heretofore that in such complex tissues as the kidney the-; cells of various structuntl comporn;nts also differ in thC'ir sensitivity to injury by such procedures. This 1nH1ld C'Xplain wh)' ;\lorgan and assoeiates 20 as ,i-ell as .Joshi and Blunwnthal23 have successfully homotransplantPd tumor and adrenal, rc,spl'c-tively, while studies with other tissues have failed_ In gcrll'ral it would appear that the more highly cl('velopecl and s1wcializcd is the biologic function of a cell, tlie more susceptible it is to injury by freezing ancl tlw lower its regenerative capacity. Thus in our experiments it has hec>n observed that whik the transitional <'Cll rpitlwlium, the mdothdial cells of vessels and the glonwruli survive clehydrntion-freezingthaw-ng ancl show the greatest n,generative capacity, the more specialized epithelium of the renal convoluted tubules is more etrnily injurrcl and shows the lowest abilit)· to regenerate. One effect of dehydration-freezing-thawing observed in the present study on renal tissue is ('Onsistent with the findings of others, namely, that dehydration-frerzing-thawing leads to a suppression of the'. reaction of tlw host tissues agairrnt the homograft_ The many studies of 20 Smith, A_ U_ and Smiks, J_: lVlicroscopic during cooling to studies of mammalian .J. Roy_ Microscopic and re-warming from Soc_, 73: 134, 195127 Klinke. J_: Direct proof that cancer and normal ce!IR live after freezing at temperature down to -255°C_ Growth, 3: 169, 1\J:39_ 28 Briggs, R_ and Jund, L_: Successful grafting of frozen and thawed mouse skin. Anat_ Rec_, 89: 7584. 1944_ 29 Blumenthal, H_ T_ and Walsh, L_ B_: Survival of guinea pig thyroid and parathyroid autotransplants previously subjected to extremely low temperatures. Proc_ Soc_ Exp_ Biol_ and Med,, 73: 6267, 1950_ 30 Billingham, R. K and Medawar, P_ B_: The freezing, drying and storage of mammalian skin. J_ Exper_ Biol., 29: 454, HJ52_

'•) •)

-t,),_)>

Loeb'' as \\'ell as the more n•cent work of Amlrescu and associates,2 2 Darcy,'Jl and Joshi ,u1d BlumenthaF" suggest that at least thP rc'spcmse of trw host to a homograft is upon the release of some substance active cC'lls rather tlmn by neerotic tissue_ ~ince in the present work some intact cl'lb ,wre prrsent in frozen homografts at ll,ast during the early period following transplantation, it \\mtld again appear that tlwir ability to call furth n reaction on the part of tlw host Imel heen in1.paircd, The fact that preservation ,vas best fflwn whol1Yth, c'itrn-C<'llular osmotic pressure is incr<'asf'cl and water is withdrawn from the cell and ultinrntely aclckcl to tbe growing crystaL The final result i~ the· development of a few large ice ,d1ich hav<' incorporatE·d all rwailable fr
434

JUAX C. BELTRAX AND HERMAN T. BLUMENTHAL

centration at high temperature can produce a high rate of injury, and 2) a prolonged exposure at high templ'rature may permit the growth of ice crystals by recrystallization prior to actual melting. Regarding the latter however, as long as such ice crystals remain extracellular, their slow redistribution is probably without significant effect. When the rate of freezing becomes sufficiently rapid, ice-crystal nuclei appear uniformly throughout the specimen and crystal formation may be predominantly intracellular. In their fornmtion and growth such crystals produce mechanical trauma, and thereby injure cells. Meryman 32 believes that with rapid freezing there is, in addition, the same potential for denaturation by high salt concentration resulting from dehydration as with slow freezing. Accordingly, storage can be conducted satisfactorily only at very low temperatures (below -50°C.). The rapidity with which destructive ice crystals ean grow in the solid state renders the thawing procedure equally if not more demanding than the freezing procedure. Luyet'1 4 has attempted to overcome the foregoing hazards by ultra rapid freezing in which a state of "vitrification" of water occurs, and by ultra rapid thawing. According to Meryman, 32 the critical temperature for water above which crystallization will occur is -130°C. He believes that the attainment of this glassy state through the supercooling of bulk water is extremely difficult, if not impossible, because of the ease and rapidity with which water is transformed into its low-energy crystal structure. However, the vitreous state can apparently be attained without difficulty by the addition of certain compounds that are effective in reducing the crystallization velocity of water. Tamman and Buchner, 35 also Lusena36 have demonstrated that relatively small amounts of certain alcohols, glycols, sugars and proteins can exert considerable effect in so retarding crystallization velocities. 11/hile the mechanism of this action is not clearly 34 Luyet, B. J. and Gehenio, F. M.: The mechanism of injury and death by low temperature. Biodynamica, 3: 33, 1940. 35 Tammann, G. and Buchner, A. Z.: Anorg. u. allgem. Chem., 222: 371, 1935. Cited by Meryman. 36 Lusena, C. V.: Ice propagation in systems of biological interest. III. Effect of solutes on nucleation and growth of ice crystals. Arch. Biochem. and Biophys., 57: 277, 1955.

