The investigation and treatment of renal bone disease.

Neutron activation analysis of a hand has been used to monitor changes in skeletal calcium content of patients undergoing maintenance hemodialysis. Assessed by this technic supplementary magnesium therapy was of value of reducing the progressive losses of skeletal calcium in a proportion of the patients receiving maintenance hemodialysis. Oral calcium supplements produced a decrease in the skeletal calcium losses of all the patients studied. 1-alpha-hydroxycholecalciferol, a potent vitamin D analogue, caused a significant increase in the calcium content of bone in three patients receiving maintenance hemodialysis; it may prove to be a safe and effective form of therapy for azotemic osteodystrophy.

The influence of repeated transfusions and cyclosporine on secondary alloantibody responses in inbred rats.

The influence of cyclosporine (CsA) on secondary and established alloantibody responses was evaluated in inbred Lewis rats and (AO x PVG)F1 hybrid rats. Lewis rats received weekly transfusions of DA whole blood for 8 weeks either with or without cyclosporine (15 mg/kg/day) after sensitization with DA splenocytes. Hybrid rats received only CsA (10 mg/kg/day) after similar sensitization. Administration of CsA did not affect the spontaneous decline in alloantibody titers against class I (RT1A) antigens, but it was associated with a significantly reduced response to class II (RT1B) antigens at the end of the study. CsA prevented maintenance of high alloantibody titers to RT1A antigens in Lewis rats transfused repeatedly following sensitization. IgG alloantibody subclass responses were also altered by CsA with significant reduction in titers of IgG1, 2a, and 2b against RT1A antigens in rats transfused repeatedly; CsA did not, however, suppress IgG2c alloantibody levels in these animals. Responses to public RT1A antigens disappeared in most animals irrespective of their treatment group, whereas those to private and public RT1B antigens persisted unless CsA was administered. The results suggest that, contrary to results obtained with other antigens, CsA does influence secondary alloantibody responses. CsA may thus prove of value in highly sensitized dialysis patients who require further blood transfusions.

Glomerulotubular function in long-term renal allograft recipients. A comparison of conventional therapy with cyclosporine.

Glomerular and tubular function were assessed, using a lithium clearance technique, in two groups of renal allograft recipients at least one year after transplantation. Group 1 comprised 14 patients receiving low-dose prednisolone and cyclosporine, and group 2, 14 patients receiving low-dose prednisolone and azathioprine. There were no significant differences in creatinine clearances between the two groups, although the clearances of lithium (which is absorbed almost exclusively from the proximal tubule) and sodium were significantly lower in the cyclosporine-treated group. Fractional lithium excretion was also significantly lower in group 1 than in group 2, but there was no significant difference in fractional sodium excretion. The absolute proximal reabsorption of sodium and water did not differ between the groups, although the fractional proximal reabsorption of sodium and water was significantly higher in group 1. In contrast, the distal reabsorption of sodium and of water was significantly lower in the cyclosporine-treated patients than in the azathioprine-treated patients; there were, however, no significant differences in the distal fractional reabsorptions of sodium and water between the two groups. In addition there was no correlation in group 1 between whole-blood cyclosporine levels or time since transplantation and any of the assessed parameters of renal function. These results indicate that tubular concentrating abnormalities in cyclosporine-treated renal allograft recipients are similar to those observed in rodent models of cyclosporine nephrotoxicity. They suggest that the pathogenesis of cyclosporine nephrotoxicity may be similar in renal allograft recipients to that in experimental models.

Alloantibody and transferable suppressor activity induced by cyclosporine and blood transfusions in the rat.

The effect of cyclosporine on the alloantibody response to blood transfusion was investigated in inbred strains of rats by IHA and CELISA; recipient animals differed from the donors at the class I (RT1A) or both class I and class II (RT1B) antigens of the major histocompatibility complex. Alloantibody titers stimulated in high responder PVGu/c animals by blood transfusions were attenuated by cyclosporine; this effect was not demonstrated in low responder PVGc rats, as alloantibody titers decreased after further blood transfusions whether or not cyclosporine was given. Cyclosporine not only reduced the initial IgM response but suppressed the subsequent production of IgG. Splenocytes from rats receiving cyclosporine and blood transfusions from donors that differed from the recipients at the class I antigen were effective in suppressing the subsequent antibody response to blood transfusion. When blood transfusions from donors which differed from the recipients at both class I and class II antigenic loci were given after splenocyte transfer, a greater degree of immunosuppression was detected than if the transfusion donor differed only at the class I locus. These data suggest that the sensitization produced by blood transfusions and the persistence or decline of the alloantibody response depend upon the responder status of the recipient. Blood transfusions given with cyclosporine are capable of inducing suppressor activity that is transferable in spleen homogenates. Subsequent alloantibody responses are influenced by the class I and class II disparities of the donor and recipient animals. If these results can be extrapolated to clinical practice, cyclosporine should be given with pretransplant blood transfusions to prevent sensitization, and the transfusion donor should differ from the recipient at both class I and class II antigenic loci.

