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Background. Delayed graft function (DGF) occurs in up to 50% of renal transplants. Hypercalcemia and hyperparathyroidism are associated with impaired renal function. Little is known on the effects of serum calcium levels on DGF. This issue was addressed in the current study. Methods. Patients receiving a cadaveric renal transplant between 1986 and 1996 were studied. Data on calcium metabolism and histologic characteristics of nephrocalcinosis, acute tubular necrosis (ATN), and acute rejection in biopsies taken within the first week were related to the occurrence of DGF. Results. The incidence of DGF in a cohort of 585 cadaveric transplants was 31%. DGF correlated independently with serum calcium levels (odds ratio [OR] 1.14 [95% confidence interval (CI) 1.04–1.26] per 0.1 mmol/L). The use of calcium channel blockers before transplantation protected against DGF (OR 0.5 [95% CI 0.29– 0.87]). In this selected group, we found an association with histologic signs of ATN and DGF. However, most of the biopsies also had features of acute rejection or nephrocalcinosis. Nephrocalcinosis was found in 12 of 71 biopsies and was not associated with serum calcium levels or the occurrence of DGF. Conclusions. In this study, serum calcium levels were independently associated with DGF. This could not be explained by the presence of microscopic nephrocalcinosis. Therefore, DGF is attributed to high intracellular calcium levels. Because calcium supplementation and vitamin D analogues are commonly used in dialysis practice, hypercalcemia influences long-term graft outcome by its effect on DGF. The pretransplant use of calcium channel blockers has a protective effect on the occurrence of DGF.

Background Rejection after pancreas-kidney transplantation may occur isolated or concurrently in both grafts. To get more insight into the cellular mechanisms underlying these rejection episodes, we compared the functional characteristics of pancreas and kidney graft-infiltrating T cells. Methods Graft-infiltrating T cell (GIC) lines were cultured from simultaneously taken pancreas and kidney biopsies from eight patients. CD4 to CD8 ratios were determined by fluorescence-activated cell sorter and cytotoxicity toward donor proximal tubular epithelial cells (PTEC) and donor spleen cells (DSC) using a standard cytotoxicity assay. Cytokine production was determined by enzyme-linked immunosorbent assay. Results CD4 to CD8 ratios were comparable between the pancreas and kidney lines for each patient, but differences were observed in cytotoxicity toward PTEC and DSC. For four of eight patients, the lysis of PTEC by pancreas GIC was less than the lysis induced by kidney GIC. This was also seen in three of five patients for lysis of DSC. The specificity of GIC lines toward mismatched donor antigens was studied for two patients and appeared to be comparable for pancreas and kidney. Most GIC lines produced interferon (IFN)-gamma (75.5+/-22.7 pg/ml), but no IL-10, indicating that the cell lines consisted primarily of Th1 and type 1 CD8+ cells. Mean production of IL-6 was 465.6+/-193.6 pg/ml. No major differences were observed between kidney and pancreas GIC for either cytokine. Conclusions We conclude that pancreas and kidney GIC lines have the same phenotype, cytokine production, and allospecificity. Differences were, however, seen for lysis of PTEC and DSC, suggesting that tissue-specific antigens might play a role.

Chronic rejection of renal allografts is a major cause of late graft loss. However, time of onset, relation with acute early rejection episodes, and risk factors are largely unknown. We undertook a cohort study of 482 consecutive patients from a single center who received a cadaveric renal allograft between January 1983 and April 1991. During the first 3 months after transplantation, 76 (15.8%) patients developed vascular rejection and 1 15 (23.9%) developed interstitial rejection. One-year graft survival of patients without rejection, with interstitial rejection, and with vascular rejection was 87.8%, 87%, and 48.7%, respectively. Five-year graft survival was 73.5% for the group without rejection, 71.4% for patients with interstitial rejection, and 34.3% for patients with vascular rejection. The adjusted relative risk of graft loss was 4.92 (95% CI 3.25-7.43) for patients with vascular rejection and 1.27 (95% CI 0.80-2.02) for patients with interstitial rejection compared with patients without early rejection, taking the time dependency of the rejection events and prognostic factors into account. The incidence of vascular rejection was increased in patients with primary nonfunction (RR 1.69, 95% CI 1.01-2.84), with 1 HLA-DR mismatch (RR 2.38, 95% CI 1.44-3.93), with 2 HLA-DR mismatches (RR 3.24, 95% CI 1.25-8.42), with a prolonged cold ischemia time (RR 1.03, 95% CI 1.00-1.06 per hr), and with 1 or more previous transplantations (RR 1.76, 95% CI 1.01-3.07). Risk of developing vascular rejection was decreased in patients using CsA as compared with azathioprine (RR 0.41, 95% CI 0.24-0.67). Early vascular rejection, occurring within 3 months after transplantation, is the most important predicting variable of both early and late graft loss. Use of CsA less HLA-DR mismatching, and in cold ischemia time of short duration possibly prevent the development of vascular rejection