understood, Meryman 32 believes it most likely that they act as impurities, creating an obstacle to the subsequent growth of crystal faces either because they become included in the oriented structure of a growing crystal face, or by acting as interfacial hydrates. In reducing the amount of ice that forms, glycerin and other substances also prevent a lethal degree of electrolyte concentration. From the foregoing, it is evident that we have utilized slow freezing in these experiments; this is particularly true as regards the interior of whole kidney. The advantages in freezing at -90 and -190°C. apparently derive from storage at a temperature sufficiently low to prevent the toxic effects of excessive concentration of electrolytes. The technique of thawing is a particularly difficult problem, as pointed out by Meryman. 32 While liquefaction of glycerin in boiling water appears to be the best of the various techniques which have been employed here, some improvements may have to be worked out in this regard before successful transplantation can be attained. Since we have dealt here solely with the transplantation of slices of renal tissue into subcutaneous pockets, the infarction-necrosis resulting from mechanical injury and/or anoxia incident to this technique basically constitutes an artefact not applicable to the transplantation of whole kidney with immediate establishment of a blood supply. Such a eonclusion is supported by the many instances of successful autografts of the latter type as well as the instances of transplantation of kidney to a unioval t"'in. The importance of the present studies derives from the obserYation that it is possible to overcome the cellular reaction which leads to the rejection of a homograft when clehydrationfreezing-thawing techniques arc employed. Furthermore, it has been shown that it is possible to freeze whole kidney and to preserve as much as 70 or 80 per cent of the renal substance. Perfection of this technique may allow not only the storage of kidney, but also the successful hornotransplantation, since this pre-treatment of the kidney will inactivate those factors responsible for the destruction of the homologous tissue. There remains, however, the problem of determining the best combination of techniques for accomplishing this end, and in this regard the present study defines specifically the problems which have to be solved.

PRESERVATION" ASD HOMOTRANSPLANTABILITY OF RE'\"AL TISS1;E SU~Il',L\.RY

The experiments in this stud? fall into three categories: 1) control auto- and homografts of kidney tissue, 2) rnodifircl auto- and homografts in which the tissue to be transplanted was subjected to various conditions of dehydrationfreezing-thawing at temperatures of 0°C., -90°C. and - l 90°C.; and 3) tissue preservation studies in which the effects of various conditions of freezing-thawing were used and the per cent of preserved tissue rstimated. The control series showed that the distinctive characteristics of the host reaction against the homograft consisted of marked lymphorytiG infiltration and intense organization. Freeze dehydration at 0°C. did not seem to influence this reaction, while at temperatures of -90 and -190°C. a markerl inhibition of the lymphocytic infiltration, a marked decrease in the host connective tissue reaction, and an increase in the proliferative t:apacity of the vascular

endothelium were noted. Certain cornporn°nts of the nephron were more sensitin' to injury by freezing than others; in this regard the collecting tubules were kast sensitin', glonwrnlal' capsule and C'nclothelium of glomeruli more sensitive, and convoluted tubules most seHsiti-l'e. The less sensitive comporwnts to dehydrationfreezing wenc those which also showed tlrn greatest activity to regenerate following transplantation. In the studies on tissue presernttion the bec:I· results wcrr observed when ,,·hole kidney \\'as tha 11·pd immersed in glycerin, frozc,n at b~, rxposurc to the temperature of boiling water until the glycerin liquefied. l.'ncler sud1 con ditions on!~- a thin surface margin showed de-generation. The authors are inddited to Dr. .\IoniR Abrams for help in carr~·ing out this study, and to l\lr. Roy c\bernathy for technical assistance.