The alloantibody response to semiallogeneic pregnancy in the rat. II. Antibodies in serum directed to multiple epitopes on conventional RT1A antigens.

Evidence for a unique class I MHC antigen (termed Pa), which is believed to be expressed on rat trophoblast during pregnancy and which stimulates alloantibody formation with unusual interstrain cross-reactivity, has been examined in inbred rats. The previously reported pattern of crossreactivity was confirmed but was not unique to antisera produced by pregnancy. Antibody blocking studies using biotinylated rat monoclonal antibodies to distinct epitopes on RT1Aa antigens suggested that antibodies present in pregnancy sera, especially from multiparous rats, reacted with several epitopes on these molecules. Moreover, a rat monoclonal antibody, 381- 1E10, directed against the putative Pa epitope was shown by synergistic lysis and cold antibody competition to be directed to the immunodominant S epitope on RT1Aa. These data argue against the existence of a distinct Pa antigen or epitope detected by pregnancy sera.

The alloantibody response to semiallogeneic pregnancy in the rat. I. Alloantibodies in sera and placental eluates directed to RT1A antigens.

Maternal alloantibodies to paternal cells were monitored by cellular ELISA, the indirect hemagglutination and erythrocyte antibody rosette inhibition assays in sera and placental eluates from primigravid and multigravid inbred rats. In primigravid animals, antibodies in sera were routinely detected only by the indirect hemagglutination assay and were of low titer; weak antibody activity was detectable only by indirect hemagglutination in 1 of the 8 placental eluates assayed from these animals. Alloantibodies in high titer were present in sera and placental eluates from multigravid rats and were found to be directed predominantly to the RT1A (class I MHC) antigens of the paternal strain. These data provide no support for the hypothesis that the difficulty in detecting maternal antibodies during a 1st pregnancy is due to their preferential binding to antigenic determinants expressed on the placenta.

Early diagnosis of rejection. Correlation of enzymuria and histological findings after renal transplantation in the rat.

One hundred renal transplants were performed between different rat strain combinations. Posttransplant renal function was monitored using serum creatinine levels, creatinine clearance rates, and N-acetyl-beta-D-glucosaminidase (NAG) enzymuria. These biochemical measurements were compared with the morphological appearances of the transplanted kidney. Rises in NAG enzymuria proved to be useful diagnostic indicators of graft rejection with a false positive rate of only 6%. In 78% of the rejection episodes, the urinary NAG level rose 2 days before any elevation in serum creatinine concentration. There was a close correlation among increasing NAG enzymuria, decreasing renal function, and the structural events of rejection--indicating that the routine measurement of tubular enzymuria would be of value in the prediction and diagnosis of renal allograft rejection.

Noncytotoxic antibodies to paternal antigens in maternal sera and placental eluates.

Noncytotoxic antibodies were detected in sera from 6 of 7 primigravid women during the first trimester of pregnancy. Such antibodies directed to antigens expressed on paternal B lymphocytes were detectable within the first 4-5 weeks of gestation. Antibody activity toward paternal B lymphocytes was also detected in 6 of 10 placental eluates, and in 3 of 10 predelivery and 2 of 10 postdelivery maternal serum samples. When B lymphocytes from umbilical cord blood were used as target cells, antibodies were detected in 5 of 7 placental eluates, and in 3 of 7 predelivery and 2 of 7 postdelivery serum samples. These antibodies also reacted with selected members of a normal B lymphocyte panel. The concept of an immunological enhancing mechanism in normal pregnancy is supported by these data.

Long-term renal allograft survival in rats preimmunized with donor strain RT1.B antigens.

Rat renal allograft survival was enhanced by active immunization with donor strain RT1.B (Ia) antigens. Lewis (LEW) rats (16) were immunized with Brown Norway (BN) lymphocyte extracts containing RT1.B, but not RT1.A antigens, prior to receiving (LEW X BN)F1 renal allografts. Group 1 (8 rats) was immunized with lymphocyte membrane fragments group 2(8 rats) was primed with lymphocyte supernatant extract. Longterm survivors (greater than 60 days; 12 animals) had a mean blood urea nitrogen of 75 +/- 31 mg% and serum creatinine of 2.0 +/- 0.8 mg% at one month. Death occurred in 90% of control allograft recipients within 10 days. Anti-BN RT1.B but not RT1.A antibodies were detected in sera from actively enhanced rats following immunization and at day 7 posttransplantation. We conclude that preimmunization with cell extracts containing donor RT1.B antigens has a protective effect on the allograft, and that the phenomenon of active immunologic enhancement can be produced without immunization to RT1.A antigens.

