Kidneys were transplanted in mongrel dogs so that renal venous drainage was into the portal system of the hosts. Thirty-one recipients were not treated, 11 were given one dose of 3 mg of azathioprine per kg, and 11 were given 2 mg of azathioprine per day. Survival was not statistically increased compared with that in three comparable series in which renal venous drainage was into the vena cava, nor were the histopathological findings favorably altered in the “portal” kidneys. The injection of semisoluble antigen into the portal vein at the same time as renal transplantation at the caval site, had an effect no different from that if the antigen were given systemically during caval site transplantation. The conclusion that drainage of grafts into the portal vein was not beneficial was reached in 20 pigs evenly divided between the portal and vena caval sites, and in 12 pairs of dog to pig or pig to dog xenografts. Thus, none of these experiments has identified an advantage of antigen delivery into the portal as opposed to the systemic venous system. © 1977 by The Williams & Wilkins Co

Benichou, J

Mazzoni, G

Porter, KA

Starzl, TE

English

PubMed

Name not available

Renal homotransplantation with venous outflow or infusion of antigen into the portal vein of dogs or pigs: Transplantation at portal site

D-Scholarship@Pitt

TRANSPLANTATION Copyright © 1977 by The Williams & Wilkins Co. Vol. 24, No.4 Printed in U.S.A. RENAL HOMOTRANSPLANTATION WITH VENOUS OUTFLOW OR INFUSION OF ANTIGEN INTO THE PORTAL VEIN OF DOGS OR PIGS TRANSPLANTATION AT PORTAL SITE! GUISEPPE MAZZONI, JOSEPH BENICHOU, KENDRICK A. PORTER, AND THOMAS E. STARZL2 Department of Surgery, Veterans Administration Hospital and University of Colorado Medical Center, Denver, Colorado, 80220, and the Department of Pathology, St. Mary's Hospital and Medical School, London W2 lPG, England SUMMARY Kidneys were transplanted in mongrel dogs so that renal venous drainage was into the portal system of the hosts. Thirty-one recipients were not treated, 11 were given one dose of 3 mg of azathioprine per kg, and 11 were given 2 mg of azathioprine per day. Survival was not statistically increased compared with that in three comparable series in which renal venous drainage was into the vena cava, nor were the histopath-ological findings favorably altered in the "portal" kidneys. The injection of semisoluble antigen into the portal vein at the same time as renal transplantation at the caval site, had an effect no different from that ifthe antigen were given systemically during caval site transplantation. The conclusion that drainage of grafts into the portal vein was not beneficial was reached in 20 pigs evenly divided between the portal and vena caval sites, and in 12 pairs of dog to pig or pig to dog xenografts. Thus, none of these experi-ments has identified an advantage of antigen delivery into the portal as opposed to the systemic venous system. There have been conflicting reports about whether or not rejection is reduced in homo-grafts transplanted so that their venous drain-age is into the portal circulation and then through the liver. Consequently, the question of portal site privilege, with or without azathio-prine treatment, was examined again in mon-grel dogs and in pigs using a kidney transplant model. In addition, the effect of donor semisolu-ble splenic antigen given via the portal versus systemic routes upon canine renal homograft survival was tested. MATERIALS AND METHODS Dog Homotransplantation Randomly selected mongrel dogs that weighed 9 to 27 kg were operated upon with IThis work was supported by Research Grants MRIS 8118-01 and 7227-01 from the Veterans Ad-ministration, Grants AM-17260 and AM-07772 from the National Institutes of Health and Grants RR-00051 and RR-00069 from the General Clinical Re-search Centers Program ofthe Division of Research 268 sodium pentobarbital anesthesia supplemented with phencyclidine hydrochloride and succinyl choline. Experiments were set up with weight-matched pairs. After bilateral nephrectomy, each dog received a kidney of its partner in the right subhepatic space (Fig. 1). The left kidney was usually used as the homograft, but ifit had a multiple arterial supply, the donor right kid-ney was used instead. Mter removal, the kid-neys were flushed free of blood with cold lac-tated Ringer's solution. The postoperative course was monitored by serial measures of blood urea nitrogen. All animals that did not live a full 5 days were excluded from the final analyses, since the early deaths were almost always associated with technical accidents, acute respiratory complications, intussusception, massive infec-tion, or hemorrhage. The number of experi-ments to obtain 53 completed portal site experi-Resources, National Institutes of Health. 