Infectious complications of either high-dose (16 mg/kg/day) or low-dose (9 mg/kg/day) cyclosporine in combination with azathioprine (Aza) (1 mg/kg/day) were studied in 128 renal transplant patients who also received low-dose prednisone (P). Three months after transplantation all patients were again randomly assigned to either continuation with CsA/P or conversion to Aza/P. During the first 3 months the number of infections was significantly lower in the CsA/P treatment than in the CsA/Aza/P group. In both groups the number of infections doubled after rejection treatment. The frequence of symptomatic CMV disease did not differ between the 2 groups. Three months after transplantation, the patient group assigned to Aza/P had a small but not significant increase of minor infections when compared with the patients who continued with CsA/P. The number of major infections did not differ between these two groups.

Background. Acute tubular necrosis (ATN) in renal allograft biopsies correlates poorly with delayed graft function (DGF). Factors involved in the pathogenesis of DGF were evaluated in biopsies in an attempt to refine the recognition of DGF. Methods. Anti-cubulin and anti-AE-1/AE-3 antibodies identified proximal and distal tubules, respectively. The terminal deoxynucleotide transferase-mediated dUTP nick-end labeling technique and active caspase-3 staining were used to demonstrate apoptosis. Antibodies against superoxide dismutase (SOD) were used as markers of the protective tubular response. Tubular regeneration was evaluated using anti-ki 67 and antivimentin antibodies. Results. Of a total of 40 biopsies, 9 were associated with DGF. ATN was seen in 16 biopsies; 5 were associated with DGF. The finding of ATN in the biopsy of a graft predicted DGF in only 56% of cases. Absence of distal caspase-3 staining predicted the absence of ATN in 87% of cases. The presence of caspase-3 predicted ATN in 54% of cases. The detection of manganese-SOD in distal tubules predicts the absence of DGF in 76% of the cases. Conclusions. The use of immunohistochemical staining on posttransplant renal biopsies improved its predictive value with respect to ATN and DGF: The absence of active caspase-3 in distal tubular epithelium predicts the absence of ATN in 87% of cases, whereas its presence predicts ATN in 54% of cases. The presence of manganese-SOD in distal tubules predicts the absence of DGF in 76% of cases.

Chronic rejection of renal allografts is a major cause of late graft loss. However, time of onset, relation with acute early rejection episodes, and risk factors are largely unknown. We undertook a cohort study of 482 consecutive patients from a single center who received a cadaveric renal allograft between January 1983 and April 1991. During the first 3 months after transplantation, 76 (15.8%) patients developed vascular rejection and 115 (23.9%) developed interstitial rejection. One-year graft survival of patients without rejection, with interstitial rejection, and with vascular rejection was 87.8%, 87%, and 48.7%, respectively. Five-year graft survival was 73.5% for the group without rejection, 71.4% for patients with interstitial rejection, and 34.3% for patients with vascular rejection. The adjusted relative risk of graft loss was 4.92 (95% CI 3.25-7.43) for patients with vascular rejection and 1.27 (95% CI 0.80-2.02) for patients with interstitial rejection compared with patients without early rejection, taking the time dependency of the rejection events and prognostic factors into account. The incidence of vascular rejection was increased in patients with primary nonfunction (RR 1.69, 95% CI 1.01-2.84), with 1 HLA-DR mismatch (RR 2.38, 95% CI 1.44-3.93), with 2 HLA-DR mismatches (RR 3.24, 95% CI 1.25-8.42), with a prolonged cold ischemia time (RR 1.03, 95% CI 1.00-1.06 per hr), and with 1 or more previous transplantations (RR 1.76, 95% CI 1.01-3.07). Risk of developing vascular rejection was decreased in patients using CsA as compared with azathioprine (RR 0.41, 95% CI 0.24-0.67). Early vascular rejection, occurring within 3 months after transplantation, is the most important predicting variable of both early and late graft loss. Use of CsA, less HLA-DR mismatching, and a cold ischemia time of short duration possibly prevent the development of vascular rejection.