Immune responses to noninherited maternal RT1A antigens in inbred rats.

Humoral responses to non-inherited maternal class I antigens (class I NIMAs) were assessed in 3 groups of inbred rats expressing the RT1u phenotype. Group 1 consisted of the progeny of (AO X DA)F1 X PVG matings; their haplotype was RT1u/c and their non-inherited maternal haplotype RT1a. Group 2 were the progeny of (AO X LEW)F1 X PVG matings the haplotype of which was also RT1u/c, but their non-inherited maternal haplotype was RT1l. Group 3 comprised 8 (AO X PVG)F1 (RT1u/c) hybrids. All rats received 2 intravenous blood transfusions (0.5 ml) from male DA (RT1Aa) donors on days 0 and 7. They were bled at weekly intervals for 6 weeks and again at 20 weeks after the first transfusion. Alloantibody responses to RT1Aa were assessed by an indirect hemagglutination assay (IHA)* and by a 51Chromium-release complement-dependent red cell cytotoxicity assay. All groups exhibited vigorous anti-class I antibody responses to the DA transfusions. No significant differences were detected, however, in antibody titers between the groups either by IHA or CDC or in the rates of decay of antibody titers up to week 20. In addition no blocking activity was found in sera obtained on day 0 from group 1 animals and tested for antiidiotypic antibody activity to cytotoxic anti-RT1Aa antibodies. In order to assess whether suppressor activity had been activated by the initial transfusions, in the animals in which class I NIMA was RT1Aa, all groups were rechallenged with a DA transfusion at week 20. All animals exhibited vigorous anamnestic responses to this challenge and no significant differences were detected between groups. In order to determine whether cellular tolerance to the noninherited maternal haplotype was present in group 1 animals, proliferative responses were assessed by one-way mixed lymphocyte cultures, using DA lymph node stimulator cells. No significant differences were detected in proliferative or kinetic responses between lymph node cells from rats the noninherited maternal haplotype of which was RT1a or from naive (AO X PVG)F1 hybrids. Peak proliferative responses to DA cells in rats the noninherited maternal haplotype of which was RT1l were similar, but maximal on day 4 as opposed to day 3. Hence in these inbred rat strains no evidence of humoral tolerance to class I NIMAs was detected. In addition there was no evidence of cellular tolerance to the noninherited maternal MHC haplotype.

The effects of rapamycin on humoral immunity in vivo. Suppression of primary responses but not of ongoing alloantibody synthesis or memory responses.

The effect of rapamycin on primary and secondary alloantibody responses to major histocompatibility complex class I antigens was investigated in inbred rat strains. Primary anti-MHC class I alloantibody responses, detected by indirect hemagglutination and complement-dependent cytotoxicity assays, were abrogated in high-responder WAG (RT1u) recipients of DA (RT1a) blood transfusions, given on days 0 and 7 of a 14-day course of rapamycin (3 mg/kg/day). Antibody class studies showed that both IgM and IgG responses were equally effectively inhibited. Moreover, when these animals were rechallenged with DA transfusions, 28 days after drug withdrawal, they exhibited donor-specific humoral unresponsiveness. Similar results were observed in cyclosporine-treated rats. In preimmunized high-responder LEW (RT1(1)) rats with high titer anti-DA class I alloantibodies, a 35-day course of rapamycin (3 mg/kg/day) had no significant suppressive effect on serum alloantibody levels when compared with untreated preimmunized control animals. WAG rats were immunized by DA transfusions and serum antibody levels then allowed to decay over 16 weeks. The animals were then challenged with a further DA transfusion given on the second day of a 14-day course of rapamycin (3 mg/kg/day). Alloantibody responses to the challenge transfusion in this group were not, however, significantly suppressed when compared with a non-drug-treated control group. The results of this study indicate that rapamycin is a potent inhibitor of primary alloantibody synthesis in high-responder rat strains, but does not significantly suppress alloantibody synthesis in animals with established humoral reactivity. These results may be of relevance if rapamycin is to be used in clinical renal transplantation, because in man similar antibodies mediate hyperacute rejection, and when they develop after transplantation are associated with very high rates of rejection.