2 Dr. Starzl was a Faculty Scholar of The Josiah Macy Jr. Foundation. October, 1977 MAZZONI ET AL. 269 B. FIGURE 1. Experimental models. A, Left kidney of canine donor is placed in the recipient right renal fossa with renal venous drainage into the inferior vena cava. B, Same as A but with renal drainage into the portal vein. ments was 80. The number of experiments to obtain 53 systemic site experiments was 84. The majority of discarded experiments in both por-tal and systemic outflow models was when aza-thioprine was given chronically. For statistical analysis, credit for duration of survival of a given animal was limited to 40 days. The autopsy tissues were fixed in 10% formalin. Light microscopic criteria of rejection were used that have been described in past publications (22). The experimental groups are outlined in Ta-ble 1. In groups 1, 3, and 5, the graft renal artery was anastomosed to the recipient renal artery, the renal vein was drained into the inferior vena cava, and end to end ureteroure-terostomy was performed (Fig. lA). In groups 2, 4, and 6, the same technique was used, ex-cept that the end of the renal vein was anasto-mosed to the side of the superior mesenteric or portal vein (Fig. IB). When used, azathioprine was given i. v., and in groups 5 and 6 conversion was made to the oral route as soon as the dogs . ate. Dog Antigen Infusion and Renal Homotrans-plantation Semisoluble antigen was prepared from the spleen of a mongrel donor according to the method of Brent and Kilshaw (4), as they modi-fied it from the method of Medawar (19), and suspended in 400 ml of heparinized lactated TABLE 1. Experimental groups with canine renal transplantation Azathio- Daily No. prine day azathio-Se- Renal venous prine ries of drainage of opera- postoper. dogs tion (mg/kg) ative (mg/kg) 1 32 Vena cava None None 2 31 Portal None None 3 11 Vena cava 3 None 4 11 Portal 3 None 5 10 Vena cava 3 2 6 11 Portal 3 2 Ringer's solution. The following day, the donor kidneys were removed at reoperation and one each was transplanted to the vena caval site (Fig. lA) of two mongrel recipients. As the transplantation was being performed, one-half (200 ml) of the heparin containing semisoluble antigen suspension was infused into the jugular vein in control animals or into a mesenteric vein tributary in the test animals. Thus, each dog received the antigen equivalent of one-half a spleen. The mg wet spleen tissue per kg recip-ient was usually about 1,600 mg, but this var-ied considerably. No immunosuppression was given. The same criteria were used to judge the results as de-scribed in the preceding section. To obtain 15 dogs with survival for at least a full 5 days (6 with systemic and 9 with portal antigen) 25 experiments were necessary. 270 TRANSPLANTATION Vol. 24, No.4 Pig Homotransplantation Landrace and large white pigs weighing 9 to 14 kg were used, with random donor recipient pairing. The conditions of anesthesia and care were essentially the same as with the dogs, except for appropriate diet adjustments. Sur-vival for 5 full days was also requisite for inclu-sion in the final analyses. To obtain 10 complete portal site experiments, 13 transplantations were required. For 10 vena caval experiments, 15 transplantations were done. The most com-mon causes of the 8 deaths before 5 days were thrombosis of the renal artery (4 examples) and pulmonary consolidation (2 examples). Previously described surgical techniques were used (17,18). These were similar to those shown for the dog (Fig. 1), with two common deviations. First, arterialization was usually by anastomosis of a Carrel patch, or an aortic graft to the recipient aorta. In portal site exper-iments, donor vena cava homografts were fre-quently