Clinically, there is an association between CMV infections and the occurrence of rejection after renal transplantation. Adhesion molecules such as intercellular adhesion molecules (ICAM) and MHC Ags are thought to be important in the induction and amplification of the rejection process. Therefore, we studied ICAM-1 and MHC expression after CMV infection or stimulation with cytokines. Cultured proximal tubular epithelial cells (PTEC) were stimulated with cytokines or infected with CMV. MHC class I, class II, and ICAM-1 expression were determined by radioimmunoassay. IFN-gamma induced class II and ICAM-1 expression. Small concentrations of IFN-alpha inhibited the IFN-gamma induced class II expression. CMV-infected PTEC displayed increased levels of ICAM-1 and class I expression. This enhancement is a direct effect of the virus on the infected cells and not mediated by soluble factors. Although MHC class II expression is not directly enhanced by CMV, infected PTEC display a normal increase of class II expression after stimulation with IFN-gamma.

Serum samples of 64 consecutive patients who underwent renal transplantation in our institution were examined for the presence of antibody-dependent cellular cytotoxicity (ADCC) activity against endothelial cells (EC). From each patient serum samples were obtained immediately before transplantation and 1 week, 1 month and 1 year thereafter. The results were evaluated in the context of tests to measure donor-specific humoral immunity against lymphocytes and monocytes, and related to parameters of presensitization, graft survival, and histology. Sera from 10 patients were positive for ADCC on a panel of HLA-typed endothelial cells. In 8 patients sera were already positive before transplantation and remained positive thereafter. In 4 patients a positive crossmatch with donor T and B cells and monocytes could be observed after transplantation. In only one patient were these crossmatches positive before transplantation. A significant correlation was found between ADCC positivity and vascular rejection (P = 0.015); in addition graft survival was significantly better in the ADCC negative group vs. the positive group (P = 0.0004). These data demonstrate the significance of allospecific anti EC antibodies for the occurrence of vascular rejection and graft loss after renal transplantation.

Fifty-nine renal transplant recipients were followed during the first 3 months after transplantation. Once weekly, cultures of urine and buffy coat for CMV were taken. Furthermore, peripheral blood leukocytes were examined by an immunocytochemical assay for immediate early antigens of CMV (IEA assay) and by a polymeric chain reaction for CMV DNA. Forty-four patients had a CMV infection; 23 of them were symptomatic. PCR was positive in 22 of the 23 patients with symptomatic CMV disease. For cultures of urine, buffy coat, and the IEA assay these figures were 23, 20, and 21, respectively. The PCR was the first test to become positive in 10 patients. For the cultures of urine and buffy coat and the IEA assay these figures were 0, 2, and 2, respectively. In the other 9 patients, 2 or more tests became positive at the same time. In the patient group with a CMV infection but without symptoms the PCR also had a good correlation with the other diagnostic techniques. These results together with its short processing time of 6 hr make the PCR a sensitive and rapid technique for monitoring CMV infections after renal transplantation.

Cellular infiltrates in 20 renal allograft biopsies taken at the time of acute rejection episodes were analyzed with antibodies reactive with monocytes (BRL antihuman monocyte monoclonal antibody), T lymphocyte subpopulations (OKT- and Leu-series of monoclonal antibodies), and B lymphocytes (heterologous antihuman IgM antibodies). For demonstration of the various mononuclear cells, an indirect immunoperoxidase technique was used. The number of monocytes in the infiltrates varied from 10-20%; the number of B lymphocytes was always below 10%. The T lymphocytes accounted for 50-90% of the total number of mononuclear cells. The OKT4/OKT8 ratios for the T lymphocytes in the graft infiltrates were correlated with the peripheral blood OKT4/OKT8 ratios measured by indirect fluorescence and flow cytometry. The OKT4/OKT8 ratios for perivascular infiltrates correlated far better with peripheral blood OKT4/OKT8 ratios (r = 0.72) than did the OKT4/OKT8 ratios for interstitial infiltrates (r = 0.58). Low or inverted OKT4/OKT8 ratios and low or inverted Leu 3a/Leu 2a ratios were associated with a high risk of irreversible graft rejection (P less than 0.001 for perivascular infiltrates).