The effect of cyclosporine on humoral and cellular alloreactivity to allogeneic pregnancy in rats.

We have evaluated the effect of a therapeutic dose (10 mg/kg/day) of the immunosuppressant cyclosporine (CsA) on humoral and cell-mediated immunity in rats during an allogeneic first pregnancy. Virgin female Lewis rats mated with DA males and treated with CsA vehicle produced a humoral response, as measured by both the erythrocyte rosette inhibition (EAI) and indirect hemagglutination assays. The capacity of Lewis splenocytes to mediate a graft-versus-host (GVH) reaction in six-week old F1 (Lewis X DA) hybrid rats was unaffected by either pregnancy or CsA. In mothers treated with CsA, however, no antibody production was detected, and a significant reduction in GVH reactivity was observed using their cells. This reduction was specific for the paternal strain. When compared with vehicle-treated primiparas, suppression of the immune response by CsA had no effect on either the number or viability of fetuses present in utero at day 20. These data suggest that antipaternal antibodies may not be essential to protect the fetus when there is concomitant suppression of the capacity of the mother's T cells to express a cell-mediated antipaternal response.

FK506--its influence on anti-class 1 MHC alloantibody responses to blood transfusions.

The influence of FK506 on in vivo alloantibody responses to major histocompatability class 1 antigens was investigated in inbred rat strains, and compared with the effect of cyclosporine. AO rats received transfusions of DA blood on days 0 and 7. From days 0 to 14 the rats also received, daily, either FK506 0.3 mg/kg suspended in saline or dissolved in olive oil, or CsA 10 mg/kg. The administration of FK506 suspended in saline at the time of blood transfusion completely abrogated the development of anti-MHC class 1 alloantibodies as detected by indirect hemagglutination (IHA)* and 51Cr release complement dependent cytotoxicity assays (CDC). Isotyping studies showed that FK506 suspended in saline suppressed IgM production and inhibited the switch to IgG production. Similar responses were seen in CsA-treated animals. In contrast, rats treated with FK506 dissolved in olive oil developed high titers of anti-class 1 alloantibodies. On days 49 and 56 the rats were challenged with further DA blood transfusions given without immunosuppression. In the groups given FK506 suspended in saline or CsA, cytotoxic antibodies did not develop; low titer antibodies were, however, detected by IHA in the animals that had previously received FK506 suspended in saline. The results indicate that FK506, in common with CsA, inhibits anti-class 1 MHC alloantibody production, and at the same time enables the development of tolerance. The vehicle in which FK506 is administered is, however, critical to its efficacy at the low doses used. These results may be of relevance to clinical transplantation as similar antibodies mediate hyperacute renal allograft rejection in man.

Reduction of sensitization induced by blood transfusion in the rat by monoclonal antibodies.

Primary and secondary alloantibody responses were monitored in (AOxPVG)F1 hybrid rats after three transfusions of DA blood; the initial transfusion was either untreated or pretreated with monoclonal antibody directed to class I antigens or other cell surface markers. Mean antibody activity in recipient sera against class I DA antigens was significantly decreased by pretreatment with the monoclonal antibodies. The most marked suppression was associated with pretreatment by antibodies to the four major nonoverlapping epitopes of the RT1Aa antigen. Subsequent transfusions of DA blood failed to stimulate a secondary response. Crossreactivity of the alloantibody reactivity with BDIX antigens was diminished by pretreating the transfusions with rat anti-RT1A antibodies and, to a lesser extent, with a mouse monoclonal antibody (OX-18) to a common class I determinant. Monoclonal antibody pretreatment had no effect on the humoral response to class II DA antigens. These studies indicate that blood transfusions pretreated with monoclonal antibodies induce a less-potent cytotoxic humoral immune response and that reactivity is most effectively suppressed by completely masking the class I antigen. This technique may prove of clinical value in preventing the sensitization caused by blood transfusions in potential transplant recipients.

Evidence that protective Fc-receptor-blocking antibodies in renal transplantation are alloantibodies not autoantibodies.

We have previously shown that the presence in pretransplant recipient sera of Fc receptor blocking antibodies detected by the EA inhibition assay is correlated with improved allograft survival. Twenty-four such sera were assessed for the presence of autoantibodies by the EA inhibition and lymphocytotoxicity assays. No autolymphocytotoxic antibodies were found, and autologous EA inhibition was noted in only one case. EA-inhibiting alloantibodies did occur, and their presence was correlated with improved allograft survival. Sera from 37 dialysis patients were also studied, and neither autologous EA inhibiting nor autologous lymphocytotoxic antibodies were present. Thus Fc receptor blocking alloantibodies that were correlated with improved renal transplant survival were not autoantibodies.

