Renal Subcapsular IsletCell TransplantationLUIS H. TOLEDO-PEREYRA, KARL 0. BANDLIEN, DEBRA A. GORDON, GERALD H. MACKENZIE, AND THEODORE A. REYMAN

SUMMARYIslet cell transplantation has been associated with ulti-mate graft rejection. This preliminary study investi-gates the use of the renal subcapsular region as a sitefor placement of canine islet cell a I log rafts. A newnoncollagenase mechanical technique was used forpreparation of the allografts. Animals in group I(N = 6) died of hyperglycemia in 4.0 ± 1.89 days(X ± SD) after pancreatectomy without subsequent is-let cell transplant. Normoglycemia and excellent sur-vival (>60 days) was obtained in pancreatectomizedanimals in group II (N = 6) and in group III (N = 6),who received an islet cell allograft to the renal subcap-sular site. Group II recipients were given no immuno-suppression, and animals in group III received minimalimmunosuppression with azathioprine. Dependence onthe islet cell allograft for maintenance of normogly-cemia was confirmed in the majority of the recipientsby nephrectomy, to remove the graft, with resulting hy-perglycemia and death. One normoglycemic animal ingroup II died on day 6 from peritonitis. One recipient ingroup II was normoglycemic at >1 mo after removal ofthe first graft by nephrectomy, followed by retrans-plantation of islet cells from a third-party donor. Twoother recipients are being studied on a long-term ba-sis, and have been normoglycemic for >6 mo and >4mo after transplantation. These studies encourage fur-ther investigation in this area for application of isletcell transplantation in man, and elucidation of the pos-sible mechanisms for prolongation of islet cell allograftsurvival at the renal subcapsular site. DIABETES 1984;33:910-14.

Progress in clinical and experimental islet cell trans-plantation has been slowed by many obstacles.Various implantation sites have been attempted forlong-term islet cell engraftment; however, graft fail-ure and/or rejection have usually been the result.1~3 De-pending on the method of preparation, the condition of thepancreas, and species used, a sufficient islet cell yield,

which will ameliorate diabetes, also has often been difficultto obtain.1"3 In an effort to improve survival and function aftertransplantation, we have investigated a new simplifiedmethod of pancreas preparation together with islet cell al-lograft placement into the renal subcapsular region. Thisarticle analyzes these preliminary findings.

MATERIALS AND METHODSIslet cell preparation. Pancreata were carefully dissectedfrom unrelated, adult mongrel dogs, flushed with cold (4°C),heparinized (10,000 U/L) Ringer's lactate until the venouseffluent was clear, and placed in saline ice. The pancreatictissue was then cut into 1-cm cubes and placed in cold (4°C)Hank's balanced salt solution (HBSS). The plunger of a 50-cc syringe was used to press the pieces of pancreatic tissuethrough a stainless steel screen (1.5-mm mesh). Processedtissue was then briefly washed twice in a refrigerated cen-trifuge (2000 rpm, 4°C) with HBSS. The resulting tissue pellet(10-15 g) was placed in a syringe for injection and the su-pernatant was discarded.Subcapsular implantation. After total pancreatectomy, asmall incision was made in the lower pole of the left kidneyof the recipient animal. A specially constructed instrumentwas inserted to separate the renal capsule from the under-lying renal cortex, thereby creating a pocket around the kid-ney. The preparation of islet cell fragments was then injectedinto the subcapsular pocket and the incision was closed witha purse-string suture (Figures 1 and 2).Experimental design. Three groups of experimental animalswere studied. All animals underwent total pancreatectomy.Group I (N = 6) animals served as apancreatic controls andwere not transplanted. Recipients in group II (N = 6) were

From the Department of Surgery (L.H.T.-P., K.O.B., D.A.G., G.H.M.), Sectionsof Transplantation and Surgical Research, and the Department of Pathology(T.A.R.), Mount Carmel Mercy Hospital, Detroit, Michigan.Address reprint requests to Dr. Luis H. Toledo-Pereyra, Department of Sur-gery, Section of Transplantation, Mount Carmel Mercy Hospital, 6071 W. OuterDrive, Detroit, Michigan 48235.Received for publication 30 April 1984 and in revised form 23 May 1984.

910 DIABETES, VOL. 33, SEPTEMBER 1984

Flush with heparinizedRinger's lactate (4*C)

FIGURE 1. Steps involved in the preparation ofpancreatic islet cell fragments for transplantation.The technique is very simple since no collagen-ase is used. As soon as the islet cell fragmentsare prepared, they are implanted into the kidneycapsule of a pancreatectomized recipient.