To investigate the comparability of the OKT and Leu monoclonal antibodies (mAbs) used for immunological monitoring, T cell subpopulations in 360 blood samples from 35 renal allograft recipients were studied by flow cytometry using both series of mAbs. To identify the activated cells in the different subpopulations, those labeled with the Leu mAbs were double-labeled with an anti-HLA-DR mAb. The percentages of T cells that stained with the Leu3a and Leu2a mAb using a direct immunofluorescence technique were significantly lower than the percentages of cells that stained with the OKT4 and OKT8 mAb using an indirect immunofluorescence technique (47.6 +/- 12.8% and 14.5 +/- 9.1% [mean +/- SD] versus 50.5 +/- 11.2% and 18.3 +/- 8.0%, respectively; P less than 0.0001). Since the difference between the Leu2a+ and OKT8+ cells was relatively greater (21%) than that between the Leu3a+ and OKT4+ cells (6%), the mean Leu3a/2a ratio was significantly higher than the mean OKT4/8 ratio (6.64 +/- 9.7 versus 3.60 +/- 2.82; P less than 0.0001). The difference between the Leu2a+ and OKT8+ cells were not attributable to a difference in the sensitivity of the direct and indirect immunofluorescence technique. A large difference between the Leu2a+ and OKT8+ cells correlated with a high percentage of DR+ cells within the Leu2a population (P less than 0.001). No correlation was found for the difference between the OKT4+ and Leu3a+ cells and the DR expression in the Leu3a+ cell population. These observations suggest that activation of some T cells of the "cytotoxic-suppressor" phenotype leads to antigen modulation in which the OKT8 epitope is not affected but the Leu2a epitope disappears or cannot be detected with the present method.

The effects of HLA-A and B matched pretransplant blood transfusions on the survival of a primary cadaveric kidney graft were studied prospectively in a group of 15 patients who had never received a transfusion and had never been pregnant. Kidney graft survival at one year was 87%, whereas a group of 14 nontransfused patients who underwent transplantation in the same center (before this study was initiated) had a graft survival of only 7%. Twenty-six patients who received a transplant in the same center just before and after each protocol patient served as controls. There were no prior pregnancies in this group; all patients had received blood transfusions from random blood bank donors. Kidney graft survival at one year was 76% for this control group, which is not statistically different from that found for the protocol group. Graft survival for the 13 contralateral kidneys from the protocol group donors was only 50% at one year. These kidneys, however, were transplanted in various other centers. From our study, prolongation of kidney graft survival could be demonstrated for patients receiving pretransplant HLA-A-and-B-matched blood transfusions. Sera screening indicated that lymphocytotoxicity might be reduced by pretransplant HLA-A-and-B-matched blood transfusions. The presence of pretransplant antibodies with specificities for HLA-A and/or B could be significantly correlated with poor graft survival.

Various rhesus alloantisera were tested for their capacity to induce enhancement of kidney allograft survival in D locus-incompatible rhesus monkeys. Six experimental groups were investigated: 1, ten allografted monkeys remained untreated; 2, six recipients were treated with Imuran and prednisolone; 3, eight rhesus monkeys were treated with a polyspecific "anti-SD" serum; 4, four animals were given oligospecific antikidney serum; 5, four monkeys were treated with oligospecific antiblood serum; and 6, nine recipients were given anti-Ia-like sera plus immunosuppressive treatment with Imuran and prednisolone. It was found that neither treatment with Imuran and prednisolone nor the administration of any of the alloantisera had a substantial effect on kidney graft survival. However, there appeared to be a difference in renal function among experimental groups. At 6 days after grafting, the mean blood urea level in the group treated with Ia-like antisera was found to be significantly lower than those of all other groups. It is discussed that this favourable effect is attributable to treatment with anti-Ia-like sera and not to the additional factors (conventional immunosuppression and partial matching for Ia-like antigens) occurring in this experimental group.