Cyclosporine-induced fetotoxicity in the rat.

Female Lewis rats mated with DA male rats were given 25 or 10 mg/kg/day cyclosporine (CsA) from the time of mating to 20 days postcoitus when autopsies were performed. At the higher dose, characteristic drug-induced pathological changes in the mother were accompanied by a striking fetotoxic effect, resulting in a high incidence of fetal mortality or in runting. In addition, fetal kidneys that could be examined showed evidence of CsA-induced proximal tubular cell damage. These abnormalities were not found at the lower dosage of CsA. The fetotoxicity observed at 25 mg/kg did not appear to be dependent on major histocompatibility differences between parental strains, since it was also observed, at the same dosage, in syngeneically mated female Lewis rats.

Immunological aspects of clinical renal transplantation.

Improved facilities for treating patients with end-stage renal failure have resulted in more elderly and debilitated patients being accepted for treatment. Renal transplantation is now the optimum form of treatment but organ procurement has failed to match clinical demand. Future developments may focus on further non-specific immunosuppressive agents. As one year survival rates for first cadaver allografts now exceed 85% in many units, the significance of new developments will be increasingly difficult to evaluate.

Identification of subpopulations of T lymphocytes and Ia positive B lymphocytes using a rosette assay based upon the biotin-avidin interaction.

Bovine erythrocytes were coated with avidin using a chromic chloride coupling technique and used successfully in an indirect rosette assay to identify and quantitate: (a) T-lymphocyte helper and suppressor subpopulations, and (b) Ia positive B-lymphocyte populations. Using mouse monoclonal antibodies to the T-lymphocyte markers OKT3, OKT4, and OKT8 it was shown that the proportion of OKT4 and OKT8 positive cells were respectively 60.9 and 39.2% of the total OKT-3 positive T lymphocytes. In a similar way, using a monoclonal anti-human Ia antibody, the percentage Ia positive cells in B-lymphocyte enriched preparations was shown to vary between 18 and 55% for normal peripheral blood and 31 and 58% for chronic lymphocytic leukaemia derived peripheral blood.

Maternal alloantibody responses during early pregnancy detected by a cellular enzyme-linked immunospecific assay.

Using a cellular enzyme-linked immunospecific assay (CELISA), we have examined sera from nulliparous women and women in the first trimester of a first or subsequent pregnancy for the presence of antibodies directed to surface determinants on peripheral blood lymphocytes from unrelated donors. Maternal antibody activity was found in sera from 1/13 nulliparae, 19/37 primigravidae, and 8/12 multigravidae. Cytotoxic antibody activity was present in 3/12 multigravidae but in no other group. Absorption with packed, pooled platelets did not remove the antibody activity from three of the primigravid sera; unabsorbed sera, however, bound equally well to T and B lymphocytes. These data suggest that the antibody detected by CELISA is not directed to any of the classical HLA antigen series (-A, -B, -C, or -DR) but may be directed to the HLA linked non-class I HT antigen system.

Comparing outcomes in renal replacement therapy: how should we correct for case mix?

The need to evaluate the effectiveness of clinical practice to justify expensive therapy in the face of financial constraints in all areas of health care delivery makes it necessary to identify groups of patients who are likely to benefit most from treatment. Various risk stratification methods have been used for analyzing survival probabilities for patients receiving renal replacement therapy. Complicated risk stratification methods produce large numbers of risk groups of small sizes, which makes comparison between individual centers difficult. We compared three simple methods of risk stratification, that divided patients into low-, medium-, and high-risk groups, in a cohort of 1,407 patients who commenced renal replacement therapy in five European countries during a 7-year period. Method 1 considered age (>55 years) and diabetes alone; method 2 used a higher age limit (>70 years) and comorbid illnesses, including those other than diabetes; and method 3 used only the number of comorbidities (none, 1, or > or =2) for stratification. Kaplan-Meier survival curves were constructed for comparison between risk groups and Cox's regression model used to assess strength of relationship with mortality. Although patient survival was significantly different between the low-, medium-, and high-risk groups using all three methods, Cox's regression analysis showed that method 2 provided the greatest discrimination between risk groups. In predicting mortality, method 2 (based on comorbidities and age) showed the highest sensitivity and specificity (84% and 80%, respectively) compared with method 1 (80% and 74%) and method 3 (64% and 82%). Validation of this approach in other populations in a prospective study is required before this method, which takes into account the influences of both age and comorbidity for risk stratification, can be used for comparing survival data and for presenting results of renal replacement therapy.