used to bridge from the renal vein to the portal vein. Usually, one donor was used for two recipients; one of these kidneys was placed at the portal and the other at the vena caval sites. All grafts were cooled with the same infu-sion technique as was used for the dogs. Heterotransplantation Acute experiments were performed with the same techniques as above by transferring pig kidneys to dogs in three pairs of experiments and dog kidneys to pigs in nine pairs. With one member of a pair, venous outflow was directed by anastomosis into the vena cava and with the other member, the anastomosis was to the por-tal vein. The outcome was judged by the gross appearance of the heterografts at successive times, by evaluation of serial biopsies, and by the volume of urine output. RESULTS Dog Homotransplantation No treatment. About one-fifth of the 32 dogs of group 1 with the systemic renal venous drainage survived for 2 weeks or longer. Two animals lived more than 1 month and one sur-vived for 46 days. Similar results were obtained in the 31 animals of group 2 with renal venous drainage into the portal vein. Two of these dogs had survival of more than 1 month and one lived for 4 months (Fig. 2). The differences be-tween groups 1 and 2 were not statistically significant. There was essentially no difference in the histopathological findings in the homografts of groups 1 and 2 (Table 2). One dose of azathioprine. A single dose of i. v. azathioprine slightly increased survival both in animals with systemic (group 3) and portal (group 4) venous drainage (Fig. 3), but this improvement was not statistically significant. There were no significant differences between groups 3 and 4. The only animal still alive at 40 days died after 44 days. The graft of that dog had systemic renal vein drainage. Histopathologically, groups 3 and 4 were in-distinguishable (Table 2). NO TREATMENT (!) z ;; 1.0 .8 ~ .6 => en z o .4 I-U <[ a: u. .2 Renal Vein to Vena Cava (32 dogs) - --Renal Vein to Portal (31 dogs) --10 20 30 TIME IN DAYS 40 FIGURE 2. Portal versus systemic renal transplantation in untreated mongrel dogs. I I October, 1977 MAZZONI ET AL. 271 TABLE 2. Pathological findings in canine renal homograftsa No. in group Rejection Total Cellular only Cellular + humoral Normal Portal vein thrombosis; no rejection Cortical necrosis; no rejection Renal artery thrombosis; infarction " The groups are defined in Table 1. 1.0 ONE DOSE IV AZATHIOPRINE (3 mg/KgJ co z > ~enal Vein to Vena Co va (II dogs) - - -Renal Vein to Portal (II dogs) --;; (}" 6 ::J U"> z <:> 4 .... u « ~ -, l~------D-------~-------r----~~ 10 20 30 40 TIME IN DAYS 32 25 6 19 4 0 1 2 FIGURE 3. Portal versus systemic renal trans-plantation in dogs with one dose of 3 mg of aza-thioprine per kg on the day of operation. Chronic azathioprine. The survival of dogs in group 6 with portal venous drainage of the transplants was not significantly different from that of the group 5 animals with systemic drainage (Fig. 4), and both were slightly better than untreated controls. One of the portal site animals lived for 49 days. Histopathologically, a protective effect of azathioprine was evident in groups 5 and 6 in that one-third of the homografts in each group was normal (Table 2). Dog Antigen Infusion and Renal Transplanta-tion The administration of semi soluble antigen by the portal and vena caval routes at the same time as orthotopic renal transplantation did not significantly influence survival, which was 11.78 ± 6.26 (SD) days after the portal infusion and 12.67 ± 2.24 (SD) days after systemic infu-sion. The histopathological findings were essen-2 3 31 11 27 8 5 2 22 6 3 1 0 0 1 0 1 10 .6 z <:> .... u « ~ 0 Groups 10 4 5 11 10 8 6 1 4 7 2 1 3 0 0 2 0 0 1 DAILY AZATHIOPRINE (2 mg/KgJ 6 11 7 3 4 3 1 0 0 Renal Vein to Vena COI,IO (10 dogs) - - -Renal Vem 10 Portal (II dogs) ---20 30 40 TIME IN DAYS FIGURE 4. Portal versus systemic renal trans-plantation in dogs with chronic azathioprine treat-ment of low doses. tially the same in the two groups (Table 3). Pig Homotransplantation The survival was almost identical to trans-plantation to the inferior vena caval outflow site versus the portal site (Fig. 5). One animal in each group lived for about 1 month. The histopathological changes were