EXPERIMENTAL DESIGN

DONOR ANIMAL RECIPIENT ANIMAL

Pancreatectomy

Total Pancreatectomy

Cut into 1 cm cubesand press throughstainless steel sieve

Injection of islet cellpreparation into

renal subcapsular region

Centrifugal washing 4"C, HBSS

Islet cell preparation ready for injection

transplanted with the islet cell preparation placed under therenal capsule. No immunosuppression was given to thisgroup after transplantation. Animals in group III (N = 6) re-ceived a renal subcapsular islet cell allograft as in group II,but were given minimal immunosuppression with azathio-prine (2.5-5.0 mg/kg/day until death or sacrifice). All ani-mals were followed with daily determinations of plasma glu-cose for the first week after transplantation, and biweekly orweekly thereafter. Renal function was periodically assessedvia serum creatinine determinations.

In several animals, insulin determinations (RIA, CambridgeMedical Diagnostics, Billerica, Massachusetts) were ob-tained from blood from the right and left renal veins, portalvein, and jugular vein in order to determine the site at whichthe pancreatic islet cell fragments were producing the in-sulin. Nephrectomies of the kidneys that received the pan-creatic islet cell fragments were performed in several ani-mals. In one case, after nephrectomy, the pancreatic isletcell fragments from a third-party donor were transplantedsubcapsularly in the remaining kidney. Insulin was deter-

mined in this case in blood obtained from the right renal,portal, and jugular veins.

Intravenous glucose tolerance tests were periodically per-formed in transplanted animals by administration of 0.5 gglucose/kg body wt. Blood samples for glucose and insulinwere taken every 10 min for the first hour and at 2 h. K-values were calculated using the method of Lundbaek.4

Student's Mest was used to compare the functional re-sponse of the transplanted groups (II and III) with the non-transplanted pancreatectomized group (I).

RESULTSAllograft function. Table 1 shows the mean plasma glu-cose values after surgery. All pancreatectomized, nontrans-planted animals in group I became hyperglycemic by thefirst postoperative day and survived between 2 and 7 daysafter pancreatectomy (X ± SD = 4.0 ± 1.89 days). Alltransplanted animals in both groups II and III were normo-glycemic on the first postoperative day and continued to

DIABETES, VOL. 33, SEPTEMBER 1984 911

SUBCAPSULAR CELL TRANSPLANTATION

STEP1ANTERIORDISSECTION

STEP 3R4NCREATICFRAGMENTSINJECTION

STEP 2POSTERIORDISSECTION

have plasma glucose values that were within normal rangeand significantly less than those of the pancreatectomizedgroup (P < 0.001). Several animals in each group underwentremoval of the islet cell transplant, which was accomplishedby removal of the kidney containing the allograft. These an-imals subsequently became hyperglycemic as describedbelow.

In group II, one animal died on the sixth postoperative dayof peritonitis, but maintained normoglycemia until death. Twoof the other five animals survived >60 days with normogly-cemia and in the remaining three animals, left nephrectomieswere done at > 4 | mo after transplant to remove the islet cellallograft. In these nephrectomized recipients plasma glu-cose values rose to hyperglycemic levels on the followingday. Two of the nephrectomized group II recipients died onthe second day after islet cell allograft removal and onerecipient was retransplanted with an islet cell allograft froma second canine donor into the subcapsular region of theright kidney. This recipient has continued to be normogly-

STEP 4COMPLETEDPANCREATICFRAGMENTSSUBCAPSULARTRANSPLANT

FIGURE 2. Renal subcapsular transplantation ofpancreatic fragments: detailed aspects of thesubcapsular implantation. Careful dissection isimportant to prevent bleeding when the capsuleis lifted for the transplant.

cemic without immunosuppression for >1 mo after trans-plantation.

Four of the five animals in group III had their islet cellallografts removed at >3 wk, > 2 | mo, >4 mo, and >4 movia left nephrectomy, became hyperglycemic on the nextpostoperative day, and died within the next 1-2 days fromhyperglycemic coma. The remaining two animals in thisgroup have been normoglycemic for >6 mo and >4 mo andwill be studied on a long-term basis.Intravenous glucose tolerance tests. The K-values for theintravenous glucose tolerance tests in animals transplantedbetween 1 and 5 mo showed a normal response to the glu-cose challenge and ranged between 2.6 and 3.4. Normal,nontransplanted, nonpancreatectomized control animalshad K-values between 2.3 and 2.6.Differential insulin determinations. Insulin levels obtainedfrom the left renal vein adjacent to the islet cell transplantwere considerably higher (17.0-150 |xU/ml) than the insulinlevels noted on the contralateral side (7.6-40 |xU/ml), in the

