Pancreas Transplantation Versus Islet Transplantation Versus Insulin Therapy in the Prevention of Nephropathy in Alloxan-Induced Diabetic Rats C.T. Spadella, S.A. Schellini, and C.E. Bacchi C LINICAL and experimental studies have shown that careful glucose control may prevent, stabilize, or reverse renal and other chronic diabetic complications. 1,2 However, the results of these studies are controversial because progression of diabetic lesions has been observed in a large number of patients after strict control of blood glucose using insulin therapy 3,4 or insulin pumps. 5 On the other hand, there are no definitive conclusions on the real effects of pancreas and islet transplantation on the preven- tion of kidney or other chronic lesions of diabetes. The study protocols are numerous, and the results are varied and conflicting. 6 In this study we addressed the kidney lesions of alloxan- diabetic rats and the long-term effect of insulin treatment and pancreas and islet transplantation as a means of prevention of these lesions. We hope to contribute to a better understanding of diabetes and the above mentioned issues. MATERIALS AND METHODS Animals and Groups Two-hundred fifty inbred male Lewis rats, approximately 3 months old, were randomly assigned to 5 experimental groups of 50 specimens each, code-named and handled as follows: NC, nondi- abetic control rats; DC, untreated diabetic control rats; I, diabetic, insulin treatment; PT, diabetic, treated with pancreas transplants from normal donor Lewis rats; and IT, diabetic, treated with pancreatic islet transplantation prepared by collagenase from nor- mal donor Lewis rats and injected into the portal vein. Each group was further divided into 5 subgroups of 10 rats each that were sacrificed after 1, 3, 6, 9, and 12 months of follow-up, respectively. Diabetes was induced by intravenous administration of alloxan (Sigma Co, St. Louis, Mo) in a single dose of 42 mg/kg body weight. Only diabetic rats showing severe diabetic state were included in the experiment. Pancreas transplantation (PT) was performed according to the original procedure described by Lee et al. 7 Islets of Langerhans were prepared by collagenase according to the procedure originally described by Moskalewski 8 and subsequently modified. 9,10 About 400 to 500 cells diluted into 2 mL of Hanks’ solution were injected into the portal vein of IT rats. Insulin was administered daily in the afternoon, using slow action porcine insulin (Monotard, Thompson Lab., USA) by subcutaneous injec- tion according to the intensity of glucosuria and ketonuria. Insulin therapy, pancreas, and islet transplantation were performed 14 days after alloxan administration. Clinical and Laboratory Analysis Seven days prior to; 4 days after; and 1, 3, 6, 9, and 12 months after insulin treatment and pancreas and islet transplantation, rats were housed in metabolic cages for 24 hours and body weight, food and water intake, urine output, blood and urine glucose levels, and plasma insulin were determined. These same parameters were also recorded for nondiabetic and the alloxan control rats. Blood and urine glucose levels were determined by the enzymatic method, and plasma insulin levels by radioimmunoassay in solid phase (DPC, Lab., USA). Histology Tissue fragments from the kidneys of 5 rats in each subgroup of the 5 experimental groups were fixed in 10% formalin, embedded in paraffin, and routinely stained with hematoxylin-eosin. Fifty glo- meruli and 50 tubules from each kidney were analysed through light microscopy by two independent researchers in a double-blind study. A total of 250 glomeruli and 250 tubules was examined for each subgroup by each of the two researchers. Glomerular base- ment membrane thickening (GBMT), mesangial enlargement (ME), Bowman’s capsule thickening (BCT), Armanni-Ebstein le- sions of the tubules (AE), and tubular lumen protein (PRO) were studied. Both glomerular and tubular lesions were scored on a scale of zero to three (0-absent; 1-minimum; 2-medium, and 3-severe). The discrepancy between the two observers was no more than 5%. Laboratory data were analysed by Morrison’s profile analysis and histology data by analysis of variance and the Student’s t-test, with the level of significance set at P  .01. RESULTS Clinical and Laboratory NC rats showed no evidence of clinical or metabolic abnormalities. DC rats presented a progressive loss of body weight, a significant increase in food and water intake, a significant decrease in urine output as well as increased blood and urine glucose levels, and significantly decreased From the Department of Surgery Ophthalmology, and Pathol- ogy, School of Medicine, University of the State of Sao Paulo, Botucatu, SP, Brazil. Research supported by FAPESP. Address reprint requests to C.T. Spadella, MD, Faculdade de Medicina de Botucatu, UNESP, 18618-970, Botucatu, Sa ˜o Paulo, Brasil. © 1998 by Elsevier Science Inc. 0041-1345/98/$19.00 655 Avenue of the Americas, New York, NY 10010 PII S0041-1345(97)01291-8 Transplantation Proceedings, 30, 327–329 (1998) 327