almost the same in the two groups (Table 4). H eterotransplantatio n No gross differences in appearance or urine production were noted between transplantation at the portal and vena caval sites with either the pig to dog transplantation in which rejec-tion occurred in a few min, or with the milder dog to pig transplantations in which rejection occurred after 30 min to 4 hr. In the pig to dog renal xenografts, general-ized and diffuse obstruction of all of the glomer-ular capillaries by aggregated platelets and 272 TRANSPLANTATION Vol. 24, No.4 TABLE 3. Histopathological findings in orthotopically placed dog renal homografts after intraoperative infusion of semisoluble antigen via the portal vein and inferior vena caval routes Portal Inferior vein vena cava Total homografts 9 6 Rejection Total 7 6 Cellular only 1 1 Cellular + humoral 6 5 Normal 0 Renal artery thrombosis; 0 infarction NO TREATMENT 10..----, '" z Renal Vein 10 'nno Cova (10 Pigs) - --Renol Vein to Porlal (10 Plgs) __ ;; ;; 7 '" ~ z o ;:: u ., 3 '" u. -, L ____ , '------"---.... ---, l~--D--r---r--D--D-~-K~D--I '0 15 20 25 30 35 TIME IN DAYS FIGURE 5. Portal versus systemic renal trans plantation in untreated pigs. TABLE 4. Histopathological findings in pig kidney homografts after transplantation to the portal vein or inferior vena caval sites Portal Inferior vein vena cava Total homografts 10 10 Rejection Total 10 10 Cellular 4 2 Cellular + humoral 6 8 Normal 0 0 clumped red blood cells was present 10 mm after transplantation. By contrast, most of the dog to pig renal xenografts appeared to be normal by light mi-croscopy at this time, although electron micros copy did reveal early platelet aggregation. About 20 min later, however, there was obvious focal and segmental blockage of the glomerular capillaries by aggregated platelets and clumped red blood cells. These changes progressed and neutrophil polymorphonuclear leukocytes be-came prominent in the lesions, until by 2 hr after transplantation, all of the glomerular cap-illary loops were occluded. There were varia-tions in the speed with which these events oc-curred in individual animals, but there were no significant histopathological differences be-tween the grafts with venous outflow into the portal vein and those with systemic venous drainage. DISCUSSION Studies in the last 15 years have suggested or demonstrated that there is a reduced response to antigens that are introduced into the portal as compared with the systemic venous system (2,6, 7,10,14,20,24,25). Partial removal of the antigens by the liver and thus a diminished host reaction to their presence has been the most common explanation. Alternatively, the portal injection could have contributed to toler-ance induction (2,8,13,16). Pursuant to such reports, it was natural in a number of laborato-ries to test whether there was an immunologi-cal advantage for kidneys (1,11,12,15,17,18, 23), hearts (3), pancreatic islets (9, 26), and parathyroid cells (21) transplanted into the portal circulation or liver. The results have been variable. In rats, all reports have described a protective effect at the portal site but usually only with easy histocom-patibility barriers (3, 21, 23, 26). Mazzoni et al. (17, 18) had positive experiments with pig kidneys but these findings were not confirmed by Hickman and Terblanche (12). Barker and Corriere (1), Fukada et al.(ll), and May et al. (15) saw no amelioration of the rejection of primary canine kidney homografts that were drained into the portal vein. However, Fukada et al. (11) thought that sensitization directed to a second kidney from the same donor was thereby reduced. The investigations herein reported failed to demonstrate any portal site privilege for pri-mary transplants either in dogs or pigs. The canine results confirmed the negative findings of previous workers (1, 11, 15), and extended these conclusions to conditions in the dog of single-dose or low-dose chronic azathioprine treatment. Furthermore, concomitant delivery of semisoluble antigen into the portal vein had an effect no different from that of systemic anti-gen, and neither route produced a prolongation of survival of transplanted kidneys. The nega-tive experiments were not different from the earlier ones by CaIne et