912 DIABETES, VOL. 33, SEPTEMBER 1984

TABLE 1Plasma glucose values (mean ± SD) noted at various times after pancreatectomy and renal subcapsular islet cell transplantation

Group Group Group

NTreatment after pancreatectomy

ImmunosuppressionMean plasma glucose (mg/dl, X ± SD)

1 day3 days5 days7 days2-3 wk4-6 wk>8 wk

None—no transplant

335.3 ± 87.7486.4 ± 156.9537.5 ± 272.2

Renal subcapsularislet cell

transplantationNone

85.8 ± 10.383.7 ± 22.3

128.6 ± 48.095.0 ± 5.685.6 ± 14.389.2 ± 13.7

102.5 ± 5.2

Renal subcapsularislet cell

transplantationAzathioprine

77.7 ± 38.470.6 ± 36.5

104.0 ±11.398.0 ± 28.288.8 ± 18.7

114.0 ± 26.598.0 ± 8.5

portal vein (0.8-40 jill/ml), or in the jugular vein (8.0-40ml). In the animal who underwent left nephrectomy and re-transplantation of islet cell fragments to the contralateral kid-ney, the insulin levels were higher in the right renal vein (17.0|xU/ml) and near the islet cell transplant than at the portalvein (1.0 |xU/ml) or jugular vein (7.6 |xU/ml) sites.Histologic findings. The islet cell preparation for injectionappeared as pancreatic fragments of various sizes with aci-nar tissue surrounding islet cells. Early histologic studiesshowed an inflammatory reaction in the renal subcapsularregion. By 3-4 mo the inflammatory exudate had clearedand was replaced by a proliferating fibrous tissue layer. Un-derlying this layer, insulin- and glucagon-producing cellswere identified by specific immunoperoxidase staining tech-niques. These cells were frequently arranged in close prox-imity to capillaries (Figure 3). Cells with secretory granulesof appropriate size and configuration typical of alpha andbeta islet cells were also identified by transmission electronmicroscopy studies.Renal function. No compromise of renal function was seenafter islet cell transplantation as evidenced by serum cre-atinine values. In addition, renal biopsies taken at death or

sacrifice confirmed normal kidney histology of the renal cor-tex after islet cell implantation.

DISCUSSIONOur study demonstrates the feasibility of the renal subcap-sular region as a site for normal function of transplantedpancreatic islet cell fragments (prepared without collagen-ase). Many techniques have been attempted experimentallyin an effort to improve islet cell allograft survival. These in-clude various methods of islet cell preparation, graft anddonor pretreatment,56 cryopreservation of islets,7 islet cellculture before transplantation,8 utilization of fetal tissue,9 andplacement of islets in special diffusion chambers to protectthem from immune recognition.10 Although the intraportal,intrasplenic, and intrap'eritoneal sites have been commonlyused for islet cell allotransplantation, several others have alsobeen described.1 These include the testis, anterior chamberof the eye, the hamster cheek pouch, the mammary fat pads,and the omentum.

The kidney subcapsular region has also been exploredas a potential site for islet cell transplantation in small ani-mals. However, our work is the first to identify the feasibility

FIGURE 3. Using immunoperoxidase stainingtechniques, specific for insulin, positive stainingcells (beta cells) were observed. In this photomi-crograph, one is located adjacent to a capillary(C), within an area of connective tissue. (Immu-noperoxidase, 40 x.)

DIABETES, VOL. 33, SEPTEMBER 1984 913

of performing subcapsular islet cell transplantation (withoutcollagenase) in large animals. Bowen et al.11 successfullytransplanted isogeneic fetal pancreas tissue beneath thekidney capsule and produced reversal of experimental di-abetes in the rat. However, Garvey and associates12 ob-served that similar allograft preparations were rejected rap-idly at this site. Pi-Sunyer and associates13 made similarobservations with neonatal pancreas tissue. Reece-Smith etal.14 attempted renal subcapsular implantation of adult ratislet allografts and found that although survival was some-what prolonged, this type of islet preparation was also re-jected. Recent work by Reece-Smith et al.14 has indicatedthat transplantation of islet cell allografts beneath the renalcapsule of diabetic rat recipients, immunosuppressed withcyclosporine (Cy A), was not prolonged. The capsule in theirstudies was not sutured; thus, possible spillage of islets frombeneath the renal capsule into the peritoneal cavity duringand immediately after transplantation could have causedsensitization of the recipients. Furthermore, there might bespecies differences. Improved survival was only obtainedby transplantation of syngeneic islet cell-kidney compositegrafts to histoincompatible diabetic recipients given Cy A.15

Subsequent studies have shown that prolonged islet cellallograft survival may be obtained by preliminary culture ofthe islet cells before implantation under the renal capsule.16

Our preliminary studies using a simplified collagenase-free method for preparation of canine islet cell allografts,with subsequent transplantation into the renal subcapsularregion, are promising. Using this new method of pancreasprocessing, normoglycemia was obtained after transplan-tation in all recipient animals. No compromise of renal func-tion was observed in any of the recipient animals. It is difficultto assess the factors that may have contributed to the im-proved survival, without evidence of rejection, in our studies.Possibly, the renal subcapsular site has more of an immu-nologically privileged character in the dog, thus allowing forislet cell allograft survival with minimal or no immunosuppres-sion.