Kidney transplantations were performed in unrelated, immunosuppressed rhesus monkeys matched for two DR antigens and given five pretransplant transfusion of whole blood. The host-donor combinations were either reactive or nonreactive in mixed lymphocyte culture (MLC) and shared up to three A and B locus antigens with their blood and kidney donors. Although previous monkey experiments had shown a definite positive influence of DR matching as well as of pretransplant transfusions on graft survival, this was not found in the current experiments (the mean survival time for the MLC responsive group was 21.3 days for the MLC nonresponsive group 28.2 days). It appears therefore that the combination of pretransplant transfusions and matching for DR antigens does not have an additive or synergistic effect on graft survival. In fact, the prominent transfusion effect demonstrated previously may have been somehow compromised by matching for DR antigens. Alternatively, the clearly positive effect of matching for two DR antigens may have been reduced or lost as a consequence of giving the blood transfusions.

The relation between urinary kallikrein excretion ( Ukal ) and rejection, graft function, and blood pressure was studied in 45 renal transplant recipients. Ukal was assayed by means of an enzymatic (amidolytic) method, as well as with a specific radioimmunoassay. In a group of 10 patients studied longitudinally from the day of transplantation till day 35 +/- 3, an increase in urinary amidolytic activity without a concomitant increase in kallikrein antigen excretion was found to precede 11 out of 14 rejection episodes. This increased amidolytic activity generally persisted for several days. It was demonstrated by chromatography using an immunoadsorbent column of antiurokallikrein that the rejection-associated esterase, or esterases, differed from urokallikrein . In 35 outpatient recipients with stable graft function, Ukal excretion was decreased compared with that of healthy controls (42 +/- 7.5 vs. 107.5 +/- 7.3 micrograms/24 hr by radioimmunoassay and 0.70 +/- 0.08 vs. 1.10 +/- 0.07 U/24 hr, using the amidolytic method); for these patients a significant correlation between Ukal excretion and creatinine clearance was found (P less than 0.02). Both in transplant recipients and in controls there was a close correlation between the results of the two Ukal assays (P less than 0.001). No significant relation between Ukal excretion and blood pressure was found, either for patients or for controls. It is concluded that acute graft rejection is accompanied by an increased excretion of nonurokallikrein esterase(s). The lower Ukal excretion in patients with stable renal function seems to be related to their reduced renal function. No relation between Ukal excretion and blood pressure levels was found.

Immunological monitoring of peripheral blood cells of renal transplant patients may be helpful for diagnostic and prognostic purposes. We therefore enumerated the percentage of Leu2a+ cells as well as the occurrence of HLA-DR activation markers within this population. Since Leu2a+Leu15+ lymphocytes display suppressor functions in-vitro, this population was included in the study. Blood samples of 50 renal transplant patients within 3 months of transplantation and 41 pretransplant samples were investigated. As controls, peripheral blood cells from 10 healthy volunteers and 20 renal transplant patients with stable graft function more than 6 months after transplantation were examined. Just prior to transplantation the percentages of Leu2a+ cells, as well as the percentages of Leu15+ and DR+ cells within this population, varied considerably. Shortly after transplantation, clinical rejection episodes and cytomegalovirus (CMV) infections correlated with decreased percentages of Leu2a+ cells (P = 0.024 and 0.016, respectively). In nine patients with a decreased percentage of Leu2a+ cells shortly after transplantation but no rejection, the percentage of Leu2a+ cells normalized within 14 days. No correlation was found between increased percentages of Leu15+ or DR+ cells and rejection episodes, although the percentages of Leu2a+Leu15+ and Leu2a+DR+ cells increased significantly shortly before rejection (P less than 0.005). A decreased percentage of Leu2a+ cells, together with increased percentages of Leu15+ and DR+ cells, was seen in association with CMV infections (P less than 0.005). From this study we conclude that low percentages of Leu2a+ cells correlate with rejection episodes or CMV infections, whereas low percentages together with increased percentages of Leu2a+DR+ and Leu2a+Leu15+ cells are associated with CMV infections, especially in patients treated with high doses of cyclosporine.