al. (5). The results in pigs were different from those October, 1977 MAZZONI ET AL. 273 observed several years ago by one of us (17,18). Failure to reproduce the earlier work might be attributable to differences in the pigs in widely separated parts of the world, even though the breeds were the same. The pig as an experi-mental animal has been criticized as an unpre-dictable experimental model. Another possibil-ity could have been the presence of inadvertent experimental bias in the earlier work, in which control experiments were carried out at a time different from the portal site experiments. The importance of concurrent controls in dog and pig experiments is also illustrated in the studies herein reported. The number of long-term survivors in untreated mongrel dog recipi-ents of portal site kidneys was so great that a therapeutic effect could have been inferred if historical control data had been accepted for comparison. Instead, it became clear that al-most 10% of the untreated animals with homo-graft drainage through either the portal vein or vena cava had survived in excess of 1 month. Under minimum treatment, these results were improved, both in terms of survival and histopathological criteria. Repeated doses of azathioprine that have been considered homeo-pathic by others had an effect. Thus, experi-ments that impute a zero effect of low-dose azathioprine alone upon survival contain a flaw in design, at least according to our data. The better picture of control data or data from low-dose immunosuppression that is emerging is in part ascribable to the culling out of experiments in which the animals fail to live 5 full days. In untreated animals, the cause of early death was almost always for anesthetic or technical reasons. These factors are usually ir-relevant or confusing in evaluating the antire-jection effects of the drug or other system being tested. Thus, the propriety of removing the ar-tifacts caused by the earlier deaths seems in-contestable. On the basis of these investigations, it seems unlikely that any specific benefit could be ex-pected in patients from renal transplantation to the portal vein. The question that remains is whether there really is an amelioration of rejec-tion in rats and mice. As already mentioned, any mitigating effects described to date have been small and seen only when there are minor histocompatibility barriers. LITERATURE CITED 1. Barker CF, Corriere IN, Jr: 1967 Ann Surg 165: 279 2. Battisto JR, Miller J: 1962 Proc Soc Exp BioI Med 111: 111 3. Boeckx W, Sobis H, Lacquet A, et al: 1975 Transplantation 19: 145 4. Brent L, Kilshaw PJ: 1970 Nature 227: 898 5. CaIne RY, Davis DR, Medawar PB, et al: 1969 Transplantation 8: 472 6. Cantor HM, Dumont AE: 1967 Nature 215: 744 7. Chase MW: 1946 Proc Soc Exp BioI Med 61: 257 8. Edelstone DI, Yonemoto RH, Hargreaves R: 1971 Surg Forum 22: 280 9. Eloy E, Kedinger M, Garaud K, et al: 1975 Lan-genbecks Arch Suppl Chir Forum 109: 114 10. Franzl RE: 1962 Nature 195: 457 11. Fukuda A, Hanaoko T, Soloway AC, et al: 1969 Transplant Proc 1: 602 12. Hickman R, Terblanche J: 1975 Transplantation 20: 338 13. Lisboa LS, Holyoke ED: 1972 Surg Forum 23: 116 14. Mandel MA, Monaco AP, Russell PS: 1965 J Immunol 95: 673 15. May AG, Bauer S, Leddy JP, et al: 1969 Ann Surg 170: 824 16. Mayer DJ, Kronman B, Dumont AE: 1965 Surg Forum 16: 243 17. Mazzoni G, DiMartino C, Demofonti A, et al: 1972 Br J Surg 59: 541 18. Mazzoni G, DiMartino C, Melis M, et al: 1971 Eur Surg Res 3: 62 19. Medawar PB: 1963 Transplantation 1: 21 20. Paronetto F, Popper H: 1964 Proc Soc Exp BioI Med 116: 1060 21. Pfefferman R, Sakai A, Kountz SL: 1976 Sur-gery 79: 182 22. Porter KA: 1964, p 299In Starzl TE (ed). Experi-ence in renal transplantation. WB Saunders Co, Philadelphia 23. Sakai A: 1970 Transplantation 9: 333 24. Triger DR, Cynamon MH, Wright R: 1973 Im-munology 25: 941 25. Triger DR, Wright R: 1973 Lancet 1: 1494 26. Ziegler MM, Reckard CR, Barker CF: 1975 Transplant Proc 7: 743 Received 23 May 1977. Accepted 27 June 1977. 

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Renal homotransplantation with venous outflow or infusion of antigen into the portal vein of dogs or pigs: Transplantation at portal site

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