It is evident that further studies will be necessary to fullyanswer these questions and provide for future applicationof these techniques to improve human islet cell transplan-tation. We are currently conducting studies to determine thesurvival of collagenase-prepared islet cell allografts placedat the renal subcapsular site. In addition, intrasplenic trans-plantation of the noncollagenase preparation is being eval-uated.

In summary, these preliminary studies indicate that pro-longed survival of canine islet cell allografts may be obtainedusing a collagenase-free technique for preparation and sub-sequent implantation under the renal capsule. These resultsmay suggest that this site may be immunologically privi-leged, since no evidence of rejection was observed.

ACKNOWLEDGMENTThis work was supported by an institutional research grantfrom the Mount Carmel Research and Education Corpora-tion.

REFERENCES1 Sutherland, D. E. R.: Pancreas and islet transplantation. I. Experi-

mental Studies. Diabetologia 1981; 20:161-85.2 Sutherland, D. E. R.: Pancreas and islet transplantation. II. Clinical

trials. Diabetologia 1981; 20:435-50.3 Toledo-Pereyra, L. H: Islet cell transplantation. In Pancreas-Basic

Concepts of Medical and Surgical Practice. In press. New York, John Wiley& Sons, 1984.

4 Lundbaek, K.: Intravenous glucose tolerance as a tool in definitionand diagnosis of diabetes mellitus. Br. J. Med. 1962; 1:1507-13.

5 Toledo-Pereyra, L. H., Gordon, D. A., and MacKenzie, G. H.: Prolon-gation of pancreatic islet cell allograft survival by graft pretreatment withantilymphoblast globulin (ALG). Am. Surg. 1982; 48:282-86.

6 Toledo-Pereyra, L. H., Gordon, D. A., and MacKenzie, G. H.: Trans-plantation of islet cells. Surg. Gynecol. Obstet. 1984; 158:49-56.

7 Toledo-Pereyra, L. H., Gordon, D. A., and MacKenzie, G. H: Cry-opreservation of islets of Langerhans. Cryobiology 1981; 18:483-88.

8 Gordon, D. A., Toledo-Pereyra, L. H., and MacKenzie, G. H.: Pres-ervation for transplantation: a review of techniques of islet cell culture andstorage. J. Surg. Res. 1982; 32:182-93.

9 Eloy, R., Doillon, C, andDubois, P.: Fetal pancreatic transplantation—review of experimental data. Transplant. Proc. 1980; 12 4 (Suppl. 2):179-85.

10Theodorou, N. A., Easterbrook, P., Tyhurst, M., and Howell, S. L:Islets of Langerhans implanted in diffusion chambers do not initiate antibodyproduction. Transplantation 1981; 31:89-90.

11 Bowen, K. M., Andrus, L, and Lafferty, K. J.: Successful allotrans-plantation of mouse pancreatic islets to nonimmunosuppressed recipients.Diabetes 1980; 30:285.

12 Garvey, J. F. W., Klein, C, Millard, P. R., and Morris, P. J.: Rejectionof organ-cultured allogeneic fetal rat pancreas. Surgery 1980; 87:157.

13 Pi-Sunyer, F. X., Woo, R., Weber, C, Hardy, M. A., and Reemtsma,K.: Selection of a practical method of pancreatic islet transplantation in therat. Surg. Forum. 1979; 30:301.

14 Reece-Smith, H., DuToit, D. F., McShane, P., and Morris, P. J.: Pro-longed survival of pancreatic islet allografts transplanted beneath the renalcapsule. Transplantation 1981; 31:305-306.

15 Reece-Smith, H, Homan, W. P., DuToit, H., McShane, P., and Morris,P. J.: A technique for transplanting pancreatic islets as a vascularized graftand prevention of rejection with cyclosporine A. Transplantation 1981; 31:442-44.

16 Lacy, P. E., Finke, E. H., Janney, C. G., and Davis, J. M.: Prolongationof islet xenograft survival by in vitro culture of rat megaislets in 95% 02.Transplantation 1982; 33:588-92.

914 DIABETES, VOL. 33, SEPTEMBER 1984